1ISBN:9788894140422

Macromolecules in Drug Delivery

ABSTRACT BOOK

with the kind support of

CRSItalyChapterWORKSHOP2017FiscianoCampus(SA),October26-28,2017

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Supramolecular assemblies for therapeutic proteins delivery Andretto Valentina, Malfanti Alessio, Mastrotto Francesca, Caliceti Paolo, Salmaso Stefano

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy. Email: stefano.salmaso@unipd.it

Background

In the last few decades, since the use of recombinant human insulin for therapy, biopharmaceutical drugs (including peptides, recombinant therapeutic proteins, enzymes, monoclonal antibodies and antibody-drug conjugates) have significantly ameliorated the efficiency of many therapeutic treatments.1 Peptides and proteins are characterized by high selectivity and pharmacological activity that make them unique for the treatment of severe diseases. However, the clinical use of these macromolecules is often limited by their physico-chemical and structural instability (e.g. aggregation, denaturation, enzymatic degradation, immunogenicity, etc.) compared to conventional small drugs. In particular, insulin stability in aqueous media is affected by conformational alterations resulting in loss of biological activity, immunogenicity and formation of insoluble fibrils.

Many efforts have been dedicated to develop several strategies to overcome the current limitations of therapeutic peptides and proteins. Two main approaches were investigated: either the modification of the protein sequence by aminoacidic mutations and by conjugation with water soluble polymers, or by dedicated formulation strategies. Advanced delivery systems, such as liposomes, polymeric microspheres and nanoparticles, are some of the available approaches used to overcome protein limitations as therapeutic agents.2

In the present study, we have explored an innovative approach for the delivery of insulin by exploiting the physical interactions between PEG based amphiphilic polymers and the therapeutic protein surface. In virtue of their peculiar physico-chemical properties, natural steroidal molecules, such as cholesterol and other polycyclic surfactants naturally occurring in the bile acids, have been largely exploited to confer amphiphilic properties to water soluble polysaccharides and synthetic polymers to generate self-assembling novel materials.3

We compare here the biopharmaceutical properties of a novel amphiphilic material, mPEG5kDa-Cholane and the commercially available mPEG5kDa-Cholesterol. These micelle-forming amphiphilic polymers have been found to physically associate to proteins and, in particular, to increase insulin solubility and stability in aqueous media, which can be very relevant for shelf-life and therapeutic performances of advanced drug delivery devices such as microinfusion pumps.

Main results The amphiphilic PEG derivatives (mPEG5kDa-Cholane and mPEG5kDa-Cholesterol) were generated by conjugating mPEG5kDa-

NH2 to either cholesterol, or 5-β-cholanic acid. Notably, the latter is a physiologic steroid derivative with no specific biological activity and a very limited toxicity. The conjugation efficiency was assessed to be >90% for both derivatives, as confirmed by colorimetric Snyder assay and 1H NMR analysis.

Initially, several physical investigations were carried out to clarify the mechanism by which the two amphiphilic polymers, mPEG5kDa-Cholane and mPEG5kDa-Cholesterol, associates with insulin. Dissolution studies showed an improvement in the protein solubility of about 85% and 73% for mPEG5kDa-Cholane and mPEG5kDa-Cholesterol, respectively, which proved the association. Isothermal Titration Calorimetry (ITC) analysis was used to assess the thermodynamic parameters related to the polymer/insulin interaction. The ITC results showed that hydrophobic forces are prevalent in the association and that the formation of this supramolecular system is energetically favoured, with similar thermodynamic parameters for both the PEG derivatives. The association studies also showed that the presence of the two amphiphilic polymers favour the monomeric form of insulin with respect to self- aggregation of insulin into multimeric entities. This results were confirmed by Transmission Electron Microscopy (TEM) images that showed how insulin alone undergoes the formation of dimeric, tetrameric and esameric aggregates.

In order to better understand how the mPEG derivatives shift the conformational equilibrium of insulin to the monomeric form, Circular Dichroism (CD) studies were undertaken with and without the two anphiphilic polymers. In the presence of the polymers, insulin α-helix content increase and this highlights the stabilization effect of mPEG- derivatives in its monomeric conformation.

Fibrillation studies carried out by Thioflavin T (ThT) fluorescence assay under different pH and temperature conditions showed that both polymers were very efficient in reducing insulin's fibrillation over time. The morphology and size of fibrils were also evaluated by imaging analysis with TEM microscopy.

Future perspective Physical PEGylation of insulin can be exploited to enhance its biopharmaceutical properties, resulting from improved

physical and colloidal stability during formulation, dissolution and storage. This promising technology is a valuable platform that can potentially be applied to other proteins and peptides.

Notably, this physical PEGylation technology avoids the generation of “new drug entities”, which can be convenient to achieve approval from the safety agencies for human use.

This insulin formulation will be tested in vitro and in vivo to extensively profile its biopharmaceutical features for a therapeutic application with microinfusion pump devices.

References

1. Mitragotri S, Burke PA, Langer R. Overcoming the challenges in administering biopharmaceuticals: Formulation and delivery strategies. Nature Reviews Drug Discovery 2014;13(9):655-72.

2. Pisal DS, Kosloski MP, Balu‐Iyer SV. Delivery of therapeutic proteins. J Pharm Sci 2010;99(6):2557-75.

3. Salmaso S, Caliceti P. Self assembling nanocomposites for protein delivery: Supramolecular interactions of soluble polymers with protein drugs. Int J Pharm 2013;440(1):111-23.

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Nanobubble-based HER2 immunotherapy through dendritic cells targeting Argenziano Monica1, Occhipinti Sergio2, Guiot Caterina3, Giovarelli Mirella2, Cavalli Roberta1

1Department of Drug Science and Technology, University of Torino 2Department of Molecular Biotechnology and Health Science

3Department of Neuroscience,University of Torino Background In the recent years continuous efforts in the field of cancer immunotherapy have led to the development of several vaccination strategies based on tumor associated antigen, such as HER2 oncogene [1]. Cancer vaccination offers distinct advantages over standard therapies such as higher specificity, lower toxicity and long-term effects due to immunologic memory. In order to correctly expand the immune response against tumor, a vaccine needs to effectively reach the dendritic cells (DCs), which play a critical role in inducing a proper immune activation [2]. Polymer nanobubbles (NBs), spherical core/shell nanostructures filled by a gas or vaporizable compounds (i.e. perfluoropentane) with sizes in the nanometer order of magnitude, are versatile multifunctional nanocarriers for the delivery of drugs and genes [3]. Through decoration of their surface with specific antibodies it is possible to convey NBs to DCs, promoting tumor antigen delivery. This work aims at designing a novel immunotherapeutic tool for the treatment of HER2+ breast cancer. At this purpose, chitosan-shelled NBs loaded with DNA vaccine and functionalized with anti-CD11c antibody to target DCs were developed. Main Results Targeted DNA-loaded chitosan-shelled NBs with sizes of about 300 nm and positive surface charge were obtained. Complexation of DNA with chitosan NBs and stability of DNA-loaded NBs were confirmed by electrophoresis on agarose gel. NBs functionalized with anti-CD11c showed high efficiency in transfecting hDCs with high selectivity for this cell type. Targeted NBs induced an increased expression of maturation marker (CD86, CD83, HLA-DR) and an increased secretion of inflammatory cytokines (IL-12 and IL-6) compared to untreated DCs. Intradermal injection of CD11c-NBs elicited migration of dermal DCs to draining lymph nodes in mice model. Additionally, intradermal injection of pHER2-loaded CD11c-NBs delayed growth of HER2+ tumors in mice by eliciting cellular and humoral immune responses. Future Perspective DCs-targeting chitosan nanobubbles loaded with tumor vaccine may provide an attractive nanotechnology approach for the future treatment of patients. This strategy might be of great importance compared to the ex-vivo loading of DCs with antigens, that is labor intensive and costly making its application limited. References 1) Occhipinti, S.; et al. Chimeric rat/human HER2 efficiently circumvents HER2 tolerance in cancer patients. Clin. Cancer Res. 2014, 20(11):2910-2921; 2) Torres Andon, F.; Alonso, M.J. Nanomedicine and cancer immunotherapy - targeting immunosuppressive cells. J Drug Target. 2015, 23(7-8):656-671; 3) Cavalli, R.; et al. New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization. Int. J. Nanomedicine 2012, 7:3309-3318.

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Non-peptidic Cell Penetration Enhancer as tools for lipoplexes formulation Balasso Anna1, Mattarei Andrea2, Paradisi Cristina2, Cavedon Cristian2, Caliceti Paolo1, Salmaso Stefano1

1Dept. of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy; 2Dept. of Chemical Sciences, University of Padova, Via F. Marzolo 1, Padova – Italy.

Email: stefano.salmaso@unipd.it

Background The use of oligonucleotide (ON) as therapeutics has emerged recently as a promising approach to treat liver and

neurodegenerative disorders, hypercholesterolemia, and, notably, also as potential anti-cancer agents. Despite the many successes obtained in different clinical trials, effective delivery of ONs in vivo at their specific intracellular site of action remains a considerable issue. In this scenario, the development of effective strategies to selectively deliver the therapeutic ONs at the target site has emerged as a major goal in the biopharmaceutical research area [1; 2].

In this context, we aim at developing a novel liposomal platform with the ability of readily condense ONs while enhancing the cell penetration capacity of the carrier resulting in improved intracellular delivery of the therapeutic ONs. At this aim, we designed a newly synthesized lipid terminated cell penetration enhancer (CPE) that was associated to the liposome bilayer. The CPE we developed is intended to mimic the naturally occurring CPEs, such as the TAT peptide, but is synthetic and devoid of peptidic backbone thus preventing possible drawbacks such as poor chemical and enzymatic stability, risk of toxicity, immunogenicity and intrinsic biological activity.

The high density of the positive charges on the CPE is expected to favour cell penetration of the nanocarrier according to a charge-charge interaction with the anionic proteoglycans located on the cell membrane [3]. Notably, the CPEs do not possess any cell type specificity and can be responsible for unspecific association to cells and tissue distribution of the carrier and the consequent side effects of the loaded drug. For this reason, the interfacial properties of liposomes were carefully investigated and the surface decoration of the nanocarrier with mPEG2kDa-DSPE or mPEG5kDa-DSPE was carefully investigated design in order to modulate the “stealth” properties of the system while providing cell penetration behaviour. Main results

The novel dendronic CPE was designed and synthesized by coupling arginines to a dendritic scaffold based on 2,2-bis(methylol)propionic acid (bis-MPA). Then, the CPE was end terminated with a dialkyl moiety to obtain a polycationic system suitable for the anchoring to the liposome bilayer.

Initially, the formulation studies were carried out to select the lipidic compositions that ensure the most efficient dsDNA loading efficiency of the lipoplexes while guaranteeing adequate colloidal stability. Lipoplexes prepared with 4 mol% of CPE (NO mPEG-DSPE) and formulated with a N/P ratio above 5 displayed high stability and dsDNA loading capacity. The DLS analyses of these formulations showed a lipoplex size of about 250 nm and a ZP of +16 mV. The lipoplexes with N/P ratios of 5 and 10 were found to load dsDNA with a 8.4- and 9.4-fold higher efficiency with respect to the corresponding CPE-free lipoplexes. In addition to that, these formulations showed great stability in HEPES buffer for more than 7 days, therefore they were selected for further studies.

The mPEG-DSPE coating was added to endow the system with “stealth” properties and to reduce the opsonization process that occurs in the presence of serum proteins. Indeed, mPEG5kDa-DSPE proved to provide a better shielding of lipoplexes compared to mPEG2kDa-DSPE when tested in medium supplemented of FBS, showing a size and a ZP values that remained constant over time up to 48 hours.

In vitro cell viability assay on MDAMB 231 cells revealed that CPE-decorated lipoplexes without PEG coating possess a moderate toxicity, while all the formulation coated with PEG were well tolerated by cells proving the PEG coating is required to generate biocompatible systems. Cytofluorimetric results of lipoplexes obtained with a 10:1 N/P molar ratio showed that the cellular uptake of the CPE-decorated lipoplexes (NO mPEG-DSPE) is significantly higher with respect to the CPE-free lipoplexes generated at the same N/P molar ratio. Moreover, it is noteworthy that, despite the overall neutral zeta potential of CPE-lipoplexes coated with mPEG5kDa-DSPE, they undergo remarkable cell uptake , suggesting that the CPE on the lipoplexes surface can still exert its function when the vesicles approach cell membrane as a consequence of its exposure.

Finally, the interaction between CPE and dsDNA was also measured by ITC analyses, revealing an exothermic association process with an affinity constant in the order of 107. Future perspective

Through dedicated formulation studies we demonstrated that the simultaneous surface decoration of lipidic vesicles with CPE and mPEG-DSPE endows dsDNA loaded liposomes, with increased stability either in buffer or in cell culture media with serum. Preliminary in vitro studies demonstrated that these carriers are devoid of toxicity on cells and that they promptly interact with cells. High level of lipoplexes association with cell were obtained also in the presence of mPEG5kDa-DSPE coating, highlighting that CPE activity is preserved.

In the light of this encouraging results we believe that these formulations is a promising platform for the improvement of ONs delivery in vivo. This platform may be suitable for the developed for the local treatment of bladder or lung cancer. Indeed, these tumors possesses a variety of barriers that must be successfully overcome by the use of carriers endowed with penetration features provided by finely tuned surface properties of the lipoplexes.

References

1. Juliano, R.L. The delivery of therapeutic oligonucleotides. Nucleic Acids Research, 2016, 44, 14, 6518-6548. 2. Rezaee, M.; Oskuee, R.; Nassirli, H.; Malaekeh-Nikouei, B. Progress in the development of lipopolyplexes as efficient non-viral gene

delivery systems. J. of Controlled Release, 2016, 236, 1-14. 3. Verdurmen, W.P.R.; Wallbrecher, R.; Schmidt, S.; Eilander, J.; Bovee-Geurts, P.; Fanghanel, S.; Burck, J.; Wadhwani, P.; Ulrich, A.S.;

Brock, R. Cell surface clustering of heparan sulfate proteoglycans by amphipathic cell-penetrating peptides does not contribute to uptake. J. of Controlled Release, 2013, 170, 83-91.

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SLN for oral insulin delivery: preliminary ex vivo/in vivo studies

Ahmadi Nahid1, Marini Elisabetta2, Muntoni Elisabetta2, Milla Paola2, Battaglia Luigi*2

1 University of Sistan and Baluchistan, Department of Chemistry 2 Università degli Studi di Torino, Dipartimento di Scienza e Tecnologia del Farmaco, via Pietro Giuria 9, 10125, Torino (Italy) +390116707142

email: luigi.battaglia@unito.it

Background Subcutaneous administration of insulin in diabetic patients is associated with the distress and difficulty of daily injections. Among alternative administration routes, the oral route seems to be the most advantageous for long-term administration, also because the peptide undergoes a hepatic first-pass effect, contributing to the inhibition of the hepatic glucose output. Unfortunately, insulin oral administration has so far been hampered by degradation by gastro-intestinal enzymes, and poor intestinal absorption, due to high molecular weight. Incorporating insulin in solid lipid nanoparticles (SLN) should have the effect of protecting the drug against proteolytic degradation and enhancing its absorption, also through the Peyer’s Patches. Insulin entrapment into SLN is not easy, because of its high molecular weight, hydrophilicity and thermo-sensitivity. In this study insulin encapsulation in SLN was obtained operating at mild temperatures with an hydrophobic ion-pairing (HIP) approach, coupled with the solvent-free fatty acid coacervation technique [1, 2, 3]. Preliminary in vitro, ex vivo, in vivo characterization of the formulations was performed. Main Results Insulin entrapment in the lipid matrix was obtained by using either sodium dodecyl sulfate HIP, or polystyrene sulfonate complexation (polymer-lipid hybrid nanoparticles - PLN); glargine insulin was also employed, owing to its modified aminoacidic sequence and solubility. Drug release in gastrointestinal medium can be modulated according to the different encapsulation strategy employed. Stearic acid SLN were compared to newly engineered stearic/oleic acid nanostructured lipid carriers (NLC): a suitable procedure was established for NLC formulation, that were preliminarily characterized through dynamic light scattering and differential scanning calorimetry (DSC – Figure 1). Bioactivity of lipid entrapped insulin was demonstrated through a suitable in vivo experiment. Selected SLN and NLC were loaded with fluorescently labelled insulins, that were in-house synthesized and characterized: ex vivo gut tied up experiments showed encouraging intestinal uptake of labelled insulin from NLC formulations (Figure 2). Future Perspectives In order to study the in vivo uptake, a pharmacokinetic approach will be followed on healthy rats, by employing nanoparticles loaded with in-house fluorescently labelled insulin, thus avoiding cross-reaction with endogenous insulin, that is typical of immunoassays. The role of gut lymphatic uptake will also be investigated through a thoracic duct cannulation experiment in healthy rats. Blood glucose responsivity assays will be performed after pharmacokinetics studies, because highly dependent on the animal model employed (healthy or diabetic) and on insulin absorption kinetic.

References:

1. Powers M.E.; Matsuura J.; Brassell J. et al. Enhanced solubility of proteins and peptides in nonpolar solvents through hydrophobic ion pairing. Biopolymers, 1993, 33, 927–932. 2. Battaglia, L.; Gallarate, M.; Cavalli, R.; Trotta, M. Solid lipid nanoparticles produced through a coacervation method. J Microencapsul, 2010, 27, 78–85. 3. Gallarate, M.; Battaglia, L.; Peira, E. et al. Peptide-loaded solid lipid nanoparticles prepared through coacervation technique 2011. International Journal of Chemical Engineering doi:10.1155/2011/132435.

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Liposomal Active Delivery of Metformin: a New Tool in Cancer Treatment?

Bellato Federica, Montopoli Monica, Vianello Caterina, Catanzaro Daniela, Brazzale Chiara, Balasso Anna, Brunato Silvia, Caliceti Paolo, Salmaso Stefano

Dept. of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy; Email: stefano.salmaso@unipd.it

Background

Metformin is a widely-used drug in the treatment of type-2 diabetes and it has recently been found to inhibit cancer cell growth in vitro by impairing the mitochondrial complex I functions [1]. However, evidences where obtained under high drug concentrations that cannot be applied in vivo. Furthermore, metformin is transported into the cytosol by the saturable transporter OCT1, expressed by many cancer cells at very low levels compared to the liver, where metformin exerts its physiologic antidiabetic effect [2].

The purpose of this work was to develop and investigate an actively-targeted metformin-loaded liposomal vehicle able to bypass the cell-saturable transporter thus enhancing the intracellular delivery of the drug at the tumor site, and to better explore the mechanisms of mitochondrial function inhibition. Notably, the liposomal platform is expected to ameliorate the drug pharmacokinetic profile, prolonging the circulation time and favouring the tumor accumulation via the Enhanced Permeability and Retention (EPR) effect. In addition, folic acid was exploited as targeting agent by its conjugation to a NH2-PEG-DSPE and its subsequent insertion in the lipid bilayer. As a matter of fact, folate receptor (FR) overexpression in cancer cells promotes cellular uptake of folate-targeted Drug Delivery Systems.

Main results NH2-PEG3.4kDa-DSPE was modified with folic acid via a standard coupling procedure [3], with a 89% conjugation efficiency.

The targeting agent was used to decorate either empty or metformin-loaded stealth liposomes. Homogenous liposomes of 220 nm (PDI=0.2) were obtained by thin layer rehydration method using Hydrogenated Soybean Phosphatidyl Choline (HSPC)/cholesterol at a 3:2 molar ratio to prepare the lipid film. Liposomes stability was also confirmed in serum-supplemented media, with only a slight size increase respect to dimension in buffer, either for empty and for metformin-loaded liposomes. Metformin loading capacity of the liposomes was found to be 55 mol% with respect to lipids, and the encapsulation efficiency was 3%.

Liposomes were decorated with 3 mol% of mPEG2kDa-DSPE with respect to lipids to generate stealth nanocarriers and with increasing density of Folate-PEG3.4kDa-DSPE (from 0 mol% to 1 mol%) to select the optimum density for liposome selective internalization by cancer cells. Liposomes decoration was found not to affect significantly neither metformin loading nor its release rate. Indeed, at 37 °C and physiological pH all formulations showed an initial metformin burst release of 25% during the first 6 hours with subsequent slow drug release profile (≈ 40% release after 6 days).

KB cells overexpressing FR were incubated for 6 hours with Rhodamine-labelled liposomes either naked or decorated with 0.1, 0.25, 0.5, 0.75, 1 mol% Folate-PEG3.4kDa-DSPE with respect to lipids. Decoration of liposomes with 0.5 mol% of Folate-PEG3.4kDa-DSPE by a post-insertion procedure resulted to provide the highest liposome internalization by KB cells. Confocal microscopy confirmed the active uptake of vesicles and their localization in the cytosol. Since folic acid is internalized by KB cells via receptor-mediated endocytosis, we postulated that folate- targeted liposomes should exploit the same pathway.

To investigate the biopharmaceutical effect of active delivery of metformin to KB cells, trypan blue exclusion test was performed to assess cell viability after 24 hours incubation of cells with targeted liposomes decorated with 0.5 mol% of Folate-PEG3.4kDa-DSPE with respect to lipids or non-targeted metformin-loaded liposomes, non-loaded liposomes or equivalent concentration of free drug. The results showed a moderate decrease of cell viability (about 30%) only for metformin-loaded folate-targeted liposomes at a 0.5 mM drug concentration, while equivalent concentration of free metformin or metformin loaded non-targeted liposomes did not induce any toxicity. Empty liposomes showed no relevant cellular toxicity. Hence, the actively-targeted liposomes enable a higher metformin cell entry in comparison to the free drug

Confocal microscopic images after mitochondrial staining with MitoTracker® Orange dye showed a slight difference in the mitochondrial network upon incubation of KB cells with targeted metformin-loaded liposomes for 24 hours, that could be due to a mitochondrial stress induced by metformin. Future perspective

In this work we have proved that metformin delivery by folate-targeted liposomes represents a novel strategy to ameliorate the drug anticancer activity and will be further exploited to better understand its mechanism of action at subcellular level. Interestingly, free metformin showed no toxic effect toward cancer cells further proving the paramount role of an efficient drug delivery system.

Ongoing studies using a mitochondriotropic targeting approaches will be explored to further enhance the organelle-specific effects.

References 1. Wheaton, W.W.; Weinberg, S.E.; Hamanaka, R.B.; Soberanes, S.; Sullivan, L.B.; Anso, E.; Glasauer, A.; Dufour, E.; Mutlu, G.M.;

Budigner, G.S.; Chandel, N.S. Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. Elife, 2014, 3, e02242. 2. Zhou, M.; Xia, L.; Wang, J. Metformin transport by a newly cloned proton-stimulated organic cation transporter (plasma membrane

monoamine transporter) expressed in human intestine. Drug Metabolism and Disposition, 2007, 35(10), 1956-1962. 3. Gabizon, A.; Horowitz, A.T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit, F.; Qazen, M.M.; Zalipsky, S. Targeting folate receptor with

folate linked to extremities of poly (ethylene glycol)-grafted liposomes: in vitro studies. Bioconjugate chemistry, 1999, 10(2), 289-298.

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Curcumin loaded nanoparticles in Alzheimer's disease Belletti D (a), Pederzoli F (a), Keller J (b), Duskey J (a), Oddone N (a), Vandelli MA (a), Tosi G (a), Grabrucker AM (b), Ruozi B

(a). (a)Department of Life Sciences, University of Modena and Reggio Emilia, Italy; (b) WG Molecular Analysis of Synaptopathies, Neurology Dept.,

Neurocenter of Ulm University, Ulm, Germany;

Background: Besides tau pathology, Alzheimer’s disease (AD) is characterized by abnormal accumulation of amyloid beta (Aβ) protein and/or its assembly into paired helical filaments and plaques. The progressive accumulation of Aβ aggregates triggers a cascade of toxic events that contributes to the progression of AD. Curcumin has been reported to display anti-amyloidogenic activity, not only by inhibiting the formation of new Aβ aggregates, but also by disaggregating existing ones (1). Unfortunately, the uptake of crcumin into the brain is severely restricted by its low ability to cross the blood-brain barrier (BBB). Therefore, novel strategies for a targeted delivery of curcumin into the brain are highly desired (2). Here, we encapsulated curcumin as active ingredient in PLGA (polylactide-co-glycolic-acid) nanoparticles (NPs), modified with g7 ligand for BBB crossing (3). Main results: Applying a single emulsion method, we obtained surface modified loaded NPs (g7-NPsCur) with a mean diameter of about 200 nm and negative Z-Potential (-22 mV). The amount of residual surfactant (PVA) was less than 8% of the total formulation mass and compatible with parenteral administration. ESCA analysis confirmed the presence of N signals indicating that the location of g7 is on the surface of g7-NPsCur, meaning an efficient surface engineering of NPs with BBB crossing ligand. g7-NPsCur showed drug loading capacity (LC) of about 3% (w/w) and an encapsulation efficiency (EE) of about 60%. The release profile of curcumin from NPs in simulated physiological condition (PBS buffer added of 50% v/v of serum, in respect to sink conditions) showed an initial massive “burst release” (about 60/70%) followed by a second phase with a controlled release. We performed in vitro experiments on primary hippocampal cell cultures demonstrating g7-NPs-Cur are non-toxic for cells at different cocentrations and able to deliver 10 uM of cur. Moreover, g7-NPs-Cur are able to reduce oxidative stress and to inhibit Aβ aggregation as well as to promote Aβ disaggregation more efficiently with respect to un-loaded free Cur. Future Prospectives: Results obtained suggest the potential role of brain targeted loaded nanoparticles in stabilizing curcumin, potentially solving the problem of administration and potentiating the effect of curcumin against Aβ related toxicity. In perspectives, considering that PLGA based carriers have already obtained FDA approval for clinical uses, it is conceivable that g7-NPs loaded with curcumin and specifically designed to transport active peptide to CNS could represent an effective therapeutic option for AD treatment. Based on the data obtaind in this study, the project will advance to in vivo experiments in animal model of AD (5*FAD) injecting different doses of g7-NPsCur and relative controls (NPsCur and Cur alone) and analyzing the amount of curcumin delivered to the brain, the effect on plaque formation and on memory losting (behaviural tests). References: 1. Yang, F.; Lim, G.P.; Begum A.N.; Ubeda O.J; Simmons M.R; Ambegaokar S.S; Chen P.P; Kayed R.; Glabe .C.G;.Frautschy A.; Cole G.M. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo J. Biol. Chem.2005, 280, 5892-5901. 2. Klinger, N.V.; Mittal, S.Therapeutic potential of curcumin for the treatment of brain tumors Oxid. Med. Cell. Longev., 2016, 2016, 9324085. 3. Tosi, G.; Costantino, L.; Rivasi, F.; Ruozi, B.; Leo, E.; Vergoni A.V.; Tacchi, R.; Bertolini, A.; Vandelli M.A.; Forni F. Targeting the central nervous system: in vivo experiments with peptide-derivatized nanoparticles loaded with Loperamide and Rhodamine-123 J. Control. Release, 2007, 122, 1-9.

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Novel albumin nanoparticles for treatment of resistant cancer cells Bessone Federica, Ferrara Benedetta, Varese Alessandra, Dianzani Chiara and Cavalli Roberta

Department of Science and Drug Technology, University of Turin, Via P. Giuria 9, 10125 Turin, Italy

Background. Resistance to chemotherapy is a major problem that limits the effectiveness of a successful treatment of cancer. Tumours may be intrinsically resistant to one or more anticancer drugs or be initially sensitive to them and acquire the resistance after repeated treatments. Moreover they can become cross-resistant to other drugs with different mechanisms of action. The anthracycline doxorubicin (DOXO) is an antineoplastic agent used in the treatment of a wide range of cancers, such as multiple myeloma, lung, ovarian, gastric, thyroid, breast, sarcoma, and pediatric cancer. In addition to problems with cardiotoxicity, DOXO can also confer resistance, in a mechanism that involves cellular transporters. ATP-dependent drug efflux pumps increase DOXO efflux via altered or increased expression. For this reason, discovery of new approaches to overcome DOXO resistance has been the focus of extensive research. Albumin based nanoparticles represent an ideal nanocarrier system because of its low cost, high availability, easy purification and better drug-loading capacity. Besides, this advanced drug delivery system can interact with both hydrophobic and hydrophilic therapeutic molecules, provide controlled release of drug and be easy modified due to the presence of functionally charged surface groups. In this work, DOXO-loaded bovine serum albumin based nanoparticles (DOXO-NPs) were prepared by a coacervation method. Glycol chitosan coated and un-coated formulations were studied and compared. Main results. The formulations were characterized measuring the size, the polydispersity index and the zeta potential by laser light scattering. Transmission electron microscopy (TEM) was used for studying the nanoparticles morphology. The formulation stability was confirmed in the short and long term. Albumin nanoparticles showed a size of about 400 nm and a negative surface charge. On the other hand, glycol chitosan coated nanoparticles had a positive charge. DOXO was encapsulated in a great extent and was released from the nanoparticles with a prolonged in vitro release kinetics. Biological assays were performed on A2780 res, an ovarian cancer cell line resistance for DOXO. Cell viability assay (MTT test) was carried out and DOXO-NPs showed an higher cytotoxicity than the free drug after 24 and 48 hours of incubation. Moreover DOXO-NPs cell uptake was evaluated by confocal microscopy. Future perspective. Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. If it could be overcome, the impact on survival would be highly important. Since DOXO-NPs showed a significant cytotoxicity on a cell line resistance for DOXO, they may represent a novel approach to improve the efficacy of cancer therapy. References: 1. Bhushan, B.; Khanadeev, V.; Khlebtsov, B.; Khlebtsov, N.; Gopinath, P., Impact of albumin based approaches in nanomedicine: Imaging, targeting and drug delivery, Adv Colloid Interface Sci, 2017, 246, 13-39. 2. Zhitnyak, I;, Bychkov, I.; Sukhorukova, I. V.; Kovalskii, A. M.; Firestein, K.; Golberg, D.; Gloushankova, N.; Shtansky, D. V., Effect of BN Nanoparticles Loaded with Doxorubicin on Tumor Cells with Multiple Drug Resistance, ACS Appl Mater Interfaces, 2017

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Influence of PEG-lipids nature and architecture on liposome behaviour

Brazzale Chiara, Poli Ilaria, Mastrotto Francesca, Caliceti Paolo, Salmaso Stefano

Dept. of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy Email: stefano.salmaso@unipd.it

Background

Several studies reported in literature have shown that conventional liposomes are rapidly removed from the blood by the mononuclear phagocyte system (MPS) and they tend to aggregate and interact with off-target cells1. In this panorama, polyethylene glycol (PEG) has emerged as polymer of choice for the surface decoration not only of liposomes but also of other drug delivery systems as it creates a steric flexible shell preventing the serum protein adsorption (opsonisation) and avoiding recognition by the immune system followed by rapid clearance from the body2.

PEG derivatives are available on the market in a wide range of structures and displaying different terminal groups. These variable physico-chemical parameters of the coating polymer could affect the liposome stability, their stealth features and, importantly, the drug release3.

In this study, we have comparatively investigated the effect of lipo-polymer architecture, molecular weight and lipophilic anchors on the colloidal stability and the pharmacokinetic behavior of liposomes.

Main results Liposomes were prepared by thin layer rehydration method and decorated with 5% mol of PEG-based lipo-polymers by post-

insertion. The PEG derivatives used in this comparative study can be grouped according to three main features: PEG chain length (2 kDa and 5 kDa), architecture (linear and branched) and nature of the lipid anchor (1,2-distearoyl-sn-glycero-3-phosphoethanolamine - DSPE, Cholesterol and Cholane). Cationic vesicles were produced by addition of 30% mol of 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP) to the conventional lipid mixture.

Liposomes were characterized in terms of size and ζ-potential by Dynamic Light Scattering and Transmission Electron Microscopy showing a mean size of 180 nm, a narrow PDI (0.1) and a neutral or positive (+13.2)surface charge for PEG-decorated liposome and cationic liposomes, respectively. Liposome cellular uptake was evaluated on HeLa cell line by flow cytometry and confocal microscopy. For this purpose, liposomal samples were labelled with Rhodamine B-1,2-diesadecanoil-sn-glycero-3- phosphoethanolamine (Rho-DHPE). Fluorescently labelled liposomes were prepared by addiction of 0.5 % mol of Rho-DSPE with respect to the total lipid content in the initial lipid mixture. Branched PEG5kDa carrying a DSPE or Cholesterol anchor have shown to be the most performing polymeric derivative in providing liposomes with stealth features.

All liposomal samples were tested in vivo on Balb/C mice which were intravenously injected via the tail vein with 200 µL of 10.0 mg/mL liposome solutions. The in vivo results confirmed the good performance of Cholesterol as anchoring agent which behaved better compared to both DSPE and Cholane. When comparing the polymeric moiety, either linear PEG2kDa or PEG5kDa or branched PEG5kDa produced liposomes with similar pharmacokinetic profiles significantly improved with respect to naked liposomes. Indeed, two hours after administration, 35-40% of these formulations were still detectable in the blood.

Future perspective The PEG ability to prevent nonspecific protein adsorption and to prolong the circulation time of liposomes was studied in

details by comparing PEG-based polymers with different physico-chemical features – i.e. architecture, molecular weight and lipid anchor.

Either the PEG architecture or length and the lipid anchor nature are paramount to provide liposomes with suitable biopharmaceutical performance. Notably, at the best of our knowledge, no attempt has been made so far to investigate in vitro and in vivo the cell association and pharmacokinetic behavior of the carrier itself rather than the encapsulated drug. This study provides important information for a rational development of efficient liposomal formulations with favorable pharmacokinetics and biodistribution profile in correlation to their physico-chemical features.

Future studies will be carried out to investigate the therapeutic performance of drug-loaded vesicles decorated with the lipo-polymers here discussed.

References

1. Nag, O. K.; Awasthi, V., Surface engineering of liposomes for stealth behavior. Pharmaceutics 2013, (4), 542-569. 2. Salmaso, S.; Caliceti, P., Stealth properties to improve therapeutic efficacy of drug nanocarriers. Journal of drug delivery 2013, 2013. 3. Drummond, D. C.; Meyer, O.; Hong, K.; Kirpotin, D. B.; Papahadjopoulos, D., Optimizing liposomes for delivery of chemotherapeutic

agents to solid tumors. Pharmacol. Rev. 1999, 51 (4), 691-744.

10ISBN:9788894140422

Aminoacid Based Polymeric Nanovectors for Anticancer Drug Delivery Brunato Silvia1, Mastrotto Francesca1, Alexander Cameron2, Mantovani Giuseppe2, Caliceti Paolo1, Salmaso Stefano1

1Dept. of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy; 2School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD – UK;

Email: stefano.salmaso@unipd.it

Background Doxorubicin is a standard of care for the treatment for a variety of cancers. Despite its wide clinical use, this anticancer drug

suffers from several limitations and drawbacks, such as poor solubility, unfavourable biodistribution and pharmacokinetic profile and low tumor tissue selectivity1. These features yield major systemic side effects and inadequate therapeutic performances. Drug delivery systems have emerged as a strategy in order to overcome all these issues as they allow for a site-specific drug accumulation and release within the tumor tissue, reducing the systemic side effects deriving from the uncontrolled drug biodistribution. For this purpose, a variety of nanovectors have been developed and among these carriers polymeric micelles obtained by amphiphilic polyaminoacidic block copolymers are gaining attention. They offer several significant advantages in terms of improved water solubility of lipophilic drugs, enhanced drug bioavailability, high biocompatibility and versatility2. Moreover, the reactive pendant group on the side-chain of polyaminoacids could be employed directly for the conjugation of drugs or imaging agents through stimuli-responsive linkages to achieve selective drug release triggered by intracellular stimuli, such as enzymes, pH, redox potential3. The site-selectivity of the drug delivery can be enhanced by carrier decoration with tumor targeting agents. In addition, the physical entrapment of another anticancer drug may enable the possibility of a synergistic therapy further increasing the treatment efficacy.

In the present work we aim to generate a library of amphiphilic di-block copolymers composed by a hydrophilic block of polyethylene glycol (PEG) and an amino acid-based block of γ-hydrazinamide-glutamic acid (hydGlu) and Leucine (Leu). It is important to highlight that hydGlu was selected for the conjugation of the anticancer drug Doxorubicin through a pH-cleavable hydrazone bond to allow for a controlled drug release once the nanocarrier has reached the intracellular acidic compartments1. Leucine was introduced as spacer between the glutamic monomers to minimize the steric hindrance of Doxorubicin and to promote the polymer self-assembly thanks to its hydrophobic nature. The goal here is to engineer a series of versatile and effective platforms for passive but also active delivery of anticancer drugs into tumor tissues, investigating their efficiency in terms of biocompatibility, biodegradability and therapeutic properties. Main results

Four mPEG5kDa-(γ-Glun-r-Leum) polymers were successfully synthesized trough Ring Opening Polymerization with the following glutamic acid γ-benzyl ester/leucine N-carboxyanhydride (NCA) monomer ratios: 13:0, 8:8, 6:10, 4:12 with monomers conversion of 90% for the Glu NCA and 70% for Leu NCA. The protecting γ-benzyl ester group of glutamic acid was converted into a γ-hydrazide with a 99% conversion yield by reaction with hydrazine hydrate. Doxorubicin was conjugated to the four mPEG5kDa-(γ-hydGlun-r-Leum) copolymers by a single step procedure. The drug conjugation resulted 34% (w/w) for mPEG5kDa-(γ-[DOXO]Glu13), 24% (w/w) for mPEG5kDa-(γ-[DOXO]Glu4-r-Leu12), 29% (w/w) for mPEG5kDa-(γ-[DOXO]Glu6-r-Leu10) and 30% (w/w) for mPEG5kDa-(γ-[DOXO]Glu8-r-Leu8). The conjugation yields achieved for mPEG5kDa-(γ-[DOXO]Glu4-r-Leu12), mPEG5kDa-(γ-[DOXO]Glu6-r-Leu10) and mPEG5kDa-(γ-[DOXO]Glu8-r-Leu8) were 100%, 87.5% and 70%, respectively. These data highlight the effectiveness of Leucine spacer between Glutamic monomers to ensure a suitable space between sequential conjugated Doxorubicin molecules. This was proved by the higher drug conjugation efficiency for the polymers containing mixed Glu/Leu monomers in comparison with mPEG5kDa-(γ-[DOXO]Glu13), that had a conjugation yield of only 50%.The morphological analyses showed that mPEG5kDa-(γ-[DOXO]Glu4-r-Leu12), mPEG5kDa-(γ-[DOXO]Glu6-r-Leu10) and mPEG5kDa-(γ-[DOXO]Glu8-r-Leu8) self-assemble into micellar nanostructures with a slightly spherical shape in a size range of 20-30 nm. mPEG5kDa-(γ-[DOXO]Glu13) associated in micelles with analogous morphology but a larger size of nearly 40 nm. As expected, the presence of Leucine monomers promotes the formation of cohesive and smaller micelles because of its hydrophobic behaviour, while its absence in the mPEG5kDa-(γ-[DOXO]Glu13) polymer may explain a looser assembly and larger size. Polymeric micelles were also characterized by the critical micellar concentration (CMC). In particular, mPEG5kDa-(γ-[DOXO]Glu6-r-Leu10) and mPEG5kDa-(γ-[DOXO]Glu8-r-Leu8) showed a decrease in the CMC values from 78.5 µM and 88.3 µM to 9.48 µM and 9.05 µM, respectively, compared to the unconjugated copolymers proving that Doxorubicin may enhance the amphiphilic properties of the polymers. Future perspectives

A polymerization strategy was set up to generate a small library of four amphiphilic block copolymers endowed of high biocompatibility and biodegradability. Doxorubicin was efficiently conjugated to all copolymers through a selectively cleavable hydrazone bond to pursue a drug controlled release in specific acidic environments. The study allow to identify the conjugate parameters that control drug loading and self-assembly.

In-vitro studies will be performed to investigate the efficacy of cell uptake of these novel “smart” nanocarriers and their intracellular trafficking. Moreover, an active targeted system based on the functionalization of the copolymers with Folic Acid has been designed with the aim of enhancing the site-specificity and release within the cancer cells, thus minimizing systemic side effects. References

1. Bae, Y., Fukushima, S., Harada, A., & Kataoka, K., Design of environment‐sensitive supramolecular assemblies for intracellular drug delivery: Polymeric micelles that are responsive to intracellular pH change. Angewandte Chemie International Edition, 2003, 42(38), 4640-4643.

2. Maeda, H., Wu, J., Sawa, T., Matsumura, Y., & Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. Journal of controlled release, 2000, 65(1), 271-284.

3. Ponta, A., & Bae, Y.,PEG-poly (amino acid) block copolymer micelles for tunable drug release. Pharmaceutical research, 2010, 27(11), 2330-2342.

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Non-uniform drug distribution HPMC matrices for zero-order release Cerea Matteo1, Maroni Alessandra1, Palugan Luca1, Bellini Marco2, Foppoli Anastasia1, Moutaharrik Saliha1, Gazzaniga Andrea1

1Università degli Studi di Milano, via G. Colombo 71, 20133 Milano, Italy, Phone: +390250324654 2Freie Universität, Kelchstraße, 31, 12169 Berlin, Germany, Phone: +49(30)838 50696

Background Hydrophilic tableted matrix systems for oral prolonged release of common cylindrical shape typically display a decrease in the rate of release over time due to i) progressive increase in the diffusional path the drug has to cover to reach the outer medium and ii) concomitant reduction of the area at the solvent penetration front. In addition, an initial burst release is observed due to the fraction of drug present on the surface of the dosage form. Because zero-order kinetics has long been sought to attain constant drug absorption rate for a predetermined period of time, thus providing the patient with constant drug levels, a novel oral prolonged release system, the NUDDMat (Non Uniform Drug Distribution Matrix) was designed, prepared and evaluated. Main Results The NUDDMat system consists of 5 overlaid layers differing in drug concentration, which decreases between contiguous layers from the inside towards the outside of the matrix according to a descending staircase function thus tending to an apparent overall linear mode (Figure 1). The nominal thickness of each layer was 315 µm aiming at an overall diameter of 4 mm. The layers were applied by powder layering onto cellulosic seed cores (850 µm diameter). Hypromellose (HPMC, Methocel® K15M, Colorcon) was employed as the swelling hydrophilic polymer for exerting a control of release. Paracetamol was used as the drug tracer. Following a preliminary set-up of the operating conditions, the powder layering process turned out feasible for the preparation of NUDDMat systems with different configurations and compositions. The final units were evaluated in terms of morphology, dimensions and in vitro release. Small spherical units were obtained, which exhibited consistent size and satisfactory mechanical characteristics. The NUDDMat system was demonstrated to reduce the initial burst effect typical of hydrophilic matrices that, as expected, was yielded by a formulation having equal overall composition but uniform drug distribution. In Figure 2 release profiles from final systems and relevant intermediate units is reported along with the release profiles from Uniform Drug Distribution Matrix system prepared by powder layering (UDDMat PL) and NUDDMat with the 5th layer with no drug and reduced thickness. The discontinuous composition of the layered matrix, varying along its cross-sectional axis, was highlighted by Raman mapping analysis. Through Durbin-Watson statistics, the novel system was confirmed to amplify the portion of linear release as compared with uniform drug distribution systems. Future Perspectives NUDDMat systems were proved to successfully contrast the impact of typical drawbacks associated with hydrophilic matrices, i.e. the initial burst, lengthening of diffusional pathway and reduction of the area at the swelling front. By exploiting powder layering, which offers major advantages because of the lack of solvents involved at reduced costs, a hydrophilic matrix system for prolonged release was designed and prepared. The simple design concept, the advantageous technique employed and the use of GRAS (Generally Regarded As Safe) materials make the delivery platform hereby proposed a valuable strategy to improve the performance of hydrophilic matrix systems for prolonged release.

References 1. Colombo, P.; Conte, U.; Caramella, C.; Gazzaniga, A.; La Manna, A. Compressed polymeric mini-matrices for drug release control. J. Control. Release, 1985,1, 283–289 2. Lee, P.I., Initial concentration distribution as a mechanism for regulating drug release from diffusion controlled and surface erosion controlled matrix systems. J. Control. Release. 1986, 4, 1–7. 3. Scott, D.C.; Hollenbeck, R.G. Design and manufacture of a zero-order sustained-release pellet dosage form through nonuniform drug distribution in a diffusional matrix. Pharm. Res. 1991, 8, 156–161

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Microfluidics-aided polyelectrolyte complexation: a new approach for tailor-made chitosan based nanoparticles

Chiesa Enrica, Dorati Rossella, Conti Bice, Modena Tiziana and Genta Ida Dept. Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia (Italy)

Background: Chitosan nanoparticles (NPs) showed promising results in drug delivery for treatment of various diseases. The considerable attention towards chitosan was owning to its outstanding biological properties, including biodegradability , biocompatibility, mucoadhesivity, bioactivity and its capacity to open tight junction [1]. Conventionally ionic/ionotropic gelation methods, based on the electrostatic interaction between amine groups of chitosan and negatively charged groups of a linker (i.e. sodium tripolyphosphate (TPP)) allow chitosan NPs synthesis by dropwise addition of the cross-linker to the chitosan solution via slow, uncontrolled bulk mixing. Despite mild conditions, conventional bulk NP synthesis methods typically resulted in unreproducible methods through the lack of control over the mixing and inducing the formation of poorly defined NPs in terms of physicochemical properties, drug loading and release [2]. Microfluidic-assisted methods have already been demonstrated to be effective technical platforms for various polymeric/lipidic NPs synthesis offering the controlled movement of small amount of reactants and their interaction in a precise volume to provide precise control of mixing and physical process at microscale [3]. Main Results: Full Factorial Design (FFD; 8, 2*3) was used to optimize the process parameters of a microfluidics-assisted method with staggered herringbone device. Total flow rate (TFR), chitosan concentration and TPP concentration were selected as factors. Particle size, polydispersity index (PDI) were the outcomes considered. The particle dispersions were characterized without any further purification or alteration by dynamic light scattering (DLS), ζ potential measurements, turbidity (at wavelength of 635nm) which is dependent on the number of particles per unit volume, compactness of NPs, molecular weight for a theoretical spherical NPs and the aggregation number of polymer chains in the NPs (NAgg). Moreover, after samples centrifugation (20 min at 16,000 rpm), the process yield was investigated through quantification of unreacted chitosan in the supernatant by colorimetric method. The models obtained by the statistical analysis carried out on runs of FFD allowed to identify the effect of each factor on the specific outcome (p value <0.05). The R-Squared statistic indicated that the model as fitted explained 95.93% of the variability in particles size; ANOVA analysis tests the statistical significance of each effect by comparing the mean square against an estimate of the experimental error. In this case, 2 effects have P-values less than 0.05, indicating that they were significantly different from zero at the 95.0% confidence level: TPP concentration (p value=0.0301) and TFR (p value=0.001). In particular increasing the TFR (from 5 to 12 mL/min) a decreasing in particles size was showed (from 304.75 ± 78.12 to 38.48 ± 4.13 nm) meanwhile increasing the TPP concentration ( from 0.3 to 0.5 mg/mL) an increase in particle size was detected (from 244.75 ± 78.57 to 371.76 ± 41.82 nm). PDI analysis experiment explained 82.50% of the variability in PDI showing 2 significant effects: chitosan concentration (p value = 0.0274) and TFR (p value = 0.0241), high chitosan concentration and high TFR allow to heterogeneous NPs (PDI > 0.4). All batches prepared showed positive zeta potential from 19 to 34 mV. Process variables also affect the process yield: keeping constant chitosan concentration (which showed no influences on yield) at 2 mg/mL a reduction in process yield was highlighted by increasing TFR (5-12 mL/min) from 62.81 ± 0.45% to 37.84 ± 2.90% and decreasing the TPP concentration (0.3-0.5 mg/mL) from 37.84 ± 2.90% to 25.51 ± 4.58%. Future Perspectives: staggered herringbone assisted ionotropic gelation method revealed a robust, versatile and well reproducible technique to precisely induce electrostatic interaction between chitosan and its cross-linker providing NPs with tailor made sizes. Further studies will be planned to investigate the effect of the process on drug encapsulation and its subsequent release. References: 1. Kumar, M.; Muzzarelli, R.A.; Muzzarelli, C.; Sashiwa, H.; Bomb, A. Chitosan chemistry and pharmaceutical perspectives. Chem Rev, 2004, 104, 6017-84. 2. Dashtimoghadam, E.; Mirzadeh, H.; Taromi, F.A.; Nystrom B. Microfluidic self-assembly of polymeric nanoparticles with tunable compactness for controlled drug delivery. Polymers, 2013, 54, 4972-79. 3. Valencia, P.M.; Farokhzad, O.C.; Karnik, R., Langer, R. Microfluidic technologies for accelerating the clinical translation of nanoparticles. Nat nanotechnol, 2012, 7, 623-9.

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Thermosensitive gels for pediatric mucositis: preformulation studies Cirri Marzia, Pieri Elena, Maestrelli Francesca, Mura Paola

Dept.Chemistry, School of Human Health Sciences, Univ. Florence, Via Schiff 6, Sesto Fiorentino I-50019, Florence, Italy

Background Oral mucositis is a significant problem in pediatric patients undergoing chemotherapeutic treatment for solid tumors. Various degrees of mucositis severity can be observed, ranging from mild sensation changes, difficulty in talking and swallowing, to multiple, ulcerative, bleeding lesions. Lesions of oral mucositis are often very painful and can compromise nutrition and oral hygiene as well as increase risk for local and systemic infections [1]. Lidocaine is widely used to induce immediate relief to the oral cavity, reducing the pain due to its anesthetic and antibacterial activity. A few studies reported the antimicrobial effect of sodium bicarbonate, being able also to enhance the lidocaine activity [2]. The aim of our study was to develop a thermosensitive gel containing lidocaine in combination with sodium bicarbonate in order to evaluate their possible synergistic effect in the treatment of oral pediatric mucositis. As a preliminary step of the whole study, preformulation studies were carried out in order to select the most suitable binary combination of Lutrol®F127 (Poloxamer 407), used as the main thermosensitive polymer, with different natural and synthetic polymers, endowed with mucoadhesive properties. Main results Sodium hyaluronate and xanthan gum were tested as natural polymers to be added to Lutrol®F127 whereas Carbopol®934 and HPMC as synthetic ones. Increasing concentrations of Lutrol®F127 (15-20-25-30% w/w) were used in combination with increasing % of the other polymers (0.25-0.5-1% w/w). Gelation Temperature of each binary system was determined by a simple tube-inverting method [3] in the range 20-40°C, at a ramp rate of 2.5°C/min. The gelation time was recorded at 37±0.5°C for those formulations showing ideal gelation Temp (Tsol-gel ≥ 28°C) both in the absence and in the presence of the drug. A gelation time less than 1 min was considered ideal for our goal. The effect of the incorporation of sodium bicarbonate was also evaluated. Regardless the type of the additional polymer, Lutrol®F127 used at 15% w/w did not lead to gelification. In the presence of the drug, the most suitable concentration of Lutrol®F127 to obtain a gelification time less than 1 min seemed to be 25% (Fig. on the left). The addition of more than 0.5% w/w of sodium bicarbonate to formulations containing the drug was not possible, due to precipitation phenomena. All the examined additional polymers, except xanthan gum, used at a specific concentration, in combination with 25% Lutrol®F127 allowed to obtain formulations containing both lidocaine and sodium bicarbonate with gelation times less than 1 min (Fig. on the right). Future perspectives These preliminary studies allowed to identify the most suitable conditions to obtain thermosensitive gels containing lidocaine in combination with sodium bicarbonate. The rheological and stability properties of the developed gel formulations will be in depth investigated as well as their in vitro performances. The most suitable formulations will be tested in vivo thanks to the collaboration with the pediatric hospital Meyer.

References: [1] Lalla, R V., Sonis, S T., Peterson, D E, 2008. Dental Clinics of North America, 2008, 52 (1), 61-77 [2] Thompson, KD, Welykyj, S, Massa, MC. J Dermatol Surg Oncol., 1993, 19 (3), 216-220 [3] Jung, Y-S, Park, W, Park, H, Lee, D-K, Na, K, 2017. Carbohydrate Polymers, 156, 403-408

14ISBN:9788894140422

Redox -sensitive nanoparticles for dual gene/chemo-therapy of cancer Conte Claudia†,‡, Stolnik-trenkic Snow†, Ungaro Francesca‡, Quaglia Fabiana‡ and Alexander Cameron†

†MTF division, School of Pharmacy, University of Nottingham, NG7 2RD, UK; ‡ Drug Delivery Lab, Dept of Pharmacy, University of Napoli, Via D. Montesano 49, 80131, Italy.

Background: Redox-responsive nanoparticles (RR-NPs) are of interest for anticancer nanomedicines, owing to the possibility to ‘design in’ selective modulation of drug release at target sites, thus strongly enhancing therapeutic effect [1]. We are investigating this concept through novel RR-NPs based on bioreducible polyethylene glycol (PEG)-poly(lactic-co-glycolic acid) (PLGA) block copolymers and cationic poly (beta-amino esters) (PBAE) for the dual delivery into cancer cells of conventional anticancer drugs and siRNA. The polymers have been modified to contain bioreducible disulfide units for the in situ delivery into cancer cells of combination drugs. In particular, we have focused our attention on docetaxel (DTX) and the TUBB3-siRNA, in order to have a synergistic effect in the treatment of lung cancer [2]. Main results: Redox-responsive PLGA-S-S-PEG block copolymer and cationic PBAE were synthesized by the combination of Ring Opening Polymerization and Michael Addition chemistries, and characterized by NMR, FT-IR, and SEC. NPs were prepared through an emulsion/solvent evaporation technique and then colloidal properties, morphology, redox-responsiveness, drug loading and release were evaluated. Biological behavior of NPs in lung cancer cells in terms of cytotoxicity, uptake, gene silencing and anticancer effect was assessed. All the polymers were obtained with a high degree of polymerization (~90%) and low polydispersity indices (~1.3). Spherical and homogenous NPs of around 150 nm with negative zeta potential and complete entrapment of both DTX and siRNA were obtained. NPs showed high stability in the most relevant simulated biological fluids whereas a fast disassembly of NP structure in conditions mimicking intracellular reducing environment and triggered drug release was found. Unloaded NPs were well-tolerated by lung cancer cells, thus completely suppressing the cytotoxicity of free PBAE due to their cationic nature. Dual-labeled fluorescent NPs were rapidly internalized into cells, and released the drugs payload intracellularly, resulting in high gene-silencing efficiency. Finally, viability of cells treated with combined DTX-TUBB3/siRNA-NPs was decreased as compared to NPs loaded only with DTX giving a 5-fold reduction in IC50 at 72 h. The enhanced anticancer effect of combined NPs was clearly evidenced by immunofluorescence staining assay of beta-tubulin. Future Perspectives: Taken together, these results demonstrate that novel redox-responsive PLGA-based NPs modified with cationic PBAE represent a promising therapeutic approach with great potential for the dual siRNA/drug therapy of lung cancer. In vivo performance of developed NPs after inhalation in a lung cancer model is now under investigation. Acknowledgments: Financial support of AIRC/Marie Curie Actions 2014 is gratefully acknowledged. References: 1. Cheng, R.; Feng, F.; Meng, F. et al. Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery. J. Control. Release, 2011, 152, 2–12. 2. McCarroll, J. A.; Gan, P.P.; Erlich, R.B. et al. TUBB3/βIII-tubulin acts through the PTEN/AKT signaling axis to promote tumorigenesis and anoikis resistance in non-small cell lung cancer. Cancer Res. 2015, 75, 415–425.

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Inhalable hybrid lipid/polymer nanoparticles for pulmonary delivery of sirna Ivana d’Angelo1, Gabriella Costabile2,3, Estelle Durantie3, Paola Brocca4, Valeria Rondelli4; Annapina Russo5, Giulia Russo5,

Agnese Miro2, Fabiana Quaglia2, Alke Petri-Fink3, Barbara Rothen- Rutishauser3, and Francesca Ungaro2.

1Di.S.T.A.Bi.F., University of Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100 Caserta, Italy. 2Laboratory of Drug Delivery, Dept. of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy.

3Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700, Fribourg, Switzerland. 4Applied Physics, Dept. of Medical Biotechnology and Translational Medicine, University of Milano, Via Vanvitelli 32, 20129 Milano, Italy

5Laboratory of Biochemistry, Dept. of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy.

Background. Nowadays, the down-regulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. Nevertheless, conceiving siRNA for lung delivery, ad hoc inhalable formulations are required: i) to enhance siRNA stability; ii) to overcome lung cellular and non-cellular barriers; iii) to increase siRNA availability at the target level [1]. With this idea in mind, here we propose hybrid lipid/polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system.

Main results A siRNA pool against the sodium transepithelial channel (ENaC) was entrapped in muco-inert PLGA/DPPC hNPs comprising or not poly(ethylenimine) (PEI) as third component. Optimized hNPs, displaying a hydrodynamic diameter of ~150 nm, a low polydispersity index and a negative ζ potential (-25 mV), encapsulated siRNA with high efficiency and displayed a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI. The siRNA formulations were stable upon aerosolization with a vibrating mesh nebulizer and showed optimal in vitro aerosol performance. Small-angle X-ray scattering analyses highlighted an excellent stability of hNPs upon incubation with cystic fibrosis artificial mucus, confirming their potential for direct aerosolization on mucus-lined airways. The fate and the cytotoxicity of siRNA-loaded hNPs upon aerosolization on a triple cell co-culture (TCCC) mimicking the human epithelial airway barrier [2], were assessed. Results confirmed that fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute pro-inflammatory effect. Thus, the effect of siRNA loaded hNPs on ENaC protein expression was evaluated. Of note, a prolonged inhibition of ENaC protein expression was observed after 72 h in A549 cells upon treatment with siRNA-loaded hNPs.

Future perspectives. Overall results, demonstrating the great potential of hNPs as carriers for pulmonary delivery of siRNA, prompt towards investigation of their therapeutic effectiveness in vivo.

References: [1] d'Angelo, I; Conte, C; La Rotonda, MI; Miro, A; Quaglia, F; Ungaro, F. Improving the efficacy of inhaled drugs in cystic fibrosis: challenges

and emerging drug delivery strategies. Adv. Drug Deliv. Rev, 2014, 75, 92-111. [2] d'Angelo, I; Costabile, G; Durantie, E.; Brocca, P; Rondelli, V; Russo, A; Russo, G; Miro, A; Quaglia, F; Petri-Fink, A; Rothen-Rutishauser,

B; Ungaro, F. Hybrid lipid/polymer nanoparticles for pulmonary delivery of siRNA: development and fate upon in vitro deposition on the human epithelial airway barrier. J Aer Med Pulm Drug Deliv, In press.

Acknowledgments: This work was supported by Compagnia di San Paolo (STAR Program, Napoli_call2013_35). G.C. is a recipient of a Short-term fellowship from International Society for Aerosols in Medicine (ISAM) (Call 2015).

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Polysulfides… a ‘break-through’ for osteoporosis? d'Arcy, Richard1, El Mohtadi, Farah2, Burke, Jason2, Rios, Julio2, Tirelli, Nicola1,2

1Istituto Italiano di Tecnologia, Genova, Italy 2University of Manchester, Manchester, United Kingdom

Introduction The word osteoporosis is derived from Greek meaning “porous bones”. As the name suggests osteoporosis is a bone degeneration disease typified by brittle, easy to fracture bones. It is commonly considered a disease of aging as it usually only manifests after middle age such as after menopause in women (due to a reduction in estrogen). In fact, 80% of those affected are women and for those aged over 45, osteoporosis contributes to more days in hospital than either diabetes, breast cancer or heart attacks[1]. Bone as an organ is under a constant state of remodeling with osteoblasts being the ‘bone producing’ and the osteoclasts being the ‘bone resorbing’ cells. In healthy conditions there is a homeostatic balance between the two conditions, however, in osteoporosis the homeostasis becomes skewed towards bone resorption due to an increase in RANKL production and a decrease in osteoprotegerin leading to increased osteoclast formation and survival[3]. Through downstream signaling, RANKL exploits reactive oxygen species (ROS) as a chemical messenger intermediate in order to stimulate the differentiation of pre-osteoclasts/macrophage-like cells into osteoclasts resulting in their increased survival through activation of the NF-kB pathway[3]. Here, we exploit an oxidation-responsive PEGylated-polysulfide in the form of star-micelles for 1) the delivery of rapamycin (an osteoclast inhibitor through the mTOR pathway) and 2) complementary inhibition of the ostoclastogensis pathway via inhibition of NF-kB pathway (inherent properties of the polysulfide micelles as ROS scavengers). Results Polysulfides could be successfully loaded with rapamycin at a loading efficiency of 85% and a loading capacity of 34% indicating they are excellent solubilizers for rapamycin. Drug release was evaluated in vitro using physiologically relevant concentrations of hydrogen peroxide as a model oxidant. Unloaded polysulfide micelles (PSMs) were first assessed and were capable of reducing both intra- and extracellular ROS levels in osteoclast progenitor cells both with and without the addition of osteoclastogenic factors (RANKL and M-CSF). What’s more, they displayed potent anti-osteoclastic properties, the reducing gene expression of classical osteoclast markers (cathepsin K and TRAP) back down to baseline levels at a PSM concentration of 20 ug/ml. Flow cytometry further confirmed a 50% reduction in cathepsin K positive cells and visual observations of cells under the microscope confirmed the absence of any multinucleated cells. SPMs loaded with rapamycin were able to reduce osteoclast-like differentiation to higher extent compared to SPM alone, while also causing down-regulation in the expression and synthesis of key osteoclast markers. No apoptotic influences are observed suggesting effects are most likely linked to biochemical events. Based on previous reports of their influences in osteoclastogenic activity, it is likely that reductions in reactive oxygen species (ROS) caused by polysulfide oxidation in the cellular environment inhibit the progression of osteoclast activity. Findings herein provide a potential basis for new bioactive drug carriers, capable of influencing the cellular environment and providing targeted release synergistically. Conclusions and Future Perspectives The PSMs were an excellent drug solubilizer for rapamycin and the unloaded vehicle displayed potent anti-osteoclastogenesis properties; PSMs were able to completely inhibit the gene expression of osteoclastic genes as well as the formation of multinuclear cells at a concentration of 20 ug/ml. Rapamycin appears to display a synergistic effect with the antioxidant micelles, most likely due to it acting on the mTOR pathway. Herein we have demonstrated an attractive drug delivery system for treating osteoporosis as evalulated using in vitro models. Future works will need to incorporate a bone targeting moiety and in vivo evaluation. References: 1. British Orthopaedic Association & British Geriatrics Society, OsteoReport 2010 2. J.A. Kanis, OI 1997; 390-406 3. W. Boyle, W. Simonet and D. Lacey, Nature 2003, 423, 337-42,

17ISBN:9788894140422

Multi-composite system for the delivery of alendronate to bone tissue Luisa S. Dolci1, Silvia Panzavolta2, Massimo Gandolfi2, Adriana Bigi2, Beatrice Albertini1, Nadia Passerini1

1Dep. of Pharmacy and BioTechnology, Via S. Donato 19/2, 40127 University of Bologna,Italy 2Dep. of Chemistry “G. Ciamician”, via Selmi 2, 40126 University of Bologna, Italy

Bakground The bisphosphonate (BPs) class drug, is widely employed for the treatment of a variety of bone disorders. In this work Sodium Alendronate (AL) was utilised as model Bisphosphonate drug. AL is often administered orally or via injection showing many side effects. Recently different release approaches have been evaluated in order to targeted AL delivery to bone. In particular calcium phosphate cements (CPCs) have been investigated as potential carries of BPs [1, 2]. The CPCs are bioactive and biodegradable grafting materials made of powders of suitable composition which, when mixed with a liquid phase, give a modulable paste which stiffens during the setting reaction and sets as primarily calcium-deficient hydroxyapatite. Unfortunately, the loading of AL interferes with the conversion of pristine powders (like alfa-tricalcium phosphate, alfa-TCP) into hydroxyapatite, increasing the setting times and worsening the mechanical properties. Therefore, the amount of AL that can be directly loaded is limited (1.8 mg / g) [1, 2]. Consequently, the drug encapsulation might overcome this drawback. The aim of this work was to develop an innovative drug delivery system potentially useful for the delivery of AL to bone tissue. In particular, we propose the use of Solid Lipid Microparticles (MPs), up to now mainly used for oral and topical drug delivery, as carrier for AL, due to the favourable biocompatibility and lower toxicity of the lipids compared with many polymers. Thus a multi-composite delivery platform consisted of a biomimetic-tricalcium phosphategelatin cement (CPCs) enriched with alendronate-loaded MPs (MPs-AL) was developed. Main results For the preparation of the MPs, the spray congealing technology was employed [3]. In particular six different excipient were considered: Stearic acid, Stearyl alcohol, Cutina® HR, Precirol® ATO 5 and Tristearin. In order to screen the effect of types, dimensions and amount of unloaded MPs on the CPCs most important mechanical properties a Design of Experiment (DoE) was employed. Then, MPs loaded with 10 % w/w of AL were produced using the different carriers. All MPs-AL exhibited a spherical shape, encapsulation efficiency higher than 90% and prevalent particle size ranging from 100-150 micron. Solid state characterization by means of DSC, HSM and X-ray powder diffraction demonstrated that encapsulation of the drug into MPs did not alter its crystal structure. MPs-AL addition to the cement provoked a modest lengthening of the setting times and of the hardening reaction leading to the complete transformation of alfa-TCP into calcium-deficient hydroxyapatite, without significantly affect the cement mechanical properties. Then MPs loaded with different concentration of AL (10%, 20% and 30%) were embedded into the CPC. The in vitro AL release studies from the multi-composite system (carried out in PB buffers at pH 7.4 at 37°C) showed that all the system allowed a controlled release of the drug over time. Therefore the results of this study demonstrated that it was possible to increase the amount of AL into the CPCs up to 10 time compared to the value previously reported [1,2]. Moreover, the use of MPs as carriers to enrich bone cement formulation with AL was a successful strategy to develop a system for the controlled local delivery of the drug. Future and prospective To value the effect of the AL release from CPCs-MPs system on cellular proliferation and differentiation, in vitro studies using osteoblasts and osteoclasts cell cultures are in progress. The obtain results suggest that this designed composite system could be useful for the delivery of other drugs (i.e. antibiotic, anti-inflammatory agents and anticancer drugs) to bone tissue. References: [1] Panzavolta S.; Torricelli P.; Bracci B.; Fini M.; Bigi A. Functionalization of biomimetic calcium phosphate bone cements with alendronate. J Inorg Biochem, 2010, 104, 1099-1106. [2] Panzavolta, P.: Torricelli P.; Bracci B.; Fini M.; Bigi A. Alendronate and Pamidronate calcium phosphate bone cements: Setting properties and in vitro response of osteoblast and osteoclast cells. J Inorg Biochem 2009, 103, 101–106. [3] Albertini B.; Passerini N.; Pattarino F.;Rodriguez, L. New spray congealing atomizer for the microencapsulation of highly concentrated solid and liquid substances. Eur. J. Pharm. Biopharm., 2008, 69, 348-357.

18ISBN:9788894140422

Enhancing anticancer activity of chemotherapeutics with antiangiogenic biodegradable nanoparticles

Diletta Esposito1, Francesca Moret2, Alessandro Venuta1, Claudia Conte1, Giovanni Dal Poggetto3, Concetta Avitabile1, Francesca Ungaro1, Alessandra Romanelli1, Paola Laurienzo3, Elena Reddi2, Fabiana Quaglia1

1 Department of Pharmacy, University of Napoli Federico II, Italy; 2 Department of Biology, University of Padova, Italy; 3 Institute for Polymers, Composites and

Biomaterials, CNR, Pozzuoli (Napoli), Italy Introduction: Combination of antiangiogenic molecules with chemotherapeutics is considered a valuable strategy to increase cancer cell killing through decreased blood supply to the tumor. Our final aim is to develop biodegradable nanoparticles (NPs) accumulating in cancer cells through folate receptor (FR) and bearing an antiangiogenic anti-FLT1 hexapeptide, which inhibits VEGF-induced endothelial cell migration and angiogenesis. Docetaxel (DTX) was selected as model of poorly water-soluble chemotherapeutics. Methods: Amphiphilic diblock poly(e-caprolactone)-polyethyleneglycol (PCL4000-PEG1500) copolymers conjugated with folate or anti-FLT1 at PEG-OH end were synthesized and characterized. These copolymers were mixed in appropriate ratios with PCL4000-PEG1000 to form core-shell NPs by nanoprecipitation. Specific uptake of NPs in HUVEC and KB (FR+) cells was assessed by fluorescence techniques and validated by competition experiments. NPs antiangiogenic properties were evaluated by endothelial tube formation assay. Cytotoxicity of DTX-loaded NPs in 2D and 3D KB cultures was assessed. Results: NPs with size around 100 nm and entrapping DTX with high efficiency were produced. anti-FLT1-NPs were internalized in HUVEC through VEGFR1 receptor and found more antiangiogenic than free peptide at equivalent concentrations. The presence of folate on NPs surface did not interfere with antiangiogenic activity, while affecting both interaction with human serum albumin and subcellular localization in HUVEC. In KB cells, DTX-loaded NPs showed a cytotoxicity significantly higher as compared with free DTX at low drug concentrations. Conclusion: We have produced biodegradable NPs with dual decoration on the surface allowing to combine the antiangiogenic properties of antiFLT1 with the targeting properties of folate. This strategy paves the way to dual functionalization of NPs based on amphiphilic polyesters as a novel approach to improve activity and selectivity of chemotherapeutics. Acknowledgements: This work was supported by Italian Association for Cancer Research (IG2014 #15764).

19ISBN:9788894140422

Hyaluronan decorated liposomes to target nifedipine in human epidermis Silvia Franzé1, Alessandro Marengo2, Silvia Arpicco2, Paola Minghetti1, Francesco Cilurzo1

1Dept. of Pharmaceutical Sciences – University of Milan; 2Dept. of Drug Science and Technology- University of Turin

Purpose: to assess the feasibility to design HA-coated flexible liposomes to enhance the skin penetration of nifedipine, chosen as model drug. With this aim the attention was focused on a) the design of HA-coated flexible liposomes and b) the evaluation of the ability of HA to drive the penetration of liposomes in the human epidermis. Methods: transferosomes (T) and transethosomes (TE) loaded with 2 mg/mL nifedipine were prepared (Table 1). The ratio between phospholipids and tween 80, selected as surfactant, was fixed at 85:15 % w/w. For the coating of HA on the surface of the vesicles, the polysaccharide was conjugated to the DPPE (DPPE-HA). The formulations were characterized in terms of particle size, z-potential, polydispersity, lipid, drug and HA contents. The thermotropic behavior was studied by modulated temperature DSC. The constant of deformability, K, of the vesicles was assessed by a dynamometer-assisted extrusion assay. The in vitro permeation through human epidermis was studied by Franz diffusion cells method determining both the NIF permeated through (Q24) and the NIF retained by the epidermis (R24).

Table 1– Lipid composition (mol/mol %) and K values of vesicles

Results: all formulations were monodispersed (PdI ≤ 0.1), with a negative ζ potential and a particle size ranging between 120 and 150 nm. The presence of the DPPE or the HA-DPPE conjugate reduced the encapsulation efficiency of nifedipine. This effect seemed to be due to a different packing of the lipids in the bilayer (confirmed by the DSC study) that led to a reduction of the capability of the nifedipine to sit in the bilayer. The DPPE also exerted a concentration dependent stiffening effect on the bilayer, which was attributed to a modification of the thermotropic pattern of the systems. The addition of the DPPE-HA further increased the rigidity of the carriers. To find a compromise between the stiffening effect of DPPE-HA on the bilayer and the need to have a certain amount of penetration driving moiety on liposome surface, ethanol was added to T2, resulting in TE1 formulation. In fact, it is known that ethanol increases the membrane fluidity and acts as skin penetration enhancer. The combined effect of ethanol and HA favored the skin penetration of the drug and its retention in the viable epidermis Conclusions: the overall results provide a proof of concept of the ability of hyaluronic acid to drive the penetration of lipid-based nanocarriers in human skin, to obtain sustained drug delivery systems with high efficiency.

Abbreviations: EPC: Egg-phosphatydilcholine; DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; *under the quantitication limit References: 1.Ennis, et al., 2016. Calcium channel blockers for primary Raynaud’s phenomenon. Cochrane Database Syst Rev. DOI: 10.1002/14651858.CD002069.pub5. 2.Franzè, et al., 2017. Tuning the extent and the depth of penetration of flexible liposomes in human skin. Mol. Pharm. 14, 1998−2009. 3.Arpicco, et al., 2013. Hyaluronic acid-coated liposomes for active targeting of gemcitabine. Eur. J. Pharm. Biopharm. 85, 373–380.

20ISBN:9788894140422

Fungal hydrophobins, proteins with potential in drug delivery Gravagnuolo Alfredo Maria (a, b), Cicatiello Paola (a), Pennacchio Anna (a), Sorrentino Ilaria (a), Politi Jane (c), De Stefano Luca

(c), Piscitelli Alessandra (a), Giardina Paola (a). (a) Dept of Chemical Sciences, University of Naples Federico II, 80126, Italy. (b) FBMH, The University of Manchester, M13 9PT, UK. Tel

+44(0)7842877559 (c) IMM-CNR, 80127, Naples, Italy

Background. After a decade of much excitement and hype, the limited clinical translation of targeted nanomedicine is prompting scientists and companies to explore new approaches in nanotechnology to overcome the hurdles towards application. One of the major difficulties in achieving efficient targeting is the immunogenicity of nanomedicines, which causes their fast clearance from the organism. Since the interactions of nanodevices with the immune system are established through their interface, the surface chemistry is a key feature determining their fate in the body. The control of the surface (e.g. the protein corona) via the development of bio-inspired smart coatings offers valid solutions to both increase the efficiency of delivery of the present nanomedicines and progress their translation to patient care on the long term. Hydrophobins are cysteine rich polypeptide chains, made of ~100 amino acids [1]. The folded protein consists of a globular, ~3 nm, and amphiphilic structure able to self-assemble at hydrophilic-hydrophobic interfaces. They are endowed with adhesive properties, and peculiar structural and functional features, and play multiple roles in the life cycle of fungi. Due to their high amphiphilic character they are able to self-assemble at hydrophobic/hydrophilic (e.g. water/air, water/solid) interfaces into ordered monolayers. In their soluble form they show astonishingly high surface activity, indicated by the reduction of the surface tension of water and by a powerful emulsification capacity, which can be useful for the formulation of stable nanodispersions of insoluble drugs in water. Moreover, upon adhesion onto various materials they can form nanostructured coatings able to reverse the hydrophilic-hydrophobic character of a surface, to work as a “primer” for the non-covalent immobilization of a second protein layer, peptides, organic or inorganic materials, and to improve the biocompatibility and chemical stability of surfaces. Therefore hydrophobins are an interesting coating for surface modification of both bulk and nanosized materials. Class I Hydrophobins can self-assemble into amyloid-like aggregates able to stably coat several materials. Interestingly, Aimanianda and colleagues have demonstrated that the amyloid layer, which coats the airborne spores of filamentous fungi, masks them to both innate and adaptive immune systems [1]. The presence of the coating prevents the production of macrophage cytokines and the recruitment of neutrophils at the site of spore germination extending the fungal survival in the host. These findings have attracted great interest on hydrophobins for the study of their immunogenicity and pathogenicity and inspired their use for the enhancement of circulation time of nanoformulated drugs in vivo. Pioneering reports show that class II hydrophobin coating on thermally hydrocarbonized porous silicon nanoparticles alters the material hydrophobicity and protein adsorption on the surface, resulting in the improvement of nanoparticle biodistribution after oral administration. However, the coating is still not optimal, for example it slowly disassembles in plasma, limiting the lifetime of these particles; therefore, the formulation of ideal hydrophobins coating is still an open question. Class I hydrophobin named Vmh2 is produced by Pleurotus Ostreatus, an edible fungus common in the Mediterranean diet [2]. We study its self-assembling behaviour for biomedical applications, its molecular engineering and we develop screenings for the identification of new hydrophobins from fungi [3]. Main Results. Vmh2 was extracted from the surface of the fungal mycelium. Its self-assembly and aggregation behaviour in water was affected by environmental conditions. Vmh2 was soluble at pH ≥ 7, however, it spontaneous aggregated into in amyloid spherical assemblies of 12 ± 2 nm and 67 ± 5 nm of hydrodynamic radii, and in amyloid fibrils over time [2]. Protein concentration, calcium ions, solvent polarity, temperature and pH affected its stability, self-assembling and aggregation into amyloid-like structures. Vmh2 self-assembling was also affected by interactions with glucose. We showed that when the stabilization of gold-PEG core-shall nanoparticles was assisted by Vmh2, the complex, 10 ± 5 nm of hydrodynamic radius, showed glucose dependent aggregation. Moreover, we produced engineered variants of Vmh2 via genetic fusion with the green fluorescent protein, or enzymes such as Glutathione S-transferase and a phenol oxidase. Vmh2 engineered at the single amino acid level, showed altered self-assembling. Finally we developed a screening for hydrophobins on a pool of 100 marine fungi allowing us to identify 6 new proteins with either good emulsification capacity or ability to stably coat surfaces [3]. They generally showed resistance to digestion with several proteases, which is advantageous for the formulation of drugs for oral administration. Future Perspectives. Bioinspired coatings based on the fungal spores would enable the production of immunological inert and biocompatible nanocomplexes amenable to nanomedical uses [1]. Moreover, the high amphiphilic character of hydrophobins and their engineered variants make them ideal biosurfactant for the formulation multifunctional core-shell nanoparticles, e.g. by the nanoprecipitation technique, for the targeted delivery of insoluble drugs. Since hydrophobins are edible proteins, the procedures for the issue of health and safety regulation would be simplified for their oral administration. References 1. Bayry, J.; Aimanianda, V.; Guijarro, J.I.; Sunde, M.; Latgé J.P. Hydrophobins—Unique Fungal Proteins. PLoS Pathogens, 2012, 8, e1002700 2. Gravagnuolo, A.M.; Longobardi, S; Luchini, A; Appavou, M.-S.; De Stefano, L; Notomista, E; Paduano, L; Giardina, P. Class I Hydrophobin Vmh2 Adopts Atypical Mechanisms to Self-Assemble into Functional Amyloid Fibrils. Biomacromolecules, 2016, 17, 954-64 3. Cicatiello, P.; Gravagnuolo, A.M.; Gnavi, G.; Varese, G.C.; Giardina, P. Marine fungi as source of new hydrophobins. International Journal of Biological Macromolecules, 2016, 92, 1229-1233.

21ISBN:9788894140422

Lysine or Glutamine site-selective PEGylation of proteins mediated by transglutaminase. A. Grigoletto, K. Maso, G. Pasut

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, Padova 35131, Italy.

Introduction Enzymatic approaches of protein PEGylation are widening their applications thanks to the advantageous obtainment of homogeneous mono-PEGylated isomers. Microbial transglutaminase (mTGase) is an enzyme that, in nature, forms protein cross-linkings between side chains of Gln and Lys residues. For protein PEGylation, mTGase allows site-specific modification with PEG-NH2 at precise glutamines of the proteins. We have already reported mTGase-mediated conjugation with PEG-NH2 to G-CSF, yielding a site selective mono-derivative conjugate involving Gln135 [1]. Interestingly, the same enzymatic reaction of mTGase, was performed using a new PEG derivative, (PEG-Z-QG), containing a Gln substrate of mTGase, in order to target the Lys residues and investigate if the selectivity of the enzyme is maintained also in this reverse conjugation method. The obtained conjugate was then compared to PEG-Q135-G-CSF isomer, in terms of biophysical characterization and pharmacokinetics [2]. In another work, an immobilized form of TGase has been studied with the aim to use it as an alternative tool for protein PEGylation that presents the advantage of a simply removal of mTGase from reaction mixture by centrifugation. The immobilized mTGase (iTGase) was characterized in term of retained activity, kinetic parameters and in different pH and temperature values. For protein PEGylation, a site-specific modification of G-CSF and α-lactalbumin (α-LA), comparing the two forms of mTGase, was then tested [3].

Results and future perspectives The mTGase-mediated conjugation of PEG-ZQG in the presence of G-CSF generated a PEG-G-CSF conjugate in which the polymer was selectively coupled to Lys41 of the protein. The PEG-K41-G-CSF conjugate was formed in high yield and the mTGase maintained high selectivity also for the lysine modification sites. PEG-K41-G-CSF was compared to PEG-Q135-G-CSF in biophysical studies, demonstrating that the two positional isomers have similar behaviors, while the pharmacokinetics in rats have exhibited comparable half-life extensions. mTGase has been demonstrated to be a valuable tool for double site-selective protein modification, either to Gln or Lys residues and can thus offer relevant opportunities in the field of selective protein derivatization, providing, at the same time two potential sites of conjugation, both with high selectivity. In the second purposed strategy, mTGase was covalently immobilized on a beaded agarose resin. The iTGase preserved more than 53% of its starting activity and revealed, as expected, a reduction of the affinity for the substrate, that is, nevertheless, counterbalanced by the increasing in the stability against different pH and temperature conditions. iTGase-mediated PEGylation of α-LA showed a selective conjugation toward only one Gln residue of α-LA, avoiding the formation of a mono and bi-conjugate mixture, achieved using the free enzyme. In the case of G-CSF, iTGase still remained selective towards only one Gln, forming PEG-Q135-G-CSF, but avoided the undesired formation of deamidated G-CSF that took place when free mTGase was used. In conclusion, iTGase has shown an increase in thermal stability and substrate selectivity, suggesting that the immobilization strategy could positively modify the conjugation outcomes and yield pure mono-PEGylated-protein conjugates. Overall, these results highlight the potentiality of mTGase in preparing site-selective protein-polymer conjugates and it would be interesting to test the applicability of these new approaches also to other therapeutic proteins.

References:

1. Mero, A.; Spolaore, B.; Veronese, F. M.; Fontana, A. Transglutaminase-Mediated PEGylation of Proteins: Direct Identification of the Sites of Protein Modification by Mass Spectrometry using a Novel Monodisperse PEG. Bioconjugate Chem., 2009, 20, 384. 2. Mero A.; Grigoletto A.; Maso K.; Yoshioka H.; Rosato A.; Pasut G. Site-selective enzymatic chemistry for polymer conjugation to protein lysine residues: PEGylation of G-CSF at lysine-41. Polym Chem., 2016, 7, 6545-6553. 3. Grigoletto A.; Mero A.; Yoshioka H.; Schiavon O.; Pasut G. Covalent immobilisation of transglutaminase: stability and applications in protein PEGylation. J Drug Target., 2017, 13, 1-9.

22ISBN:9788894140422

Chitosan effect on thermodynamic properties of SPAN/TWEEN monolayers Hanieh Patrizia N1*, Rinaldi Federica2, Bordi Federico3, Sennato Simona3, Marianecci Carlotta1, Carafa Maria1

1Drug Chemistry and Technologies Dpt; 3 Physics Dpt, University "Sapienza”, Rome, Italy; 2Center for Life NanoScience – IIT@Sapienza, Rome, Italy - *patrizianadia.hanieh@uniroma1.it

Background Chitosan is a natural polysaccharide derived from chitin, used for biomedical applications and in pharmaceutical and

biotechnology industries, as fat reduced, bactericide agent and wound healing material and coating agent for stabilization of vesicular systems [1]. In all these uses, its interesting properties such as biocompatibility, biodegradability and non-toxicity are exploited. While the efficacy for some of them is proven, little is known about the molecular-level interactions involved in these applications. More interestingly, owing to its cationic character in acidic solutions, chitosan may interact with the negatively charged surface of biomembranes, and therefore understanding such interactions is important for its use.

Main Results In this work we have employed Langmuir monolayers as membrane model to probe the interactions of chitosan with

monolayers formed by Tween20 or Span20 non-ionic surfactants, mixed to Cholesterol and anionic DCP, to mimic the composition of niosomal vesicles whose efficacy as drug delivery systems has been widely explored [2]. The aim of this investigation is to elucidate the controlling factors useful for a rational design of a chitosan-coated drug delivery system and understand in which conditions chitosan decoration of the vesicle can improve the niosomal structural properties and drug delivery efficiency.

The surface pressure–area isotherms of equimolar Span 20/Chol and Tween 20/Chol monolayer have been measured at 25°C. Chitosan was dissolved in acetate buffer solution (0.03 M, pH 4.5) at different concentrations up to 0.45 mg/mL, where it is not surface-active. To understand the role of the different forces involved in the polycation-monolayer interaction, we also considered the effect of the addition of the anionic lipid Dicetylphosphate (DCP) to form charged Tween20/Chol/DCP films, with molar ratio 1/2/1.

Our study revealed that chitosan strongly adsorbed at monolayers surface inducing a film expansion both at low and at high surface pressure, where it interacts not only superficially but also inserted to a certain degree into the film. In certain conditions, depending on both chitosan concentration and on the different hydrophilic-hydrophobic balance of the surfactants, modification of the monolayer phase transition has been observed.

Future Perspectives Elucidating the molecular organization of chitosan-surfactant monolayers could be useful to better understand the release rate

of model drugs from niosomes and their stability in vitro and in vivo. Acknowledgements The financial support of IIT and MIUR (FIRBRBFR12BUMH) is acknowledged

References: [1] M. Rinaudo, Chitin and chitosan: Properties and applications. Prog. Polym. Sci., 2006, 31, 603-632. [2] C. Marianecci, L. Di Marzio, F. Rinaldi, C. Celia, D. Paolino, F. Alhaique, S. Esposito and M. Carafa, Niosomes from 80s to present: the state of the art. Adv. Colloid Interface Sci., 2014, 205, 187-206.

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Neuronal alpha-synuclein silencing by siRNA-loaded RVG liposomes

Michele Schlich1, Francesca Longhena2, Gaia Faustini2, Caitriona M. O’Driscoll3, Chiara Sinico1, Anna Maria Fadda1, Arianna Bellucci2, Francesco Lai1

1 Dept. Life and Environmental Sciences, University of Cagliari; 2 Dept. Molecular and Translational Medicine, University of Brescia

3 School of Pharmacy, University College Cork. Background: In the last decade, RNA interference (RNAi) has been exploited to knock down the expression of genes involved in several pathologic conditions, being often reported as a revolutionary therapeutic approach for cancer, as well as neurodegenerative and infective diseases. However, the clinical translation of RNAi therapeutics such as small interfering RNA (siRNA) is hindered by their unfavourable pharmacokinetic properties, such as poor enzymatic stability in vivo and low intracellular distribution, an essential feature for their mechanism of action [1]. The inclusion of siRNA in nanocarriers could significantly increase their efficacy ensuring protection from enzymatic degradation, prolonged circulation and increased cell uptake [2]. With this in mind, we developed a novel lipid-based nanocarrier able to load, protect and deliver a siRNA specifically designed to reduce neuronal α-synuclein expression in vitro. Numerous evidence points out that the accumulation of α-synuclein oligomers in Lewy bodies plays a pathogenic role in Parkinson's diesase (PD), as well as in other pathologies collectively called synucleinopathies. Thus, strategies aimed at reducing α-synuclein levels have been suggested as potentially relevant for PD treatment. Main results: In the present work, anionic, PEGylated liposomes decorated with a rabies virus glycoprotein (RVG)-derived peptide, were loaded with a preformed complex of siRNA and protamine. The RVG peptide is known for its ability to trigger the receptor mediated transcytosis of macromolecules and nanocarriers through the interaction with the nicotinic acetylcholine receptor, expressed at the luminal side of brain capillaries [3]. The nanoparticles, obtained through a modified film hydration method, were characterized for their ability to load, protect, and deliver the functional siRNA to mouse primary hippocampal and cortical neurons as well as their efficiency to induce gene silencing in these cells. Results showed that the RVG decoration significantly increase the liposomal siRNA uptake by neurons as compared to untargeted vesicles. Accordingly, a marked knock down (over 70% decrease compared to untreated control) of alpha synuclein could be evidenced by immunocytochemisty in neurons treated with RVG-liposomes. With the aim of future in vivo applications, the developed nanocarriers were evaluated for their stability in serum, showing no sign of aggregation and efficiently protecting the siRNA from nucleases for over 2 hours. Future perspectives: As witnessed by the recent literature, the reduction of alpha synuclein expression is receiving growing interest as a potential therapeutic option for PD. The use of exogenous RNAi for this purpose is intriguing and challenging at the same time because of the cited delivery issues. The present research was oriented at obtaining a safe, simple and reproducible liposomal siRNA formulation, able to prove its efficacy in vitro as a first step of a broader scope. Indeed, our results, together with essential features such as PEGylation and a the inclusion of a brain targeting mojety, indicate that RVG-decorated liposomes may be an ideal tool to achieve efficient in vivo α-syn gene silencing in animal models of PD.

References [1] Haussecker, D., Current issues of RNAi therapeutics delivery and development. J. Control. Release 2014, 195, 49–54. [2] Lorenzer, C.; Dirin, M.; Winkler, A.-M.; Baumann, V.; Winkler, J., Going beyond the liver: Progress and challenges of targeted

delivery of siRNA therapeutics. J. Control. Release 2015, 203, 1–15. [3] Alvarez-erviti, L.; Seow, Y.; Yin, H.; Betts, C.; Lakhal, S.; Wood, M.J.A., Delivery of siRNA to the mouse brain by systemic

injection of targeted exosomes. Nat. Biotechnol. 2011, 29, 341–345.

24ISBN:9788894140422

Mucoadhesive S-preactivated thiolated glycolchitosan for drug delivery Perrone Mara1, Lopalco Antonio1, Laquintana Valentino1, Lopedota Angela1, Cutrignelli Annalisa1, Franco Massimo1, Bernkop-

Schnürch Andreas2, Denora Nunzio1 1Dpt of Pharmacy –Drug Sciences, University of Bari “Aldo Moro”, Bari, Italy

2Dpt of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck,Austria

Background This work continues and improves our previous studies on thiolated glycol chitosan (GC) used as mucoadhesive drug delivery system. Here, we have described the synthesis and characterization of two novel S-preactivated thiolated GC polymers. Therefore, GC was thiolated by covalent attachment of glutathione (GSH) and N-acetyl cysteine (NAC), respectively. The immobilized thiol groups were preactivated by disulfide bond formation with 2-mercaptonicotinic acid (2-MNA). Their improved mucoadhesive properties have been evaluated using in vitro as well as ex-vivo methods. Main Results GSH and NAC were covalently attached to GC by the formation of amide bonds between glycine carboxylic acid groups or cysteine carboxylic acid groups of GSH and NAC and primary amino groups linked to the C-2 of pyranose ring of GC backbone. Hence, reactions were carried out in an acidified solution at pH 5.0 [1]. The carboxylic acid moieties were activated using EDAC prior to the coupling reaction. Subsequently, the attached thiol groups were protected by disulfide bond formation by using 2-MNA [2]. The obtained GC-NAC-MNA and GC-GSH-MNA conjugates were characterized regarding content of free thiol groups by Ellman’s assay and content of MNA residue, biocompatibility, swelling/erosion behavior and mucoadhesive properties, compared to thiolated and unmodified GC [3]. The thiolation and S-protection of GC influenced slightly the biocompatibility of the polymers. A concentration dependent toxicity was observed among the modified polymers. Cell viability, performed using a CaCo-2 cells model, dropped down to approximately 80 % at the highest concentration of 2 mg/mL after 24 h whereat viability remained over 85 % at a concentration of 0.02 mg/mL for both S-preactivated conjugate polymers. On the contrary, the mucoadhesive properties of S-preactivated conjugates, evaluated by tensile and rotating cylinder studies, appeared to be influenced by the introduction of 2-MNA residue. In particular, in terms of time of adhesion, the S-preactivated conjugates remained attached on freshly excised porcine mucosa for 3.5 h and 3.25 h for GC-NAC and GC-GSH, respectively (Fig. 1). Similar results were achieved with tensile studies. Indeed, the conjugates resulted in a 1.6- and 1.5-fold increase in total work of adhesion and 1.5- and 1.3-fold increase in maximum detachment force, in comparison to the thiolated conjugates, GC-NAC and GC-GSH, respectively (Fig. 2). Furthermore, water-uptake studies showed a weight gain in the first hour for the S-protected polymers, followed by disintegration in the next two hours of the experiment. Future Perspectives The introduction of 2-MNA on thiolated GC changed the polysaccharide characteristics by improving mucoadhesive properties. This work represents the first study describing GC-NAC-MNA and GC-GSH-MNA conjugates as potential platform useful to realize appropriate mucoadhesive drug delivery systems, suitable to prolong the residence time of drugs. Further studies will be performed using these new mucoadhesive polymers with a model drug.

References: 1. Denora, N.; Lopedota, A.; Perrone, M.; Laquintana, V.; Iacobazzi, R. M.; Milella, A.; Fanizza, E.; Depalo, N.; Cutrignelli, A.; Lopalco, A.; Franco, M. Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates. Acta Biomaterialia. 2016, 43, 170–184. 2. Perrone, M.; Lopalco, A.; Lopedota, A.; Cutrignelli, A.; Laquintana, V.; Douglas, J.; Franco, M.; Liberati, E.; Russo, V.; Tongiani, S.; Denora, N.; Bernkop-Schnürch, A. Preactivated thiolated glycogen as mucoadhesive polymer for drug delivery. Eur J Pharm Biopharm, 2017, 119, 161-169. 3. Perrone, M.; Lopedota, A.; Liberati, E.; Russo, V.; Cutrignelli, A.; Laquintana, V.; Pereira de Sousa, I.; Franco, M.; Tongiani, S.; Denora, N.; Bernkop-Schnürch, A. Natural dendrimers: Synthesis and in vitro characterization of glycogen-cysteamine conjugates. Eur J Pharm Biopharm. 2017, 115, 168-176.

25ISBN:9788894140422

Nanopolyplexes bearing folic acid as a new therapeutic nanoplatform towards cancer cells Malfanti Alessio1, Scomparin Anna2, Brazzale Chiara1, Pozzi Sabina2, Balasso Anna1, Ben Shushan Dikla2, Mastrotto Francesca1,

Salmaso Stefano1, Satchi-Fainaro Ronit2, Caliceti Paolo1 1Dept. of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy.

2Department of Physiology and Pharmacology, Sackler School of Medicine,Tel Aviv University 69978 Tel Aviv – Israel. Email: alessio.malfanti@phd.unipd.it; paolo.caliceti@unipd.it

Background: Gene therapy is defined as the treatment or prevention of human diseases by introduction of new genetic material, such as therapeutic oligonucleotides (e.g. siRNA, miRNA, etc), into specific cells.1 In the last few decades the research community has invested substantial resources in the delivery of therapeutic oligonucleotides (ON). Unfortunately, due to their anionic nature, high molecular weight and structural fragility, ONs administration suffers from poor bioavailability and biological efficiency. These macromolecules can in fact undergo fast degradation by serum nucleases, negligible transmembrane transport, off-target profile and also elicit immunogenic response.2 So far, cationic based nanocarriers (including polymers and lipids) are the most promising methods to protect and deliver ON. In this work, we have designed a novel cationic polymer for ONs (i.e. siRNA) complexation driven by coulombic interactions. The polymer have been further functionalized with folic acid to endow the system with targeting properties towards tumor cells. The obtained complexes, here referred as nanopolyplexes, has been found to improve the pharmacokinetic and pharmacodynamics parameters of ONs generating a valuable tool for selective cancer treatment. Main results: Nanopolyplexes were prepared by simply self-assembling our cationic polymer in the presence of siRNA. siRac1 was used as a therapeutic siRNA for its ability to inhibit cell migration, metastathization and cell invasion3. Electrophoresis gel shift assay was used to investigate nanopolyplexes formation at different nitrogen/phosphate (N/P) ratios starting from 1 up to 10. The results showed that complete complexation of the siRNA was achieved at the N/P ratio of 3. The nanopolyplexes with 3 and 5 N/P ratio were characterized by Dynamic Light Scattering (DLS) showing a mean diameter size of 80 nm and a surface charge of +4 mV. Nanopolyplex formed at N/P ratios of 3 and 5 were further investigated showing a satisfactory serum stability and remarkable siRNA protection from nucleases. The strength of the interaction, thus the stability of polymer/siRNA interaction, was confirmed by the high heparin concentration (2.5 IU/mL, about 16 fold the heparin concentration in blood stream which is 0.15 IU/mL4) required to achieve siRNA displacement from the nanopolyplexes. The hemolytic profile of polyplexes at 3 and 5 N/P ratios was assessed by measuring red blood cells (RBCs) lysis and negligible hemolysis was observed. Nanopolyplexes formulation was then further modified to provide the system with targeting ability towards breast cancer cells. To this aim, the cationic polymer was functionalized at one chain end with folic acid and polyplexes were formed by adding 25 % w/w of the folate-conjugated polymer to the formulation (folate-targeted nanopolyplexes).The The human adenocarcinoma cell line expressing folate receptor, MDA-MB-231, was selected as model for the in vitro and in vivo studies and a N/P ratio of 3 was used. Silencing and toxicity studies were initially performed. Folate- targeted nanopolyplexes showed a gene silencing about two folds higher compared to naked polyplexes (86% vs 49%, respectively). Both formulations were found to have high biocompatibility (cell viability >80% for all the concentration tested, 125-500 nM siRac1 equivalent dose). The ability of the nanopolyplexes to inhibit cell migration was assessed by wound healing assay on MDA-MB-231 cell line. The gap closure was observed after 12 hours and the results showed a 40% inhibition of the cell migration when cells were treated with 500 nM siRNA equivalent dose for folate-targeted nanopolyplexes. On the contrary, naked nanopolyplexes inhibited cell migration of only 15%. The cell uptake of nanopolyplexes was evaluated by confocal analysis and it was observed that the differences between the targeted and non-targeted system in the silencing activity do not arise from the cellular uptake efficiency. To explain the silencing results, intracellular trafficking studies were performed. Early endosomes and lysosomes were immunostained and the results showed that folate-targeted nanopolyplexes did not co-localize with early endosomes and lysosomes. Conversely, naked nanopolyplexes fully co-localized with early endosomes. mechanism of cell internalization was evaluated by immune staining the caveolin-1 and clathrin heavy chain, responsible of caveolae and clatrhin-mediated uptake, respectively. Cell internalization was observed to be caveolae- mediated for folate-targeted nanopolyplexes while was fully clathrin-mediated for naked nanopolyplexes. These results highlighted the involvement of folic acid on the caveolae mediated uptake resulting in a better gene silencing. The systemic toxicity of folate-targeted and naked nanopolyplexes at a siRNA concentration ranging from 1 to 6 mg/kg was evaluated on Nu/Nu female mice by stepwise escalation dose intravenous injection until maximum tolerated dose for short period. No toxicity was found for all the doses used for both formulations. Finally, folate- targeted nanopolyplexes accumulation in the tumor was investigated observing a ~500-fold and ~65-fold increase in Rac1 siRNA tumor accumulation in comparison with saline or scramble siRNA (siEGFP) treatment, respectively. It was possible to detect a linear correlation between the size of the tumor and the polyplexes accumulation. Future perspectives: We have design and characterized a new polymer able to interact by coulombic interaction with siRNA. Future perspectives will involve the use of different ONs or further combination with anticancer drugs in order to improve the anticancer efficacy. References

1. Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S., Design and development of polymers for gene delivery. Nature reviews. Drug discovery 2005, 4 (7), 581.

2. Geary, R. S.; Norris, D.; Yu, R.; Bennett, C. F., Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Advanced drug delivery reviews 2015, 87, 46-51.

3. Bid, H. K.; Roberts, R. D.; Manchanda, P. K.; Houghton, P. J., RAC1: an emerging therapeutic option for targeting cancer angiogenesis and metastasis. Molecular cancer therapeutics 2013, 12 (10), 1925-1934.

4. Engelberg, H.; Dudley, A., Plasma heparin levels in normal man. Circulation 1961, 23 (4), 578-581.

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Hyaluronic acid decorated liposomes containing disulfiram-copper: a new platform against pancreatic cancer stem cells?

Alessandro Marengo1, Stefania Forciniti2, Barbara Stella1, Franco Dosio1, Nicolas Tsapis3, Elias Fattal3, Marta Palmieri2, Silvia Arpicco1

1Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P. Giuria 9, 10125, Torino 2Dipartimento di Scienze della Vita e della Riproduzione, Università degli Studi di Verona, Strada Le Grazie 8,37134, Verona

3CNRSUMR 8612, Institut Galien Paris-Sud, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France Pancreatic ductal adenocarcinoma (PDAC) is a highly lethally disease showing the worst prognosis of any major malignancy (3% 5-year survival). In most cases it has been diagnosed in an advanced state for which there are little or no effective therapies. Increasing evidences indicate the crucial role of small subpopulations of cells, called cancer stem cells (CSCs), in pancreatic tumor formation, metastasis and progression. Pancreatic CSCs were discovered in 2007 by Li et al1. They identified a small populations of cancer cells (0.2-0.8 % of tumor mass), CD44+CD24+ESA+ with 100 fold higher tumorigenic potential in comparison to CD44-

CD24-ESA- cells populations. Currently, CSCs are considered the main obstacle in the eradication of pancreatic cancer. Conventional therapies destroy the tumor bulk but not CSCs; consequently CSCs remain viable after therapy and re-establish the tumor. Based on these evidences, the aim of this work was to prepare liposomes for the active targeting of anticancer drugs against receptors over-expressed on pancreatic CSCs. To this end, disulfiram-copper complex (DSF-Cu) was chosen as anti-CSC agent, based on its well documented anti-CSC activity2.To overcome the insolubility of DSF-Cu in biological medium and to maximize its delivery on CSCs we developed liposomal formulations decorated with hyaluronic acid (HA) able to recognize and target the CD44 receptor overexpressed on pancreatic CSCs surface. Liposomes containing DSF-Cu were prepared using a ion gradient method; this method provides the formation of the DSF-Cu complex directly inside the liposomes ensuring high encapsulation efficiency. To obtain an active targeting against CD44 receptor we decorated liposomes with HA-phospholipid conjugate previously synthesized by an amino reductive reaction between the amino group of 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and the aldheydic group of HA (4800 and 17000 Da) generated after the sugar ring opening. HA decorated liposomes showed a more negative zeta potential in comparison to non decorated ones, due to the presence of HA on their surface and were stable for 35 days in storage conditions (4°C).The cryo-TEM analysis revealed the presence of needle-shaped structures in the aqueous core of liposomes suggesting the precipitation of the DSF-Cu complex inside the liposomes in form of crystals. In vitro tests on PANC-1 CSCs demonstrated the strong anticancer activity of the liposomal formulations: PEGylated liposomes showed IC50 value of 80 nM and the decoration of liposomes with HA further reduced the IC50 value to 30 nM suggesting the effectiveness of the HA as targeting agent. The anticancer activity of all formulations was superior in CSC model respect to the parental ones although the CSCs are considered more resistant. In vitro tests conducted in presence of N-acetyl cysteine (NAC), a potent antioxidant, revealed a ROS mediated cell death; indeed in presence of NAC the anticancer activity was completely abolished. In conclusion, these results indicate that the encapsulation of DSF-Cu into HA decorated liposomes could be a promising platform to target and eliminate pancreatic CSCs. References: [1] Li, C; Heidt, D.G; Dalerba, P; Burant, C.F; Zhang, L; Adsay, V; Wicha, M; Clarke, M.F; Simeone, D.M; Identification of pancreatic cancer stem cells, Cancer research, 2007, 67, 1030-1037. [2] Liu, P; Brown, S; Goktug, T; Channathodiyil, P; Kannappan, V; Hugnot, J.P; Guichet, P.O; Bian, X; Armesilla, A.L; Darling, J.L; Wang, W; Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells, British journal of cancer, 2012, 107, 1488-1497.

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Development of Anticancer Polymeric Conjugates based on Hyaluronic Acid and PEG for Ovarian Cancer Combined Therapy

Gabriele Martinez1, Antonella Grigoletto2, Isabella Montagner1, Antonio Rosato1, Gianfranco Pasut1,2

1Veneto Institute of Oncology IOV-IRCCS, Padua, Italy. 2Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, Padova 35131, Italy.

Background: Drug delivery systems (DDSs) has been proposed as promising approach to overcome issues related to small drugs and to achieve drug targeting to the site of action. Polymeric conjugation to small drugs could be a convenient way to overcome problems such as low water solubility, a short in vivo half-life, and low specificity [1]. The selective delivery achievable by a non-targeted DDS formulation in a tumor site is mainly due to its preferential accumulation by ‘Enhanced Permeability and Retention effect’ (EPR). Furthermore, the addition of a targeting agent to the polymeric carrier can increase the selectivity towards specific cells, tissues or organs. The target of this project is ovarian cancer (OC), one of the leading causes of deaths from gynaecological malignancies worldwide. To hit this tumor it has been proposed a combination chemotherapy based on two targeted drug-polymer conjugates specifically designed for intravenous and intraperitoneal administration respectively. This will improve the effectiveness of combined intravenous-intraperitoneal OC chemotherapy We developed two different anticancer polymer-drug conjugates: one based on hyaluronic acid (HA) for i.p. administration, to obtain a conjugate able to specifically interact with the CD44 receptors that are overexpressed in many tumors [2], and the second based on PEG derivatives bearing the folic acid (FA), which interacts with the FA receptors, also overexpressed in tumors. The carboxylic groups of HA can be easily modified to obtain reactive groups suitable for drug coupling. Diaminocyclohexan platinum (DACHPt) is an anticancer drug for the treatment of ovarian cancer (OC). A chelating group, (3-aminopropyl) malonic acid, was linked directly or through a disulphide linker to HA’s carboxylic group. The malonate moiety is used to chelate DACHPt to HA. Two different HA molecular weights were investigated, 50 and 200 kDa. In the case of PEG, a heterobifunctional NH2-PEG5k-COOH has been used to allow the coupling of FA at the -COOH terminus and Paclitaxel (PTX) at the –NH2 terminus. To increase the FA/PEG ratio, a dendron structure was synthetized at polymer’s end. FA, recognized by FA receptors, presents two carboxylic groups, γCOOH and αCOOH that can be modified to achieve conjugation to PEG. The γCOOH is essential for the binding to FA receptors so its modification will prevent its targeting properties. In this case it is extremely important that all the molecules of FA are coupled to PEG through the αCOOH thus preserving the FA receptor binding and avoiding the obtainment of non-targeted nanoconjugate fractions, which might have an unspecific toxicity. Such issue was faced by preparing an FA-α-cadaverine-NH2 intermediate in which the α-isomer was selected through accurate synthesis optimization and extensive purification of crude reaction mixtures. Main results: Four different HA-DACHPt conjugates were developed and fully characterizeed. The conjugates are divided in two groups, the first based only on the (3-aminopropyl)malonic acid linker, 1) HA50-Pt and 2) HA200-Pt, and the second having a disulphide bond in the linker, 3) HA50-SS-Pt and 4) HA200-SS-Pt. The Pt loading, determined by atomic absorption spectroscopy, was: 20.3% (wt/wt) for HA50-SS-Pt, 22.0% (wt/wt) for HA200-SS-Pt, 2.1% (wt/wt) for HA50-Pt and 9.4% (wt/wt) for HA200-Pt. The higher drug loading of the conjugates with the disulphide linker is due to the different chemical strategy of conjugation. Conjugates were not degraded, in terms of size decrease, by incubation at different pH values (pH 5.5, 6 and 7.4) while were very susceptible to enzymatic degradation by hyaluronidase. The drug release was tested in aqueous physiological conditions. Conjugates with disulphide bridge showed a massive dug release under a reductive media by glutathione. The conjugates were tested in A2780 cell line, sensitive to Oxaliplatin but not-overexpressing the CD44 receptor that binds HA, and SKOV-3 cell line that are resistant to Oxaliplatin but overexpress the CD44 receptor. The results were very interesting for all conjugates that outperformed or equalled the activity of Oxaliplatin in both cell lines. HA200-Pt was particularly promising being more than four times more active than the free drug in the resistant cells. The fact that both HA conjugates based on the polymer at 200 kDa showed better activity than the conjugates based of the HA at 50 kDa suggested that the binding to the CD44 receptor is stronger for 200 kDa HA with respect to the 50 kDa HA. Furthermore, HA-DACHPt conjugates induced higher degree of apoptosis than the free drug. PTX-PEG-FA and PTX-PEG-(FA)3 conjugates were synthetized and characterized. PTX loading and conjugate purity were assessed by RP-HPLC methods. The conjugates were characterized confirming the ability to form micelles in water. Future Perspectives: The assessment of in vivo biodistribution and pharmacokinetics, the evaluation of in vivo therapeutic activity, and the in vivo combined activity. References 1. Duncun, R. Polymer conjugates as anticancer nanomedicines. Nature Rev Cancer, 2006, 6, 688-701. 2. Sneath, R.J.S.; Mangham, D.C. The normal structure and function of CD44 and its role in neoplasia. J Clin Pathol Mol Pathol, 1998, 51,191-

200.

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Intracellular delivery of a protein-antibody system in endothelial cells Maso Katia1, Muro Silvia2, Pasut Gianfranco1

1Dept. Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131, Padova, Italy 2Fischell Dept. of Bioengineering, University of Maryland, 5115 Plant Sciences Building, 20742, College Park (MD), USA

Background. Monoclonal antibodies (mAbs) are largely exploited in targeted therapies thanks to their high affinity and unique specificity for antigens that are associated with characteristic markers of diseased tissues. However, most mAbs are not sufficiently potent to be therapeutically active on their own and are used in combination with other drugs. From this evidence, a great innovation has been achieved by combining the targeting activity of mAbs to the pharmacological activity of potent small drug molecules in a unique entity, thus creating the well-known therapeutic class of antibody-drug conjugates (ADCs). As in the case of the two ADCs now on the market, Adcetris and Kadcyla, drugs have been traditionally coupled in a random way to lysines or cysteines of mAbs giving a heterogeneous final product composed of multiple ADC’s isoforms that can have different stability and activity [1]. Nowadays, research is focused on the development of site-selective conjugation techniques to create new ADCs with improved efficacy and properties. Here, we proposed a new approach based on a non covalent interaction between the Fc region of a mAb, that represents the targeting moiety, and a protein, that should act as drug carrier, aiming to achieve a higher degree of homogeneity and to create a versatile drug delivery system. We used Protein G, a surface receptor derived from some Streptococci C and G strains, that is employed in mAbs industrial purification processes because of its high affinity (Ka = 107-109 M-1) for the Fc of human IgGs and of a variety of other mammalian IgGs such as mouse IgGs [2]. Main Results. Recombinant 22.8 kDa Protein G was site-specifically PEGylated at the N-terminus with a PEG20kDa whose free end was exploited for dye (or drug) coupling. The polymer acts like a linker between the protein and the payload and ITC analysis assessed that, after PEGylation, Protein G affinity for IgGs was preserved. In this work, we investigated the capacity of this non covalent protein-antibody system to be internalized by human umbilical vein endothelial cells (HUVECs). The endothelium is a specialized tissue constituted of different endothelial cells (EC) subtypes and associated with a wide variety of pathological conditions such as ischemia, thrombosis, inflammation and vascular oxidative stress. Therefore, this tissue represents a promising therapeutic target [3]. One of the most studied EC surface determinants is the intercellular adhesion molecule-1 (ICAM-1) that is constitutively expressed at low levels but whose expression can be induced by cytokines and proinfiammatory factors such as TNFα, IL-1 and INFγ. As targeting antibody, we used a mouse IgG2a against human ICAM-1 receptor, from here called aICAM. A series of in vitro experiments on HUVECs seeded on gelatin coated coverslips were carried out to study receptor binding, mechanism of internalization and lysosomal trafficking of Alexa488-PEG-Protein G targeted by aICAM. AlexaFluor488 was used to visualize the samples during fluorescence microscopy analysis. To induce ICAM-1 expression on HUVECs surface, cells were treated with a TNFα solution for at least 16 hours. The results demonstrated that aICAM targeted systems bound specifically to activated HUVECs, and that, after binding, were internalized through CAM-pathway, a non classical route of endocytosis that relies on the interaction between the targeting antibody (aICAM) and the receptor (ICAM-1). Alexa488-PEG-Protein G alone and targeted by an aspecific IgG did not retain any binding capacity and were not internalized. Since the labelling dye was tethered to Protein G through PEG linker, these results proved that during receptor binding and subsequent uptake by cells, the protein-antibody system maintained its stability acting like a unique structure. Intracellular trafficking was then followed staining lysosomes with Texas Red dextran 10 kDa. It appeared that the percentage of the internalized Alexa488-PEG-Protein G that co-localized within lysosomes increased over time, revealing that proteins were moved from endocytic vesicles to lysosomes for degradation. Future Perspectives. In conclusion, we suggested an alternative to ADCs approach creating an antibody-protein system based on a non covalent interaction that may guarantee control over conjugation site and antibody-drug ratio. This system is very versatile since different mAbs can be potentially used depending on the target, thanks to Protein G ability to bind Fc that is the conserved region among IgGs. In human endothelial cellular models, it was proven the possibility to achieve an active delivery of cytotoxic drugs inside cells. However, further and deep investigations will be necessary to evaluate the in vivo stability of the system in order to verify that it maintains its integrity also during blood circulation for longer periods of time. References: 1. Shen, B. Q.; et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol., 2012, 30, 184-189. 2. Lund, L. N.; Christensen, T.; Toone, E.; Houen, G.; Staby, A.; St Hilaire, P. M. Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry. J Mol Recognit., 2011, 24, 945–952. 3. Ding, B.; Dziubla, T; Shuvaev, V. V.; Muro, S.; Muzykantov, V. R. Advanced drug delivery systems that target the vascular endothelium. Mol Interv., 2006, 2, 98-112.

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Tuning Mannose Receptor function via specific targeted ligands

Salama Alan1, Martinez-Pomares Luisa2, Mantovani Giuseppe3, Mastrotto Francesca4 1Centre for Nephrology, Royal Free Campus, University College London, London WC1E 6BT United Kingdom

2School of Life Science, University of Nottingham, Nottingham NG7 2UH, United Kingdom; 3School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, United Kingdom

4Dept. of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy,

0498275695; Email: francesca.mastrotto@unipd.it

Background The biological roles of glycans have been extensively investigated over the years spanning from modulatory properties to

specific recognition to glycan binding proteins (GBP).1 Acquired knowledge about glycans physico- chemical properties essential for interaction with GBPs together with the development of advanced analytical techniques, promoted research into ligand density and multivalency to identify biologically relevant interaction between “synthetic glycans” and specific receptors.2 Among the GBPs, Mannose Receptor (MR) has gained particular interest. MR is primarily expressed by populations of macrophages, immature dendritic cells, endothelial cell subsets and kidney mesangial cells. The biological function of this receptor encompasses pathogen recognition, clearance of endogenous molecules, promotion of antigen presentation, modulation of cellular activation and trafficking, and it also provides the link between innate and adaptive immunity.3 Despite MR is involved in many physiopathological events, the full elucidation of its role is non-trivial due to the lack of selective modulators in vivo (including anti-MR mAbs). In this study, we have generated libraries of macromolecules selectively targeted to mannose receptor (MR) specific domains and we have deeply investigated their binding and uptake profiles by varying targeted domain, molecular weight and binding unit density. Interestingly, the results revealed the ability of CR-directed polymers to temporarily inhibit uptake of MR ligands and therefore to modulate its biological function paving the way for a better understating of its contribute in several diseases.

Main results Libraries of fluorescent polymers targeted to either the carbohydrate lectin-like (CTLD) or cysteine-rich (CR) domains of MR

and displaying increasing percentage of binding units in the backbone (0, 33, 66 and 100%) were synthesized via Atom Transfer Radical polymerisation techniques and “click chemistry”. As control, non-targeted polymers with analogous structures were used in all the experiments performed. The polymer internalisation profiles in CHO/ CHO-MR+ or WT/MR--macrophages were assessed by flow cytometry varying concentration and incubation time. CTLD-targeted polymers showed linear correlation between binding units % and uptake, while the opposite trend was observed for CR-targeted polymers. Polymer affinity for CTLD4-7- and CR-Fc chimeras or soluble MR (sMR) was evaluated via ELISA and surface plasmon resonance (SPR), respectively. ELISA assay confirmed the specificity of each polymer to the targeted domain while, surprisingly, SPR results revealed that CR-targeted polymers display higher affinity for the receptor in comparison to the CTLD-targeted polymers, and highlighted that increased density of binding units leads to lower binding constant (KD). We therefore decided to further investigate the different internalisation mechanism by looking at the total or surface expression of MR in CHO-MR+- cells or WT macrophages after treatment with CR o CTLD-targeted polymers using western blot and flow cytometric analysis, respectively. No difference in total expression was detected while a remarkable reduction of the MR surface expression (<50-80%) was observed when cells were pre-treated with CR-targeted polymers. Competition assays and time course experiments were performed in MR+ cell lines to elucidate the ability of CR-directed polymers to inhibit uptake of MR ligands and identify the post-treatment time window for effectiveness. The inhibitory effect was found to last for 16 to 24 hours depending on the polymer molecular weight. These data were further confirmed in in vivo competition experiments. Confocal colocalization analysis showed that CR-targeted polymers sequestrate the receptor within the cells for prolonged time. The use of CR-targeted polymers is currently under investigation for the treatment of inflammatory diseases and the results indicate their ability to prevent renal damage in an in vivo unilateral model of kidney ischemia-reperfusion injury.

Future perspective Polymers designed to selectively target the CR domain block MR functionality by sequestrating the receptor within the cells.

Accordingly, treatment with CR-specific polymers diminishes MR surface expression while the total amount remains unvaried. This can be explained by the high affinity and the pH-independent binding to the receptor of this class of material. MR is well known to be involved in a range of diseases, therefore we generated a valuable tool for its activity modulation. Ongoing studies are exploiting the use of CR-targeted polymer for the treatment or prevention of ischemic damages. The mechanism by which the MR blockage results in protection from damage in vivo is still unidentified, thus dedicated studies have been planned to elucidate it.

References

1. L. J. Varki A, in Essentials of Glycobiology. 2nd edition., ed. C. R. Varki A, Esko JD, et al., Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, NY, 2009, ch. 6.

2. S. Sattin and A. Bernardi, in Carbohydrate Chemistry: Volume 41, The Royal Society of Chemistry, 2016, vol. 41, pp. 1-25. 3. L. Martinez-Pomares, Journal of leukocyte biology, 2012, 92, 1177-1186.

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Single- and multi-compartment capsules for delivery of nutraceuticals Melocchi Alice (1,2), Parietti Federico (2), Maroni Alessandra (1), Zema Lucia (1), Gazzaniga Andrea (1)

(1) Università degli Studi di Milano, Dip. Scienze Farmac., via G. Colombo 71, 20133 Milano, Italia, +390250324654; (2) Multiply Labs, 1760 Cesar Chavez St., 94124 San Francisco, US-CA.

Background

Dietary supplements have reached huge market shares, particularly in the US where consumers are becoming more demanding about bioefficacy and release performance of nutraceuticals. Hence, exploitation of drug delivery technologies in this area would enable the design of innovative products, able to fulfill increasingly complex customer needs.

Capsular devices have recently been proposed for the preparation of novel drug delivery systems (DDSs), as the performance of the final product is determined by the composition and design features (morphology and thickness) of the shell only [1-3]. Particularly, capsules able to release their content at different time points were attained using both fused deposition modeling (FDM) 3D printing and injection molding (IM). While the former would enable product personalization and fabrication on demand, the latter would allow its manufacturing on a larger scale. The aim of this work was thus to evaluate the viability of such systems for pulsatile release of caffeine, a supplement of high current interest that could greatly benefit from a deferred release onset after administration. The feasibility of FDM as a rapid prototyping tool vs IM in the development of the devices was also investigated. Main results

Capsular devices were designed to be characterized by the presence of eiher a single internal cavitiy or two separate compartments (Figure 1). The different systems were composed of i) a body and a cap, having same thickness and composition, or ii) two hollow parts and a middle part, acting as a joint and a partition, which may differ in thickness and composition.

Starting from promptly-soluble and swellable/erodible polymers (i.e. Klucel™ LF, KLF; Affinisol™ 15cP, AFF; Kollicoat® IR, KIR), IM and FDM were carried out using a BabyPlast 6/10P and a MakerBot Replicator 2 [1,2]. While a mold already available was employed for IM, FDM was perfomed starting from purposely-developed computer-aided design (CAD) files. By combining capsule parts having same or different composition and design features, devices provided with a single or double inner compartments were obtained. These systems were proved able to yield two-pulse release patterns: immediate release followed by delayed-onset release of caffeine, or successive release pulses thereof, based on the nature of the polymeric components of the capsule parts. Comparable results were obtained from printed and molded devices having same composition, thus supporting the possibility of exploiting FDM as a prototyping tool to speed up and simplify the R&D phases of IM processes. By way of example, Figure 2 shows the release profiles of single (top)- and double (bottom)-compartment capsular devices, having same composition and design features, fabricated via both FDM (left) and IM (right). Future persepctives

Capsular devices able to yield one or more release pulses of caffeine at different time points were succesfully developed and manufactured by FDM and IM. The potential of such systems should further be explored considering other polymeric materials to achieve diversified release performance that could be of utmost interest for personalization. FDM was proved to be a useful rapid prototyping tool that would help in the identification of critical composition and design features of the molded systems. In perspective, the usefulness of this techique as an R&D tool should be confirmed by considering other dosage forms/DDSs.

References

1. Gazzaniga, A.; Cerea, M.; Cozzi, A.; Foppoli, A.; Maroni, A.; Zema, L. A novel injection-molded capsular device for oral pulsatile delivery based on swellable/erodible polymers. AAPS PharmSciTech., 2011, 12, 295-303.

2. Zema, L.; Loreti, G.; Macchi, E.; Foppoli, A.; Maroni, A.; Gazzaniga, A.; Injection-molded capsular device for oral pulsatile release: development of a novel mold. J. Pharm. Sc., 2013, 102, 489-499

3. Melocchi, A.; Parietti, F.; Loreti, G.; Maroni, A.; Gazzaniga, A.; Zema, L. 3D printing by fused deposition modeling (FDM) of a swellable/erodible capsular device for oral pulsatile release of drugs. J. Drug. Deliv. Sci. Technol., 2015, 30 Part B, 360-367.

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Poly(methyl methacrylate) salt to design orodispesible films Musazzi U.M., Selmin F., Franzè S., Gennari C.G.M., Rocco P., Minghetti P., Cilurzo F.

Dept. Pharmaceutibcal Science, Università degli Studi di Milano, via G. Colombo, 71 - 20133 Milan (I) Background To design orodispersible films (ODF), the selection of a film-forming polymer is the most critical step because all proposed materials exhibit peculiar issues related to the mechanical properties, manufacturing process or drug loading ability. Moreover, the anatomy of the oral cavity limits the size of the dosage form (6 cm2) and the drug loading (< 100 mg). Poly(sodium methacrylate, methyl methacrylate) (NaPMM), obtained by alkali neutralization of a pharmaceutical-grade approved poly(methyl methacrylate), was proposed to design drug delivery systems intended for buccal administration [1-3]. NaPMM showed promising properties to design ODF exhibiting very fast dissolution rate, low swelling profile and good palatability. This work aims to evaluate its possible use to design ODF prepared by casting technique, using PEG400 as plasticizer. The impact of the polymer/plasticizer ratio and the residual water expressed as loss on drying (LOD) on film properties was investigated on placebo ODF. Moreover, the drug loading capacity was assessed using ketoprofen and paracetamol, as model drugs. Main results Placebo ODF containing PEG400 in the 10-30 % w/w range and 10-15 % LOD were homogeneously transparent in appearance, easy-to-handle and disintegrated within 30s. Figure 1 exemplifies the possible tensile patterns of placebo ODF. At low strain a linear region, associated to the reversible deformation, was evident. Increasing the strain, the behaviour shifted from elastic to plastic, the curve lost linearity and the deformation became irreversible, until the maximum force was reached. Then, three profiles were identified increasing the PEG concentrations and/or LOD values (Figure 1). First, the maximum force value was followed by a significant reduction of the stress, indicating local reduction of the cross section, namely “necking”, that propagated along the length of the sample until rupture (code N). Secondly, the film can show a tear behaviour characterized by a slow and smooth decrease of cross section upon increasing the tensile stress (code T). Finally, the elongation at break at values higher than 200% indicates formulations characterized by a ductility unsuitable for packaging (code D). NaPMM plasticized by 20% PEG400 allowed designing ODF containing 125 mg paracetamol on a surface of about 6 cm2, maintaining acceptable tensile and biopharmaceutical properties. In case of ketoprofen (pKa=4.45), both 25% or 50% loaded ODF swelled without disintegrating and the disaggregation medium pH shifted from 7.6 to 4.4. ATR-FTIR spectroscopy evidenced that this feature was attributed to a partial protonation of NaPMM (Figure 2) since the intensity of anti-symmetrical (1560 cm-1) and symmetrical vibrations (1300-1400 cm–1) of the carboxylate groups underwent a significant depression in presence of ketoprofen (red line) with respect to the placebo film (blue line). However, the presence of 5% surfactants (e.g., Tween 80 and Span 80) allowed 25% ketoprofen loaded ODF to disintegrate within 30 s without compromising the film mechanical properties. Future perspectives The overall data underline the versatility of NaPMM to produce ODF, even if further studies have to be performed to evaluate the in vivo performances.

References: 1. Cilurzo, F.; Minghetti, P.; Selmin, F.; Casiraghi, A.; Montanari L. Polymethacrylate salts as new low-swellable mucoadhesive materials, J Controll Rel, 2003, 88, 43-53. 2. Cilurzo, F.; Selmin, F.; Minghetti, P.; Rimoldi, I.; Demartin, F; Montanari, L. Fast-dissolving mucoadhesive microparticulate delivery system containing piroxicam. Eur J Pharm Sci, 2005, 24, 355-361. 3. Cilurzo, F.; Selmin, F.; Minghetti, P.; Gennari, C.G.M.; Demartin, F., Montanarti, L. Characterization and physical stability of fast-dissolving microparticles containing nifedipine. Eur J Pharm Sci, 2008, 68, 579-588.

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Challenges in development of enzyme loading nanoparticles Pederzoli Francesca*(1), Belletti Daniela (1), Tomanin Rosella (2), Duskey Jason (1), Forni Flavio (1), Vandelli Maria Angela (1),

Ruozi Barbara (1), Tosi Giovanni (1) (1) University of Modena and Reggio Emilia, Department of Life Science, Modena, Italy.

(2) Istituto di ricerca pediatrico "Città della Speranza", Padova, Italy. Background: Advances in biotechnology resulted in the rise of numerous therapeutic proteins and peptides. Unfortunately, clinical applications of these agents are hampered by numerous obstacles to their successful delivery. Intrinsic physico-chemical and biological properties, including large molecular size, poor permeation through gastrointestinal membrane, poor stability at low pH values of gastric fluid or due to presence of proteolytic enzymes lead oral delivery of proteins and peptides to be highly challenging [1]. For these reasons, a carrier or delivery system for therapeutic proteins and peptides would be ideal [2]. Drug delivery systems can be advantageous to improve and ameliorate disease therapy by: (1) enhancing protein/peptide solubility; (2) controlled release of protein/peptide molecules; (3) sustained release instead of burst release effect, avoiding undesirable side-effects; (4) improved biodistribution of protein/peptides and (5) target the diseased tissue in vivo [3]. Here we present our results on the development of a modified double emulsion method specifically designed to deliver therapeutic enzymes. Main Results: We initially encapsulated a model enzyme (beta-glucosidase, B-glu) in polymeric NPs by investigating the impact of the formulative process on the activity of the enzyme. The results showed that some conditions (e.g. sonication and contact with organic solvent) represent the most stressful factors leading to the lost of enzyme activity after the encapsulation process. Aiming to better preserve the stability of the loaded enzyme, modification on the double emulsion method were applied. In particular, the use of stabilizer molecules (e.g. proteins, sugar, surfactannt) during formulation procedure was evaluated. All the formulations were purified and characterized in terms of shape, morphology, size, surface charge, architecture/internal structure. Enzyme loading was evaluated after extraction and analytical quantification and its stability was monitored by enzymatic activity assay. Among the stabilizer molecules added during the formulation procedure, the bovine serum albumin (BSA) showed to be the best in preserving enzyme activity. The use of different amounts of BSA in the enzymatic solution during NP-formulation process did not impair NP features in terms of size and Z potential. However, the use ofsolutions with high concentration of BSA (20% w/v) lead to a lower recovery at the end of the formulation process (tab.1). The best encapsulation efficiency was reached when NPs were formulated with 5% and 10% BSA. The release kinetic experiments revealed that the enzyme released by BSA-NPs was significantly more active than the enzyme released by Bglu-NPs without BSA (fig.1). On these bases, we therefore planned to re-formulate new brain-targeted NPs (g7-NP) loaded with two recombinant enzymes used for the therapy of two different lysosomal storage disease (iduronate 2-sulfatase and galactosylceramidas). Preliminary in vitro results have shown a partial improvement in terms of encapsulation efficiency and enzymatic activity of both these enzymes. Future Perspectives: We aim to improve the in vivo behaviour of enzyme-loaded NPs, with respect to an efficient transport of the enzyme to the CNS and to the mantainence of the enzymatic activity in the brain tissue of pathological mouse model.

References: 1. Patel, A.; Cholkar, K.; Mitra, A.K. Recent developments in protein and peptide parenteral delivery approaches. Ther. Deliv., 2014, 5, 337–365. 2. Salvalaio, M; Rigon, L.; Belletti, D.; D’Avanzo, F.; Pederzoli, F.; Ruozi, B.; Marin, O.; Vandelli, M.A.; Forni, F.; Scarpa, M.; Tomanin, R.; Tosi, G. Targeted Polymeric Nanoparticles for Brain Delivery of High Molecular Weight Molecules in Lysosomal Storage Disorders. PLoS ONE, 2016, 11, e0156452. 3. Tan, M.L.; Choong, P.F.M.; Dass, C.R. Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery. Peptides, 2010, 31, 184–193.

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Structure Analysis of Drug Delivery Systems with Small Angle X A. Pichler, M. Medebach, C. Invigorito, S. Lanteri, R.Pellegrino

Anton Paar GmbH, Straße 20, 8054 Graz, Austria - Phone nr. +43 - 316 - 257 - 2885 Background Small-Angle X-ray Scattering (SAXS) draws increasing attention in the field of pharmaceutical engineering. SAXS is a versatile technique used for shape and size characterization of nanostructured materials between 1 nm and 200 nm. Biological samples, like proteins or viruses are already well known to be investigated with SAXS. Other interesting examples of applications in pharmaceutical research are drug delivery systems such as drug loaded vesicles, where size and shape parameters of the vesicle and the drug are found. Furthermore it is possible to measure the viscosity of liquids with very little sample volume and obtain the internal surface of granulate powders, which correlates with the tablet hardness and therefore with the dissolution properties of the tablet. Main Results In this contribution we present select applications of biological samples, employing multifunctional laboratory Small and Wide Angle X-ray Scattering (SWAXS) systems, the SAXSpoint and the SAXSpace. Both instruments enable SAXS and WAXS studies at ambient and non-ambient conditions, GI-SAXS, in-situ tensile SWAXS experiments and satisfy both beginner and advanced users with a wide range of dedicated sample stages, full experimental flexibility and highest resolution. The system provides simple operation, short measurement times and excellent angular resolution. This is achieved by a smart beam formation concept which includes a brilliant X-ray source, advanced X-ray optics and optimized scatterless collimation while maintaining a laboratory-friendly compact system size and small footprint. Future Perspectives Different scattering studies on biological and pharmaceutically relevant samples were performed on Anton Paar SAXS systems. Some of the samples required high resolution, i.e. a very low minimum scattering angle in order to resolve large structural dimensions. The unique sample-positioning mechanism enables WAXS measurements to determine crystallinity without re-aligning any part of the SWAXS system. The presented studies clearly show that high-resolution and high-quality SWAXS data can be obtained with laboratory SWAXS systems.

34ISBN:9788894140422

Oral treatment of phenylketonuria: microparticulate-based formulation Pereira de Sousa Irene, Leroux Jean-Christophe

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland, +41 44 633 04 97

Background: Phenylketonuria (PKU) is a hereditary disorder affecting the metabolism of phenylalanine (Phe) due to a deficiency in the enzyme phenylalanine hydroxylase. It is characterized by elevated blood Phe concentration, which can lead to severe intellectual disability in newborns. The current strategy to prevent this devastating consequence is limited to a strict, life-long, Phe-restricted diet, which implies major lifestyle disturbances. To this end, two strategies for PKU treatment have been developed: Kuvan, a synthetic enzyme cofactor only active on milder forms of PKU, and Pegvaliase, an enzyme replacement therapy intended for subcutaneous administration showing significant drawbacks (e.g. development of antibodies against the enzyme and polymers of the formulation) [1]. The aim of this project is to design an efficient Phe-metabolizing system suitable for oral delivery that can bypass the issues arising with injectable enzyme replacement formulations. Main Results: Mesoporous silica particles (MSPs) were synthesized following a procedure previously described, using a Poloxamer P-123 - mesitylene mixture as template and tetraethylorthosilicate as silica precursor [2]. MSPs with a hydrodynamic volume of 16 ± 3 µm and a zeta potential of -15 ± 3 mV were obtained. To enlarge the MSP's pore diameter a hydrothermal treatment at 170 °C for 5 h in teflon lined autoclaved was performed and in the obtained particles phenylalanine ammonia-lyase (PAL, 310 kDa) was encapsulated by impregnation method. The MSP's pore diameter could be enlarged to 34 nm (MSP-L) starting from 13 nm (MSP-S) resulting in a greater enzyme loading of about 5% and 2%, respectively. PAL encapsulation in MSP-L could be further improved by raising the PAL:MSP mass ratio; in fact, 1:10, 2:10, 4:10 ratios led to a loading of 5%, 16% and 38%, respectively. Upon encapsulation, PAL enzymatic activity was maintained. Finally, the particles were coated with chitosan in order to shield the payload from intestinal protease. The formation of the coating was visually assessed by using rhodamine-conjugated chitosan, which did not result in a significant increase in particle size Future Perspectives: MSPs with a size in the micrometer range and large pore diameter were designed. PAL was efficiently encapsulated in the formulated particles and its activity was preserved. Therefore, a Phe-metabolizing system could be obtained. Further investigations will be focused on identifing the optimal coating conditions to allow the permeation of Phe into the carrier and at the same time to hinder the permeation of intestinal digestive enzymes. Acknowledgements: This work was supported by ETH Zurich Postdoctoral Fellowship and Marie Curie Actions for People COFUND program.

References: 1. Blau, N; Longo, N. Alternative therapies to address the unmet medical needs of patients with phenylketonuria. Exp. Opin. Pharmacother, 2015, 16, 791-800 2. Lei, C; Soares, TA; Shin, Y; Liu, J; Ackerman, EJ. Enzyme specific activity in functionalized nanoporous supports. Nanotechnology, 2008, 16(12), 125102

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Water soluble Chitosan-MCD derivatives: NPs vs. macromolecular complex Piras Anna Maria*, Fabiano Angela*, Chiellini Federica^, Zambito Ylenia*

*Dep. of Pharmacy, University of Pisa, Via Bonanno 33, 56126 Pisa, Italy,+39 0502219704 ^Dep. of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, 56124 Pisa, Italy

Background. In order to enhance drug adsorption across mucosal tissues (i.e. GI tract, eye, lung, etc.), different formulation strategies are commonly investigated, such as using mucoadhesive polymers, cyclodextrin inclusion complexes, or mucoadhesive polymeric nanocarriers. In a recent report[1], the solubilization ability of metyl-β-cyclodextrin (MCD) and the mucoadhesive properties of water soluble quaternary ammonium chitosan (QA-Ch) were merged into a QA-Ch-MCD derivative obtained by covalently grafting through a 10-atom long spacer. QA-Ch-MCD formed stable inclusion complexes with the model drug dexamethasone (DEX), showing improved properties with respect to the physical blend of polymer and MCD/DEX complex. From the above considerations, the actual advantages/disadvantages of the soluble macromolecular carrier QA-Ch-MCD were investigated in comparison to the relevant supramolecular nanosized carrier, obtained by ionotropic gelation of the same drug complex. Main Results. The QA-Ch-MCD derivative was prepared starting from a depolymerized low molecular weight chitosan further functionalized with 2-diethylaminoethyl chloride. The obtained derivative was characterized by ATR-FTIR and 1HNMR , resulting in 24.4%wt of grafted MCD. The phase solubility profile of the macromolecular complex QA-Ch-MCD/DEX was determined. Nanoparticles (NPs) suspensions were obtained by ionotropic gelation of the QA-Ch-MCD/DEX complex in presence of sodium tripolyphosphate, leading to 8.7% ±0.5 of loading efficiency. Both NPs and QA-Ch-MCD/DEX complex were characterized in terms of size and Zeta potential, showing a diameter of 299±32nm (P.I. 0.049) and 2.7±0.4nm (P.I. 0.048), and Zeta potential of 11.5±1.1 mV and 6.7±0.6 mV, respectively. FITC labeled QA-Ch-MCD/DEX solutions and relevant FITC labelled NPs suspensions were submitted to in vitro studies of water-assisted transport through mucus, evaluation of DEX permeation through excised rat intestine, and ex-vivo mucoadhesivity studies. The macromolecular complex showed greater mucoadhesion and lower transport ratio through mucus, with respect to the relevant NPs. Concerning drug permeation through excised rat intestine, the macromolecular complex provided a faster and greater drug permeation, correlating also with increasing amounts of permeated FITC-labelled carrier. Future Perspectives. The outcome of the performed studies suggests a correlation between the physical properties of the investigated carriers and their role in promoting the permeation of DEX through the intestinal mucosal tissue. In this specific case, the use of NPs seems not to provide any advantage over the use of the simpler macromolecular complex. Additionally, NPs are generally preferred for the formulation of labile drugs; meanwhile, cyclodextrins are known to slow down the degradation rate of labile drugs and stabilize macromolacular drugs such as small proteins and peptides. Future investigation will then comprise the application of the developed carriers to drugs liable to degradation. References: 1. Piras, A.M.; Zambito, Y. et.al. A water-soluble, mucoadhesivequaternary ammonium chitosan-methyl-β-cyclodextrin conjugate forming stable inclusion complexes with dexamethasone. Journal of Material Science: Materials in Medicine, 2017. Submitted .

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Neurofuzzy-logic based methodology as a new approach for the manufacturing of oil-loaded core-shell microcapsules by prilling technique

Rodríguez-Dorado Rosalía1,2, Landin Mariana3, Aquino Rita Patrizia1, Del Gaudio Pasquale1

1Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Italy 2PhD Program in Drug Discovery and Development, University of Salerno, Italy

3Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Spain r.rodriguezdorado@studenti.unisa.it

Background: Microencapsulation of hydrophobic compounds into a hydrophilic matrix is a useful approach to protect and delivery bioactive substances subjected to degradation from environmental factors. In fact, different techniques and specific polymeric excipients allow the encapsulation of active pharmaceutical ingredients (APIs) with poor bioavailability, undesired taste or volatile while enabling the controlled release of the active ingredient. The main challenge to enhance biopharmaceutical properties of the API is the selection of a proper matrix or reservoir system. Literature data have demonstrated the possibility to encapsulate oils in Ca-alginate microcapsules by an inverse gelation process but both homogeneous particle size and high oil content were difficult to achieve [1, 2]. As an alternative, in the present work a new inverse gelation approach carried out by prilling technique [3], using artificial intelligence systems to optimize the variables involved in the process, has been developed to obtain core-shell microcapsules loaded with sunflower oil as model carrier of hydrophobic active ingredients. Results: Inverse gelation process was achieved directly at the co-axial nozzle of the prilling apparatus using calcium chloride/sunflower oil emulsion (W/O) and an alginate solution that were pumped through the inner and annular nozzle, respectively. A database of 43 formulations was generated and modeled by the commercial neurofuzzy logic software FormRules® v4.03 with the variables set for the preliminary experiments used for producing the batches: the percentage of alginate, the concentration of CaCl2 in the emulsion, the amounts of surfactants (Tween™ 85 and Span® 85) as well as the flow rates and frequency of vibration. Those variables were introduced as inputs and three categorized characteristics of the produced particles (shape, oil content and oil distribution) were used as outputs. The optimized parameters obtained after FormRules modeling proved the efficiency of the neurofuzzy logic model. In fact, spherical double-layered beads with a narrow size distribution (1.1 mm) were obtained without breaking during the prilling process when the optimized variables were used. Furthermore, fluorescent microscopy assays pointed out the homogeneous oil distribution into the core of the particles and the presence of a thin alginate shell (95 µm). Future perspectives: The purpose for future studies will be the manufacturing of the optimized core-shell particles by prilling technique loading an hydrophobic API into the oily phase to subsequently submit the microcapsules to a drying process through supercritical fluid technology in order to eliminate completely the oily phase, thus allowing the encapsulation of the API by the alginate layer and obtaining a novel aerogel formulation loaded with an hydrophobic drug. References [1] Martins E.; Renard D.; Davy J.; Marquis M.; Poncelet D. Oil core microcapsules by inverse gelation technique. Journal of Microencapsulation, 2015, 32(1), 86-95. [2] Abang S.; Chang E. S.; Poncelet D. Effects of process variables on the encapsulation of oil in Ca-alginate capsules using an inverse gelation technique. Journal of Microencapsulation, 2012, 29(5), 417-428. [3] Del Gaudio P.; Auriemma G.; Russo P.; Mencherini T.; Campiglia P.; Stigliani M.; Aquino R. P. Novel co-axial prilling technique for the development of core-shell particles as delayed drug delivery systems. European Journal of Pharmaceutics and Biopharmaceutics, 2014, 87, 541-547.

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Glutathione Responsive Nanosponges for Controlled Ailanthone Delivery Tannous Maria1-3, Caldera Fabrizio1, Argenziano Monica2, Dianzani Chiara2, Trotta Francesco1, Bassil Bassem2, Cavalli Roberta2

1Department of Chemistry, University of Turin, via P. Giuria 7, 10125 Turin, IT 2Department of Drug Science and Technology, University of Turin, via P. Giuria 9, 10125 Turin, IT

Background: Cyclodextrins (CDs) are naturally occurring encapsulating agents that have highly specific interactions with many therapeutic agents. The benefits of cyclodextrins have been studied extensively, and approved to be used as drug delivery systems by regulatory authorities. Cyclodextrin-based nanosponges present a further development in this field. Many drugs with different structures, solubility and pharmacological activities have been incorporated in nanosponges and good results were obtained in their delivery. Generally due to their characteristics such as low water solubility and bioavailability, the delivery of anti-cancer drugs represent several challenges. To overcome these limitations, cyclodextrin based formulations were designed to help improve wetting, solubility, stabilization, protection, taste masking of drugs. Stimuli-responsive cyclodextrin nanosponges offer an additional advancement in anticancer drug delivery due to their ability to release a loaded drug in response to several stimuli as light, temperature, redox and pH variations. Glutathione (GSH) a principal cellular antioxidant found in chemoresistant tumor cells. GSH-responsive nanosponges (GSH-NSs) have been designed as a promising tool for targeted intracellular drug release. Ailanthone a natural compound, extracted from Ailanthus Altissima, displaying antimalarial and antitumor activities and exhibiting no inhibitory effects human cytochromes and no significant hepatotoxicity was chosen as a potential anticancer drug model. The aim of this work was the development of a beta-cyclodextrin based glutathione responsive nanosponges for the delivery of ailanthone. Main results: GSH-NSs were obtained by a one-step synthesis, by reacting cyclodextrin, cross-linking agent and 2-hydroxyethyl disulfide, to get disulfide bridges in the polymer matrix. Top-down methods were used to obtain nanoscale GSH-NSs of about 200 nm. They had negative surface charge and were able to load ailanthone with an encapsulation efficiency of about 98%. In vitro release kinetic studies showed a prolonged release profile over time and the amount of drug released from NS was proportional to the GSH concentration in the receiving medium. Biological assays demonstrated that ailanthone-loaded NS display a higher anti-proliferative effect than free drug, in cell lines PANC-1 pancreatic carcinoma and B-16 melanoma. Future perspective: Glutathion responsive nanosponges might be considered a promising nanocarrier for intracellular triggered release of anticancer drugs. Additionally, the use of natural compounds, such as ailanthone, with therapeutic activity could be further exploited due to their confirmed biological functions in their natural context and possible biocompatibility. References: 1. Trotta, F., Caldera, F., Dianzani, C., Argenziano, M., Barrera, G., & Cavalli, R. Glutathione Bioresponsive Cyclodextrin Nanosponges. ChemPlusChem, 2016, 81(5), 439-443. 2. Daga, M., Ullio, C., Argenziano, M., Dianzani, C., Cavalli, R., Trotta, F. et al. GSH-targeted nanosponges increase doxorubicin-induced toxicity “in vitro” and “in vivo” in cancer cells with high antioxidant defenses. Free Radical Biology and Medicine, 2016, 97, 24-37. 3. Peng, S., Yi, Z., & Liu, M. Ailanthone: a new potential drug for castration-resistant prostate cancer. Chinese journal of cancer, 2017, 36(1), 25.

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Hyaluronic acid-click-riboflavin nanogels as drug delivery carriers Zoratto Nicole, Manzi Giuliana, Sabia Rocchina, Villani Claudio, Coviello Tommasina, Matricardi Pietro, Di Meo Chiara

Department of Drug Chemistry and Technologies, ‘‘Sapienza’’ University of Rome P.le Aldo Moro 5, 00185, Rome, Italy, chiara.dimeo@uniroma1.it

Background: In the recent years, nanohydrogels (NHs) have gained considerable attention thanks to their unique characteristics, that combine the favourable properties of hydrogels such as high water content, hydrophilicity and soft consistency with those of nanoparticles, including nano-size dimensions, long circulation time in the blood stream, easy penetration into tissues and the chance to passively/actively target the desired site [1,2]. Among NHs, those composed of natural polymers typically show the advantage to be non-toxic, biocompatible, biodegradable and have a low immunogenicity [2]. Hyaluronic acid (HA), thanks to its ability to interact with several cell-receptors, plays an important role in the cell proliferation, migration and differentiation (e.g. CD44 is the main targeted HA receptor over-expressed on the surface of several cancer cells and activated inflammatory cells). Therefore, HA has been already employed for the formation of NHs through its covalent linkage with hydrophobic moieties [2, 3]. In this context, click-chemistry is emerging as an attractive technique to chemically modify polymers with a wide range of different molecules in a simple way with high selectivity and specificity and by using mild reaction conditions. For this purpose, new “click” HA-Riboflavin derivatives (HA-c-Rfv) were synthetized by "click" Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction. The resulting amphiphilic polymers spontaneously self-assemble in aqueous media, forming NHs that can be used to deliver both hydrophobic and hydrophilic drugs. Main results: HA was first functionalized with propargyl moieties in order to provide the polymer chains with alkyne groups, making HA tuneable for click-chemistry reaction. Then, the azido-hexyl derivative of riboflavin-tetrabutyrate (Rfv) was synthetized and covalently coupled to the HA-alkyne derivative. HA of different molecular weights (Mw) and degrees of substitution (DS) have been studied and finally, HA (Mw of 220 kDa) with a propargyl feed of 40 mol/mol % and a Rfv feed of 40 mol/mol % (HA-c -Rfv 40/40) derivative was selected as the most interesting one thanks to its ability to form NHs in the size-range of 150-200 nm with a negative ζ-potential (-50 mV). In particular, NHs were formed by using the autoclaving process that exhibits the advantage to spontaneously form sterile NHs in one-step. Click-NHs suspension showed high stability in water at 4°C (over two months), and for 48 hours in glycerol/simulated intestinal fluids media (pH 7.4) at 37°C. Piroxicam, dexamethasone and paclitaxel were chosen as model hydrophobic drugs for the encapsulation into NHs by film-hydration process. The drug-loading tests showed that the apparent water solubility of the model drugs increase of 12, 3 and 25-fold respectively, confirming the ability of NHs to act as solubility enhancers. Moreover, the drug-loaded NHs were stable in water suspension for two months and, by adding dextrose as cryoprotectant, it was also possible to re-constitute the NHs formulation after freeze-drying and subsequent re-hydration. Finally, a HA-c-Rfv derivative bearing an excess of propargylic portions (HA-c-Rfv 60/40 mol/mol%) was further derivatized with PEG-N3 chains by azide/alkyne “click” reaction thanks to the presence of the free propargyl moieties that were still available for the “click” coupling reaction. In this way, PEGylated NHs were obtained, with the aim to improve the blood circulation of these nanocarriers, thus opening new avenues for a wide range of functionalization strategies. Future Perspectives: Click chemistry reactions emerged as an important synthetic strategy to easily functionalize the HA polymer chains through fast and quantitative reactions with good yields of recovery. Moreover, HA-c-Rfv click-NHs showed the ability to entrap a wide range of hydrophobic drugs, offering the chance to be effectively employed as a drug delivery system. In this regard, future studies will be focused on the investigation of the drug release profile from these nano-carriers and on the evaluation of their invitro and in vivo biocompatibility. References: [1] Dalwadi, C.; Patel, G. Application of Nanohydrogels in Drug Delivery Systems: Recent Patents Review. Recent Patents on Nanotechnology, 2015, 9, 17-25. [2] Di Meo, C.; Montanari, E.; Manzi, L.; Villani, C.; Coviello, T.; Matricardi, P. Higly versatile nanohydrogel platform based on riboflavin-polysaccharide derivatives useful in the development of intrinsecally fluorescent and cytocompatible drug carriers. Carbohydrate Polymers, 2015, 115, 502-509. [3] Montanari, E.; De Ruggeris, M.C.; Di Meo, C.; Coviello, T.; Alhaique, F.; Matricardi, P. One-step formation and sterilization of gellan and hyaluronan nanohydrogels using autoclave. Journal of Material Science: Materials in Medicine, 2015, 26-32.