BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine9 (2013) 1203–1213

Research Article

Thermo- and pH-sensitive dendrosomes asbi-phase drug delivery systems

Mohsen Adeli, PhDa,b,⁎, Ali Kakanejadi Fard, PhDa, Fatemeh Abedi, Msa,Beheshteh Khodadadi Chegeni, Msa, Farhad Bani, Msc

aDepartment of Chemistry, Faculty of Science, Lorestan University, Khoramabad, IranbDepartment of Chemistry, Sharif University of Technology, Tehran, Iran

cInstitute of Biochemistry and Biophysics University of Tehran, Tehran, Iran

Received 13 January 2013; accepted 28 May 2013

nanomedjournal.com

Abstract

Fully supramolecular dendrosomes (FSD) as bi-phase drug delivery systems are reported in this work. For preparation of FSD,amphiphilic linear-dendritic supramolecular systems (ALDSS) have been synthesized by host-guest interactions between hyperbranchedpolyglycerol having β-cyclodextrin core and bi-chain polycaprolactone (BPCL) with a fluorescine focal point. Self-assembly of ALDSS inaqueous solutions led to FSD. They were able to encapsulate paclitaxel with a high loading capacity. The dendrosome-based drug deliverysystems were highly sensitive to pH and temperature. They were stable at 20–37 °C and pH7–8, but dissociated and released drug attemperatures lower than 20 °C or higher than 37 °C and pH lower than 7 quickly. Dissociation of FSD building blocks by temperature or pHresulted in inclusion complexes between the released drugs and polyglycerol as the secondary drug delivery system.

From the Clinical Editor: This paper reports on the development of a pH- (below 7) and temperature- (below 20 °C or above 37 °C)sensitive delivery system using supramolecular dendrosomes for more specific delivery and release of drugs using paclitaxel as a model.© 2013 Elsevier Inc. All rights reserved.

Key words: Biphase-drug delivery systems; Dendrosomes; Controlled release; Linear-dendritic copolymers; Supramolecules

Dendrosomes are a type of supramolecular systems that arecreated by molecular self-assembly of amphiphilic blockcopolymers in aqueous solutions. In these systems, at least oneblock of amphiphilic copolymers is a dendritic polymer. Theyhave spherical topology with hollow structure consisting of ahydrophilic core which is surrounded by a bi-layer membrane.These supramolecular systems are able to encapsulate anddeliver hydrophobic and hydrophilic therapeutic agents andsmall guest molecules. Due to the hydrophilicity of the core of

Abbreviations: FSD, Fully supramolecular dendrosomes; ALDSS,Amphiphilic linear-dendritic supramolecular systems; PTX, Paclitaxel; β-CD,beta-cyclodextrin; β-CD-g-PG, hyperbranched polyglycerol with acyclodextrin core; PG, Polyglycerol; IR, Infrared; NMR, Nuclear magneticresonance; DLS, Dynamic light scattering; UV, Ultraviolet; TEM, Trans-mission electron microscopy; AFM, Atomic force microscopy; GPC, Gelpermeation chromatography; Mn, Molecular weight; BPCL, lipid-like bi-chain polycaprolactone; CAC, critical aggregation concentration; DLC, Drugloading content; DLE, drug loading efficiency.

This work is supported by National Nanotechnology Initiative financially.⁎Corresponding author.E-mail addresses: adeli@sharif.edu, mohadeli@yahoo.com (M. Adeli).

1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2013.05.013

Please cite this article as: Adeli M, et al, Thermo- and pH-sensitive de2013;9:1203-1213, http://dx.doi.org/10.1016/j.nano.2013.05.013

dendrosomes and hydrophobicity of their membrane, hydrophil-ic and hydrophobic guest molecules encapsulate inside their coreand membrane respectively.1–12

Recently, stimuli-responsive dendrosomes have been pre-pared and their application as drug delivery systems has beenalso investigated. These systems are able to release therapeuticagents in the target sites by using a stimuli factor such astemperature, pH, glucose, light or enzyme.13–17

Unique properties for dendrosomes are the result ofsupramolecular interactions between their building blocks,amphiphilic copolymers. Amphiphilic linear-dendritic copoly-mers in which hydrophobic and hydrophilic blocks are attachedby supramolecular interactions should lead to fully supramolec-ular dendrosomes with new properties. One of the easiest ways toprepare amphiphilic linear-dendritic supramolecular copolymers,is to design dendritic copolymers which are able to forminclusion complexes with linear chains or polymers in aqueoussolutions.2,3

Recently hyperbranched polyglycerol with a cyclodextrincore (β-CD-g-PG) has been synthesized by our researchgroup.18 Incorporation of cyclodextrin in the core of PG confers

ndrosomes as bi-phase drug delivery systems. Nanomedicine: NBM

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host-guest property in its structure so that it is able to encapsulatehydrophobic guest molecules in aqueous solutions.19–21 Host-guest interactions between β-CD-g-PG and a guest moleculewith a hydrophobic tail should lead to an amphiphilic linear-dendritic supramolecular copolymer which is able to formdendrosomes in aqueous solutions.11

Herein we report amphiphilic linear-dendritic supramolecularsystems (ALDSS) consisting a bi-chain polycaprolactone andhyperbranched PG as linear and hyperbranched blocks respec-tively. Linear and hyperbrached blocks were attached togetherby host-guest interactions. Self-assembly of ALDSS in aqueoussolutions led to fully supramolecular dendrosomes (FSD) with ahigh capacity to encapsulate and transfer paclitaxel. FSD weresensitive to the best regions of pH and temperature. Controlledrelease of encapsulated drugs is possible through stimulating ofdendrosomes by pH or temperature factors. The released drugswere again encapsulated by β-CD-g-PG, which was the productof the destruction of dendrosomes, and a secondary drug deliverysystem with a smaller size was produced.

Methods

Materials and instruments

Fluorescein, ε-caprolactone, sodium methoxide, methanol,chloroform, paclitaxel (PTX), 1-pyrene carboxaldehyde and ionexchange IV (weakly acidic cation exchange) were purchasedfrom Merck. β-cyclodextrin and glycidol were purchased fromSigma Aldrich. Dialysis bag (Mn cutoff 2000) was providedfrom Sigma-Aldrich (St Louis, Missouri). Infrared (IR) spectrawere obtained using a Nicolet 320 FT-IR. 1H NMR spectra wererecorded in CDCl3 on a Bruker DRX 400 (400 MHz).Ultraviolet (UV) spectra were recorded on a Shimadzu (1650PC) scanning spectrophotometer. Ultrasonic bath (Model: 5RS,22 KHZ, Made in Italy) was used to disperse materials insolvents. Fluorescence experiments were performed with a RF-5301 PC spectrofluorometer with a thermostate cell unit. Theparticle size and zeta potential of materials were determinedusing dynamic light scattering (DLS) (zetasizer ZS, MalvernInstruments). Molecular weights were determined using KnauerGel permeation chromatography (GPC) equipped with SmartinePump 1000 with a PL aquagel-OH mixed-H 8-μm columnconnected to a differential refractometer, with water as themobile phase at 25 °C. Surface imaging studies were performedusing atomic force microscopy (AFM) in ambient condition andon silicon surface. The Transmission Electron Microscopy(TEM) images were obtained using a LEO 912AB electronmicroscope with accelerating voltage of 200 kV.

Preparation of β-CD-g-PG

β-CD-g-PG was synthesized via an anionic ring openingmultibranching polymerization according to reported procedurein literature.18 Briefly β-CD (0.5 g, 0.5 mmol) was added tomethanol (3 ml suspension of sodium methoxide (0.78 g,14.4 mmol)) and mixture was added to a polymerization ampuleequipped with a magnetic stirrer and vacuum inlet and stirred for1 h at room temperature for partial deprotonation of β-CD. Then

methanol was vaporized using vacuum oven for 2 h at 60 °C.Glycidol (6.17 ml, 92 mmol) was added to the deprotonated β-CD gradually at 100 °C for 2 h and mixture was stirred at120 °C for 12 h. Then it was cooled and dissolved in methanoland precipitated into acetone and neutralized by filtration overcation-exchange resin. Product as a yellow and viscouscompound was dried using vacuum oven at 80 °C for 6 h. Theyield of reaction was 87%.

IR (cm-1): O-H (3380), C-O-C (1116), C-H (2921). 1H NMR(ppm): O-H (protons of cyclodextrin and polyglycerol) (4.3-3.6),anomeric proton of cyclodextrin (5.2). GPC(g/mol): (3638).DLS(nm): (2.8).

Preparation of bi-chain polycaprolactone (BPCL)

Polymerization of ε-caprolactone by fluorescein sodium saltas an initiator was led to bi-chain polycaprolactone (BPCL) withfluorescein focal point. In a typical reaction, fluorescein (0.5 g,1.32 mmol) was added to a polymerization ampule equippedwith a magnetic stirrer and vacuum inlet. Then toluene solutionof Sn(Oct)2 (1.0 × 10−3 M) (1 ml) was added to polymerizationampule and mixture was stirred at 50 °C for 30 min. Solvent wasevaporated by vacuum oven at 60 °C for 30 min. ε-caprolactone(2.82 ml, 26 mmol) was added to polymerization ampule andmixture was stirred under vacuum for 1 h at 60 °C. Thepolymerization ampule was sealed and heated at 120 °C for 12 hwhile contents were stirred vigorously. Finally, it was cooledand the contents of ampule were dissolved in chloroform.The solution was filtered and the product was precipitated inn-hexane as a white solid compound. The yield of reactionwas 90%.

IR (cm-1): O-H (3429), C-H (2947–2864), C = O (1728), C =C (1590). 1H NMR (ppm): aromatic protons (7.4-6.9), O-H (4.9),C-H (OCH2) (4.06), C-H (OHCH2) (3.64), C-H (CH2 COOAr)(2.64), C-H (CH2 COO) (2.31), C-H (CH2CH2COO) (2.029), C-H(CH2CH2O) (1.64), C-H (CH2CH2O) (1.26). DLS(nm): (16.5).

Preparation of ALDSS

Host-guest interactions between fluorescein focal point ofBPCL and cyclodextrin incorporated in the core of PG were ledto ALDSS. In a typical reaction, BPCL (0.068 g, 0.03 mmol)was dissolved in dichloromethane (200 μL) and the resultedsolution was added to 5 ml phosphate-buffered saline (PBS).Then dichloromethane was evaporated by vacuum distillation.Solutions of β-CD-g-PG (2.3 × 10−2 M in PBS) were added toBPCL dispersed in PBS, and then mixture was sonicated for 2 hat room temperature.

Preparation of dendrosomes

The critical aggregation concentration (CAC) of ALDSS inorder to produce dendrosomes was determined by fluorescencespectra and using of 1-pyrene carboxaldehyde as a hydrophobicprobe.22–24 Typically, PBS solutions of ALDSS, preparedaccording to explained method, were stirred at 35 °C for 3 hand then cooled to room temperature gradually. Then fluores-cence spectrum of each solution was recorded in the presence ofprobe. Concentration of BPCL in PBS solutions of ALDSS wasvaried from 5 × 10−6 to 1 × 10−2 mol/L while the concentrationof 1-pyrene carboxaldehyde was fixed to be 3 × 10−6 mol/L.

Fig. 1. (I) Preparation of a) β-CD-g-PG and b) BPCL by anionic and cationic ring opening polymerizations respectively. (II) IR spectra of a) β-cyclodextrin andb) β-CD-g-PG. (III) 1H NMR spectra of β-CD-g-PG in the D2O. (IV) GPC diagram shows 3638 (Mn) molecular weight for β-CD-g-PG. (V) IR spectra of a)FLO, b) ε-caprolactone and c) BPCL.

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Preparation of PTX-Loaded dendrosomes

The membrane of dendrosoms can encapsulate hydrophobicdrugs such as PTX. Typically, PTX (0.06 g, 0.07 mmol) wasdissolved in dichloromethane and added to PBS solutions ofALDSS (2.5 × 10−3 M), then dichloromethane was evaporatedby vacuum distillation. Resulted mixture was sonicated at 37 °Cfor 3 h and cooled to room temperature gradually, then dialysisagainst PBS for 1 h.

For determination of drug loading content, the PTX-loadeddendrosomes were dissolved in methanol. Then UV–vis spectraof solutions were recorded at 200–800 nm and concentration ofPTX was determined using calibration curve.25

Drug loading content (DLC) and drug loading efficiency(DLE) were calculated according to the following formulas:

DLC ¼ weight of loaded drug=weight of polymerð Þ � 100DLE ¼ weight of loaded drug=weight of drug in feedð Þ � 100

Determination of the size and shape of dendrosomes

Size distribution of ALDSS and dendrosomes was measuredby dynamic light scattering (DLS). In these experimentsconcentration of ALDSS was 1.6 × 10−3 mol/L and water wasused as solvent.

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Morphology of ALDSS and dendrosomes was determined bytransmission electron microscopy (TEM) and atomic forcemicroscopy (AFM). For TEM experiments an aqueous solutionof ALDSS with 1.6 × 10−3 mol/L concentration was used. Also,for AFM experiments an aqueous solution of ALDSS with1.8 × 10−3 mol/L concentration was prepared and dropped onholder and then solvent was evaporated.

Examination of the pH-sensitivity of dendrosomes

In order to study the stability of ALDSS in the differentpHs, PBS solutions of ALDSS (1.3 × 10−3 mol/L) with pH7.4,6.5, 6 and 5.5 were prepared and UV–vis spectra of thesesolutions was recorded at ambient conditions. Intensity of theUV–vis absorbance of solutions was assigned to the stabilityof dendrosomes.

Examination of the temperature-sensitivity of dendrosomes

In order to study the effect of temperature on the stability ofALDSS, a PBS solution of ALDSS (1 × 10−3 mol/L) withpH7.4 was prepared and its UV–vis spectra was recorded at 25–40 °C range. Increased or diminished intensity of UV–visabsorbance was assigned to the stability and instability ofdendrosomes respectively.

Controlled release of encapsulated PTX by dendrosomes

The in vitro release behaviour of PTX-loaded dendrosomes wasinvestigated at 37 °C. Briefly 0.5 mL of the PTX-loaded dendro-some solutions (in PBS with pH7.4 or 5.5) was transferred into adialysis bag (Spectrapor, MW cutoff 2000 gmol/1). The dialysisbag was immersed into 30 mL of BPS with pH7.4 or pH5.5 at37 °C. At selected time intervals, the amount of released PTX wasdetermined by UV–vis spectrometer using calibration curve.

Results

In order to prepare dendrosomes amphiphilic linear-dendriticsupramolecular systems (ALDSS) were synthesized. For prep-aration of ALDSS, polyglycerol with a cavity in its central pointand bichain polycaprolactone with a aromatic ring in its focalpoint were synthesized.

β-CD-g-PG was synthesized via anionic ring openingpolymerization according to reported method in literature.18

Fig. 1, Ia shows synthesis of β-CD-g-PG schematically. Brieflyβ-cyclodextrin (β-CD) was mixed with sodium methoxide inmethanol and it was deprotonated partially. Then solvent wasevaporated and glycidol was added to deprotonated β-CD invacuum gradually. Mixture was stirred at 120 °C for 12 h.Product was dissolved in methanol and precipitated in acetone.

Fig. 2. (I) 1H NMR spectra of BPCL in the CDCl3. (II) Host-guest interactions bof the intensity of UV–vis absorbance of the fluorescein focal point of BPCL uponCD-g-PG. BPCL was dispersed in BPS with pH7.4 and fixed in 0.0005 g (3.8(2.7 × 10−2 M) and was added in 5 ml volumes to BPCL solution. (IV) Selcarboxaldehyde or b) paclitaxel leads to dendrosomes with encapsulated guest mocaboxaldehyde was increased abruptly upon addition of aqueous solution of ALDin PBS pH7.4 was determined through plotting of the ratio of the intensity of pyrenthe logarithm of concentration.

β-CD-g-PG was obtained as a yellow and viscose compoundin 90% yield. IR spectra of β-CD and β-CD-g-PG are shown inFig. 1, II. In the IR spectrum of β-CD-g-PG, three absorbancebands corresponded to the O-H, C-O-C and C-H groups areexhibited at 3380, 1116 and 2921 cm-1, respectively.

A broadening in the C-O absorbance band of β-CD-g-PG incomparison with that for β-CD is due to the attachment ofpolyglycerol to the functional groups of cyclodextrin.

In the 1H NMR spectrum of β-CD-g-PG (Fig. 1, III), signalsof protons of cyclodextrin and polyglycerol are overlapped at4.3-3.6 ppm. In this spectrum a weak signal at 5.2 ppm isassigned to the anomeric protons of cyclodextrin.

According to GPC experiments, the molecular weight (Mn)of β-CD-g-PG was estimated 3638. Monomodality of GPCdiagram showed that compound is pure and free fromcontaminants (Fig. 1, IV).

BPCL was synthesized via cationic ring opening polymeri-zation of ε-caprolactone (Fig. 1, Ib). The chemical structure ofBPCL was evaluated by the IR and 1H NMR spectra which areshown in Figs. 1V and 2I. In Fig. 1, IV, IR spectra of fluorescein,ε-caprolactone and BPCL are compared. In the IR spectrum offluorescein, two absorbance bands corresponded to the O-H andC = C bonds are exhibited at 3383 and 1635 cm-1 respectively.In the IR spectrum of ε-caprolactone, absorbance bandcorresponded to the C = O functional group is appeared at1735 cm-1. Appearance of the absorbance bands of the endhydroxyl, aliphatic C-H and carbonyl groups of polycaprolac-tone chains at 3429, 2947–2864, and 1728 cm-1 respectivelytogether with C = C bonds of fluorescein and 1590 cm-1 provessuccessful synthesizing of BPCL (Fig. 1, IVc).

Presence of the signals of aromatic rings of fluorescein togetherwith signals of polycaprolactone chains in the 1H NMR spectra ofBPCL confirm preparation of BPCL (Fig. 2, I). The ratio of peakarea of aromatic protons (7.4-6.9 ppm) to the endmethylene groupsof polycaprolactone chain (3.7 ppm) in the 1H NMR spectra ofBPCL, showed that number of polycaprolactone chains connectedto fluorescein focal point is two. Also molecular weight of BPCLwas estimated 2610 through the peak area ratio ofmethylene groupsof polycaprolactone backbone (−CH2OCO-,4.2 ppm) to that for itsend methylene groups (−CH2OH, 3.7 ppm).

Host-guest interactions between flourescine focal point ofBPCL and cyclodextrin core of β-CD-g-PG was led to ALDSS(Fig. 2, II). ALDSS are supramolecules, because hydrophobiclinear and hydrophilic dendritic blocks are attached together bynoncovalent interactions.

Formation of ALDSS could be understood visually, becauseBPCL was dissolved in PBS upon addition the β-CD-g-PGgradually. However host-guest interactions between BPCLand β-CD-g-PG leading to ALDSS were investigated byspectroscopy methods. As it can be seen in Fig. 2, III, BPCL

etween focal point of BPCL and β-CD-g-PG lead to ALDSS. (III) Increasingaddition of β-CD-g-PG, proves host-guest interactions between BPCL and β-× 10−5 M) concentration. β-CD-g-PG was dissolved in BPS with pH7.4

f-assembly of ALDSS in aqueous solutions in the presence of a) pyrenelecules between hydrophobic chains. (V) a) Fluorescence intensity of pyreneSS, where formation of dendrosomes was occurred. b) CAC for dendrosomese carboxaldehyde fluorescence at 402 and 383 wavelengths (I402/I383) versus

Fig. 3. (I) Fluorescence spectra of a) BPCL and b) dendrosomes in PBS pH7.4 and room temperature (25 °C). (II) DLS diagrams for β-CD-g-PG, BPCL, ALDSS and ADL (concentration has been fixed at1.4 × 10−3 mol/L and 7 × 10−4 mol/L respectively). DLS experiments have been performed at PBS pH7.4 and at room temperature (25 °C). (III) a) Contrast and b) topographic AFM images of FSD on siliconsurface. (IV) Intensity of the absorbance of FSD was decreased by changing pH from 7.4 to 5.5. Inset shows photograph of dendrosomes when pH is 7.4 and 5.5. Concentration of ALDSS in examined solutions was1.5 × 10−3 mol/L. (V) Intensity of the UV–vis absorbance of dendrosomes in PBS pH7.4 was decreased by going out of the 20–37 °C temperature range, proving the dissociation of building blocks ofdendrosomes and precipitation of BPCL. Inset shows photograph of dendrosomes solutions in PBS pH7.4 at 25 and 40 °C. Concentration of ALDSS in examined solutions was 1.1 × 10−3 mol/L.

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shows a λmax in UV–vis spectra (spectrum b), due to thepresence of flourescine in its structure. Upon addition the β-CD-g-PG to a constant concentration of BPCL intensity of its λmax

was increased (spectra c to j), proving the host-guest interactionsbetween these two segments and preparation of the ALDSS.

Since linear blocks of ALDSS are hydrophobic, the secondtype of noncovalent interactions in aqueous solutions ishydrophobic-hydrophobic interactions between these blocks.Due to lipid-like structure of ALDSS, it is expected that theycreate liposome-like nanobjects (dendrosomes) in aqueoussolutions upon passing their critical aggregation concentration(CAC). Pyrene carboxaldehyde was chose as a probe to determinethe CAC of ALDSS by fluorescence spectra (Fig. 2, IVa).

CAC of ALDSS was determined 1.2 × 10−3 mol/L in PBS.On the other words, below this concentration ALDSSs were notaggregated and they were in their individual forms and there wasjust the first type of noncovalent interactions (host-guestinteractions) between BPCL and β-CD-g-PG, while higherthan this concentration second type of noncovalent interactionsbetween BPCL chains were led to dendrosomes (Fig. 2, V).

Fig. 4. (I) TEM images of FSD a) without and b) with encapsulated PTX.(II) In vitro release profiles for encapsulated PTX from FSD at 37 °C and inPBS a) pH7.4 and b) pH5.5 at 37 °C.

Discussion

The main goal of incorporation of fluorescein in the BPCLstructure was, not only preparation of a lipid-like structure but alsoconferring fluorescence property in the whole structure and finallyin dendrosomes. Since dendrosomes are designed as stimuli-responsive anticancer drug delivery systems, this property could beused to follow them in the body. Fig. 3, I shows that both BPCL anddendrosomes are able to emit fluorescence light upon excitation.

Dynamic light scattering (DLS) was used to evaluateformation of ALDSS and their self-assembly to create dendro-somes. While diameter of β-CD-g-PG in aqueous solutions wasbelow 5 nm, due to its dense structure,[18] and also diameter ofBPCL dispersed in PBS was 16.5 nm, addition of β-CD-g-PG toBPCL in PBS led to aggregates with 300 nm diameter.11 Thisdiameter is much higher than that for ALDSS, which would bethe sum of diameter of BPCL and β-CD-g-PG or somethingaround. Therefore it can be found that higher than CAC the self-assembly of ALDSS leads to dendrosomes with 300 nmdiameter (Fig. 3, II). Interestingly with decreasing of theconcentration of ALDSS, size or diameter of objects decreasedramatically. This result proves that formation of dendrosomes ishighly depend on the concentration of building blocks and theyare fully supramolecular systems (Fig. 3, II (ALD)).

Fig. 3, III shows AFM images for dendrosomes formed inaqueous solutions. These images and many other recorded AFMimages prove the formation of dendrosomes with a cavity inside.According to these images the average size of dendrosomes isaround 400 nm and average thickness of their wall is 50 nm. Sincethe calculated length for a ALDSS is around 23 nm, the thicknessof the shell of dendrosomes, which is twice, should be 46 nm.However 4 nm difference between the calculated and observedthickness is the result of the flexible structure of dendrosomes ornoncovalent interactions between their building blocks probably.

It has been proved that host-guest interactions between BPCLand β-CD-g-PG and therefore the created ALDSS are highly

sensitve to pH. Interestingly BPCL was completely dissociatedfrom β-CD-g-PG and ALDSS were broken down to theirbuilding blocks in pH5.5 (Fig. 3, IV (Inset)). Fig. 3, IV showsUV–vis spectra of dendrosomes with concentration fixed at1.5 × 10−3 mol/L in PBS. As pH was changed from 7.4 to 5.5intensity of the UV–vis absorbance of BPCL decreased,confirming that denrosomes are destroyded by dissociation ofALDSS. Since pH in tumor tissues is 5.8–7.6, dendrosomesconsisiting of ALDSS are promising systems in order to releaseanticancer drugs into these tissues selectively.26

ALDSS and therefore dendrosomes were higly sensitive totemperature. They were stable at 20–37 °C temperature rangeand below than 20 °C or higher than 37 °C they were diassiciateto their building blocks (Fig. 3, V (Inset)).

Fortunately, the upper transition temperature of dendrosomesfalls exactly at normal body temperature and therefore increasingthe temperature of the target region in body (tumor) by a stimuli

Fig. 5. (I)Hydrolysing of the focal point of BPCL, due to the sensitivity of esteric bonds between fluorescein and polycaprolactone chains to pH. (II) Increasingof the intensity of UV–vis absorbance of the PTX, upon addition of β-CD-g-PG proves host-guest interactions between PTX and β-CD-g-PG.

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factor leads to collapsed dendrosomes and finally release theencapsulated drugs.

Due to the unique properties of the prepared dendrosomes,especially their sensitivity to pH and temperature, they weresubjected to encapsulate and controlled release of paclitaxel(PTX) in vitro. PTX is one of the most potent anticancer drugs inthe treatment of different types of solid malignant tumors.However poor physical stability of this drug in the aqueousenvironments decreases its clinical applications. Therefore,preparation of PTX delivery systems with high stability isgreat of interest.27

Dendrosomes were able to encapsulate PTX with a highloading capacity (Fig. 2, IVb). Drug loading content (DLC) anddrug loading efficiency (DLE) of dendrosomes was evaluatedusing UV–vis spectroscopy at 200–800 nm. According to theseexperiments DLC and DLE of dendrosomes for PTX was 63%and 86% respectively.

Size and morphology of dendrosomes without and withencapsulated PTX was investigated by TEM. Based on TEMimages, size of dendrosomes was 400 nm and the thickness oftheir shell was 50 nm (Fig. 4, Ia). Although inside, cavity, ofdendrosomes should be hydrophilic medium, TEM imagesshowed that this cavity occupied by PTX molecules fully (Fig. 4,Ib). Since dendrosomes are formed by supramolecular in-

teractions, replacing of the encapsulated water molecules intheir cavity by PTX leads to the increased entropy of the wholesystem and diminish unfavourable interactions between watermolecules and PTX.28

The effect of the stimuli factors, pH and temperature, on therate of release of PTX from PTX-loaded dendrosomes wasinvestigated in vitro.29–31

Fig. 4, II shows the profile of release of PTX fromdendrosomes at pH7.4 and 5.5 at 37 °C. It is obvious that therate of release of PTX from dendrosomes at pH5.5 is much fasterthan that at pH7.4, indicating a pH-dependent drug releaseprofile. At pH7.4, the rate of release of PTX is slow and less than20% of the encapsulated PTX is released after 40 h. However themost interesting aspect of the release profile of PTX at pH5.5 isthe fast diminishing of the accumulative release percent after arapid release. On the other words, FSD show a three-step releasefor PTX in low pH. In the first step, PTX release fromdendrosomes up to 80% during 2 h. In the second step a 40%diminish in the released drug is observed and the third step showa constant concentration for the released drug, 40%.

The first step, fast release of PTX in acidic environment,could be attributed to the instability of ALDSS and thereforeFSD in this medium. Dissociation of the host from the guest istriggered by hydrolysing of the focal point of BPCL, due to the

Fig. 6. (I) Effect of pH on the stability of host-guest interactions between PTX and β-CD-g-PG. It can be seen that the UV–vis absorbance of PTX/β-CD-g-PGsystem diminish around 3 and 5% in pH5.5 and 7.4 respectively. This result shows that PTX and β-CD-g-PG system PTX and β-CD-g-PG is stable in thesemediums. (II) It can be seen that how FSD can be used as two-stage drug delivery systems for targeting anticancer drugs to tumors. They are stable in a mediumwith pH7.4 (for example blood vessel) and destruct with decreasing the pH (for example tumor site) and secondary drug delivery system produced in tumors. Thesecondary drug delivery system will be up taken by cells in the next step. (III) In vitro release profiles for encapsulated PTX from FSD in PBS pH7.4 and at a)37 °C, b) 38 °C and c) 39 °C. (IV)Supramolecular hydrogel at I) 25 °C and II) 40 °C. Increasing of the concentration ofALDSS in PBS, pH7.4, to 1.9 × 10−2 mol/Lleads to supramolecular hydrogel the transition of which temperature to a liquid state is 40 °C.

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sensitivity of the esteric bonds between fluorescein andpolycaprolactone chains to pH (Fig. 5, I). Irreversibility of thehost-guest interactions with increasing of the pH to 7.4 is areason for this suggestion.

However, it is well-known that PTX is not soluble in aqueoussolutions and therefore it should not get out from the dialysis bagsolely. After dissociation of dendrosomes and releasing of theencapsulated PTX molecules, a secondary drug delivery system

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comprising PTX as guest and β-CD-g-PG as host (PTX/β-CD-g-PG) is created18 and the host-guest system transfer from thedialysis bag and actually this system is considered as the releaseddrug in the release profile. Recording the UV–vis spectra of thereleased PTX, λmax of β-CD-g-PG was also observed whichconfirm the host-guest interactions between these two segmentsand transferring PTX from dialysis bag as an inclusion complex.

In order to prove this idea, host-guest interactions betweenPTX molecules and β-CD-g-PG were investigated and it wasfound that PTX molecules form inclusion complexes with β-CD-g-PG efficiently. As it can be seen in Fig. 5, II, the intensity ofthe UV–vis absorption of a fixed concentration of PTX in PBSincreases upon increasing the concentration of β-CD-g-PG. Thisexperiment proves that there are host-guest interactions betweenPTX molecules and β-CD-g-PG in aqueous solutions.

Two explanations could be proposes for the second step of therelease profile. An explanation is instability of the PTX/β-CD-g-PG host-guest system at low pH and precipitation or decompo-sition of PTX.

In order to evaluate this suggestion, PTX/β-CD-g-PG host-guest system was prepared and its stability at pH7.4 and 5.5 wasinvestigated. It was found that this system is almost stable at bothpHs (Fig. 6, I). Therefore this suggestion is rejected. The secondexplanation is based on osmotic pressure. Since osmotic pressureis a colligative property, it depends on the number of particlesand not their sizes. When FSD are dissociated, the osmoticpressure of the solution inside dialysis bag increases suddenly,leading to transferring the PTX/β-CD-g-PG systems to theoutside solution. Osmotic pressure of the outside solutionincreases with transferring of the hot-guest systems form thedialysis bag. When all dendrosomes dissociated, some PTX/β-CD-g-PG systems transfer from the outside to the inside solutionto induce a balance between the osmotic pressure of thesesolutions (Fig. 5, II).

This bi-phase drug delivery system with a pH-dependent andthree-step release profile is of particular interest to achieve thetumor-targeted PTX delivery. Therefore the prepared FSD mayrepresent a highly promising approach to release PTX insidetumors quickly, increasing the osmotic pressure of the tumor andtherefore forcing the uptake of the released PTX by the cellsusing secondary drug delivery system (Fig. 6, I).

FSD were also subjected to release of the encapsulated PTXat different temperatures to see whether this factor is effective forcontrolled release of encapsulated PTX or not.32

Fig. 6, III shows the release profile of PTX fromdendrosomes at 37 °C, 38 °C and 39 °C. Clearly the rate ofrelease of the encapsulated PTX from dendrosomes at normalbody temperature is much lower than that at higher tempera-tures. It can be found that even a degree of increasedtemperature leads to a much faster release of PTX from FSD.Targeting of the PTX-loaded dendrosomes to tumor sites andthen one or two degree of temperature shocking would lead torelease of PTX in that site selectively. A diminish in the releaseddrug after several hours could be assigned to the samemechanism explained for pH sensitivity and formation of thesecondary drug delivery system.

It is worthwhile to mention that self-assembly of ALDSS isalso sensitive to concentration. With increasing of the concen-

tration of ALDSS in aqueous solutions to 1.9 × 10−2 mol/L,network-like self-assemblies or a supramolecular hydrogels wereformed (Fig. 6, IV(I)). Since all interactions between segmentswere noncovalent and they have been assembled by several typesof supramolecular interactions, hydrogels were thermoreversible.The transition temperature for hydrogel from solid to aqueoussolution was 40 °C (Fig. 6, IV(II)).

In this work we successfully developed a novel type of drugdelivery systems based on fully supramolecular dendrosomes.Upon dissociation of the FSD by stimuli factors such astemperature or pH, a secondary drug delivery system based oninclusion complexes were formed. In these systems the releasedanticancer drugs are never free and therefore they will createminimum side effects and optimum therapy.29

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