International Journal of Pharmaceutics 495 (2015) 276–289

Lipid-polyethylene glycol based nano-ocular formulation ofketoconazole

Shilpa Kakkara, Sankunny Mohan Karuppayilb, Jayant S. Rauta, Fabrizio Giansantic,Laura Papuccid, Nicola Schiavonee, Indu Pal Kaura,*aUniversity Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study, Panjab University, Chandigarh 160014, Indiab School of Life Sciences, SRTM University, Nanded, 431606 Maharashtra, IndiacDepartment of Translational Medicine and Surgery, Eye Clinic, University of Florence, Viale Morgagni 85, 50134 Florence, ItalydDepartment of Experimental and Clinical Biomedical Sciences, Section of Experimental Pathology and Oncology, University of Florence, Viale Morgagni, 50,50134 Florence, ItalyeDepartment of Experimental and Clinical Biomedical Sciences, Section of Experimental Pathology and Oncology, University of Florence, Viale Morgagni, 50,50134 Florence, Italy

A R T I C L E I N F O

Article history:Received 15 June 2015Received in revised form 24 August 2015Accepted 26 August 2015Available online xxx

Keywords:Drug deliveryFungal eye infectionSolid lipid nanoparticlesPosterior eyePermeationOcular safety

A B S T R A C T

Ophthalmic mycoses including corneal keratitis or endophthalmitis affects 6-million persons/year andcan cause blindness. Its management requires antifungals to penetrate the ocular tissue. Oral use ofKetoconazole (KTZ), the first broad-spectrum antifungal to be marketed, is now restricted to life-threatening infections due to severe adverse effects and drug-interactions. Local use of KTZ loadednanocarrier system can address its toxicity, poor solubility, photodegradation, permeation andbioavailability issues.Solid lipid nanoparticles (SLNs) comprising Compritol1 888 ATO and PEG 600 matrix, were presently

prepared using hot high-pressure homogenization. Employing extensive characterization: TEM, NMR,DSC, XRD and FTIR, it is proposed that SLNs comprise of a polyethylene glycol (PEG) core into which KTZ isdissolved. PEG endows the lipid matrix with amorphousness and imperfections; rigidity; and, stability toaggregation, on storage and autoclaving. PEG is a simple, cost-effective and safe polymer with superiorsolubilizing and surfactant-supporting properties. Without its inclusion KTZ could not be loaded intoSLNs. It ensured high incorporation efficiency (70%) of KTZ; small size (126 nm); and, better permeationinto the eye. Pharmacokinetic studies indicated 2.5 and 1.6 fold higher bioavailability (AUC) in aqueousand vitreous humor, respectively. Biocompatibility and in vitro (both in corneal and retinal cell lines) andin vivo (in rabbits) ocular safety is the other highlight of developed formulation.

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1. Introduction

Local delivery to the eye, though a preferred route with lowerincidence of systemic side effects, cannot address internal eyediseases (Sigurdsson et al., 2007; Bucolo et al., 2012; Geroski andEdelhauser, 2000; Ahmed and Patton, 1985; Hughes et al., 2005).Nanostructured systems may however deliver drug successfully tothe posterior segment of the eye (Kaur and Kakkar, 2014). Solidlipid nanoparticles (SLNs), comprising of a nanosized lipidic corestabilized by a layer of surfactants is now attaining popularity as asuitable system for ocular delivery (Leonardi et al., 2014; Seyfoddinet al., 2010). Both the nano size, and the lipidic nature of SLNs helps

* Corresponding author.E-mail addresses: indupalkaur@yahoo.com, indukuips@gmail.com (I.P. Kaur).

http://dx.doi.org/10.1016/j.ijpharm.2015.08.0880378-5173/ã 2015 Elsevier B.V. All rights reserved.

to improve ocular bioavailability of encapsulated drug; both due toprolonged ocular retention and improved permeation (Hippal-gaonkar et al., 2013; Mohanty et al., 2015; Seyfoddin et al., 2010).

Fungal infections of the eye, though less common thaninfections with bacteria and viruses, are usually more severeand may lead to loss of vision. With an increased immunocom-promised population, including HIV infected persons, patientsundergoing surgeries/transplants, and those receiving chemother-apy, the ophthalmic mycosis is howsoever on the rise. Efficientadministration of appropriate antifungal therapy can help topreserve vision, provided the agent reaches the affected tissue insufficient concentration (Kaur et al., 2008b).

Ketoconazole (KTZ) is a broad spectrum antifungal agent, withhigh lipo-solubility (log P = 4.74) (Logua et al., 1997) but a shortocular half life (elimination half life is 19 min in aqueous humorand 43 min in cornea) (Zhang et al., 2008) and very poor solubility

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(0.04 mg/ml). It is recommended to be administered orally at adose of 100 to 400 mg every 12 h (Müller et al., 2013). However, it isassociated with low oral bioavailability (Baxter et al., 1986) andsevere adverse effects like nausea, vomiting, gastrointestinaldisturbance, hepatitis, gynecomastia, adrenal cortex suppression(O’Brien, 1999) and hepatotoxicity (FDA, 2013; Sharma et al., 1993).Its absorption from the gut is dependent on the gastric pH.Significant drug–drug interactions are also reported upon its oraladministration (Thomas, 2003).

High lipophilicity of KTZ may promote permeation, however itslarge molecular weight (531.44 Da), tends to impede its transportacross the biological membranes. Similarly, high lipid solubilityalthough can help passage across corneal epithelium, however, itspassage through the aqueous corneal stroma will be compromised(Barar et al., 2008) following topical administration. Furthermore,its limiting water solubility (0.04 mg/ml) makes it difficult topresent KTZ in a solubilised form on the corneal surface, latterbeing an important pre-requisite for ocular formulations.

Presently SLN ocular dispersion of KTZ was designed to providean alternate topical route rather than an oral route of administra-tion, which is associated with significant adverse effects. Furtherintent of the study is to develop a nanocarrier system which canpermeate upto the posterior eye in an intact form so as to carry theincorporated drug along with it. Fungal infection of the internal eyeinvariably leads to vision loss within a short span and achievinghigh drug concentration in the vitreous is difficult post oraladministration due to the existence of blood-aqueous and blood-retinal barriers. Developed KTZ-SLN system was extensivelycharacterized and confirmed to permeate the cornea via ex vivocorneal permeation studies. In vivo pharmacokinetic profile ofKTZ-SLN was compared with the corresponding free drugdispersion. In vitro and in vivo safety and autoclavability of thedeveloped KTZ-SLN system was confirmed. The dispersion wascomprehensively developed as a suitable ocular formulation interms of pH, refractive index, osmolarity, stability on keeping (bothentrapment efficiency and particle size were evaluated upto 1 year)and preservation against contamination (using challenge test).Though a well established preservative benzalkonium chloride(BAK) was employed, but it is important to confirm that physicaladsorption or interaction with any of the components does notcompromise the activity of BAK.

2. Methods

2.1. Materials and selection of components

Ketoconazole was a kind gift from Torrent Pharmaceuticals Pvt.Ltd., H.P., India; Compritol1 888 ATO was a gift sample fromGattefosse, France; and Phospholipon 90 G (soya lecithin) wasgifted by Lipoid, Germany. Phosphotungstic acid (PTA) and BAKwere procured from Sigma–Aldrich, USA. All other reagents used inthe study were of analytical grade.

Compritol1 888 ATO was presently chosen as the lipidcomponent, because it is reported to result in stable SLNformulations with small particle size (Kaur et al., 2008a; Kakkaret al., 2011; Bhandari and Kaur, 2013); is safe for ocular use (Gokceet al., 2009); and, due to varying chain lengths of its componentsforms imperfect crystals, which can accommodate more amount ofdrugs (Muller et al., 2000).

We have employed Tween 80 as a surfactant and a simplepolymer like PEG (Israelachvili, 1997) to achieve small size, highloading and significant entrapment. Both these agents arepermeation enhancers (Kaur and Smitha, 2002; Jiao, 2008; Youshiaet al., 2012) and P-gp efflux inhibitors (Jiao, 2008; Zhang et al.,2003; Werle, 2008). PEG, although a polymer, is known to supportsurfactant and co-surfactant properties of other surface active

agents and decrease gelation of SLNs (Mehnert and Mäder, 2001;Yu et al., 2004).

The co-surfactant Phospholipon 90G is a phospholipid, which isreported to produce spaces in the lipid helping achieve higherentrapment efficiency (Attama et al., 2008). Moreover, itsantioxidant nature (Judde et al., 2003) will protect KTZ againstphotodegradation (Souto and Muller, 2005).

2.2. Preparation of solid lipid nanoparticles (KTZ-SLNs)

Although KTZ is a lipophilic molecule, it was not soluble inCompritol1 888 ATO. To improve drug loading and entrapmentefficiency it is important that the drug is incorporated into SLNs ina soluble form (Mehnert and Mäder, 2001) and for that purpose anoptimized concentration of surfactants may be employed (Soutoet al., 2006). We tried a series of surfactants and cosolvents viz.transcutol P, propylene glycol, PEG 300 and PEG 600 (details shownelsewhere). Latter was chosen, as the most suitable solvent as KTZwas significantly soluble (3.2 mg/ml) in it. It may be highlightedhere that it was not possible to solubilise and entrap KTZ withinSLNs without the use of PEG 600 (Table 1 of Supplementary data).SLN dispersions in the absence of PEG were unstable and tended tothrow out the drug. The finalized formulation based on entrap-ment efficiency and stability was prepared as described below.

The non-aqueous phase comprising molten Compritol1 888 ATO(2.5%), KTZ (0.25%) and PEG 600 (4.2%) was heated to 75 �C withcontinuous stirring and added all at once to the hot aqueous phase.Latter comprised of 5.8% Tween 80 and 0.4% Phospholipon 90 G inwater, heated to the same temperature (F17, Table 1; Supplementarydata). The mixture was stirred at 8000 rpm for 8 min using a highspeed stirrer (WiseTis homogenizer) to result in a pre-emulsion.Latter was passed through a high pressure homogenizer (Emulsi-Flex-C3, Canada), applying pressure of 1000 bar for three cycles. Theformed o/w emulsion was allowed to cool to room temperature toresult in SLN dispersion (IPA 2074/Del/2014).

Note: The mentioned percentage values represent concentra-tion of these components in the final SLN dispersion and notindividual phases.

2.3. Preparation of KTZ suspension (KTZ-SUS)

KTZ (0.25% w/v) was added to water containing 0.5% (w/v)Tween 80 as a dispersing agent and stirred for 3 h. This suspensionwas used as a control for KTZ-SLNs.

2.4. Characterization of SLNs

The developed SLNs were characterized in terms of transmis-sion electron microscopy (TEM), particle size, total drug content(TDC), entrapment efficiency (EE), zeta potential, pH, osmolarity,refractive index (RI), fourier transform infrared spectrometry(FTIR), differential scanning calorimetry (DSC), X-ray diffraction(XRD) and nuclear magnetic resonance (NMR) spectroscopy(details given in Table 2 of Supplementary data).

2.5. Ex vivo corneal permeability studies

Ex vivo corneal permeability studies were performed using themembrane diffusion technique at 37 � 0.2 �C, under mixing con-ditions using a magnetic stirrer, as described previously (Aggarwalet al., 2004). The preparation (0.2 ml) to be studied was placed on thecornea. Freshly prepared glutathione bicarbonate ringer (GBR)containing 2% sodium lauryl sulphate (SLS) was used as the diffusionmedium (Li et al., 2008). SLS was used in the diffusion medium(20 ml) to solubilizeKTZ and maintain sink conditions. Aliquots werewithdrawn at regular intervals from the sampling port and an equal

278 S. Kakkar et al. / International Journal of Pharmaceutics 495 (2015) 276–289

quantity of fresh media was replaced to maintain a constant volume.Samples were analyzed spectrophotometrically at 291 nm aftersuitable dilutions. The method was validated for linearity (25–200 mg/ml), accuracy and precision before analytical estimations(details not included). The apparent corneal permeability coefficient(Papp) of both KTZ-SLN and KTZ-SUS were determined, as reportedpreviously (Aggarwal et al., 2004).

An isolated cornea obtained as above, was fixed with a 8% w/vformalin solution, dehydrated with an alcohol gradient andmounted into paraffin blocks. Thin cross sections (<5 mm) ofthe corneal tissue were cut, stained with Schiff’s base (Thiel et al.,2002) and observed microscopically (Nikon eclipse 80i, Japan), tocheck for integrity of the corneal layers.

2.6. Stability studies

2.6.1. Under refrigerated conditionsStudies were carried out to investigate the leaching of drug from

SLNs storedin tightlyclosed,ambercolored,screwcapped vials, at 2–8 �C for 12 months. Samples were withdrawn periodically andanalyzed for total drug content and the entrapment efficiency.

2.6.2. PhotostabilityPhotostability of the samples was also confirmed by exposing

KTZ-SLN and corresponding free KTZ-SUS to an illumination of1.2 million lux hours and an integrated near ultraviolet energy of200 W h2 for 10 days at 25 �C in a photostability chamber (Binder,Germany), in clear glass containers as per ICH guidelines.

2.6.3. Stability upon sterilizationKTZ-SLNs were sterilized by autoclaving at 121 �C (15 psi

pressure) for 20 min. Any change in terms of size and % entrapment(leakiness) was noted post sterilization.

2.7. Antifungal activity

Sabouraud agar medium was seeded with a suitably dilutedinoculum of Candida albicans so as to result in approximately100CFU/petriplate (i.e. 30 ml medium). The media was poured intosterile petri plates (30 ml/plate) and allowed to solidify. Four wells(1 cm2) were bored into each plate using a stainless steel cylinder.Various concentrations (in a specified volume of 100 ml) of KTZ-SLN or KTZ-SUS were placed in wells against the control (blankSLNs and 0.5% Tween 80 in water, respectively). The plates wereincubated at 25 �C for 48 h and diameter of the zone of inhibition

Fig. 1. TEM micrographs of KTZ-SLNs at (a) �15,000

around each well was measured using a scale. Statistical analysisusing ANOVA at level of significance of p < 0.05, was conducted onthe results.

2.8. Antibiofilm efficacy

Activity of the developed formulation against planktonic andbiofilm growth of C. albicans (ATCC 90028) was studied usingstandardized protocols. Minimum inhibitory concentrations forplanktonic growth were established using guidelines of ClinicalLaboratory Standards Institute (CLSI, 2002). Efficacy of KTZ-SLNs,KTZ-SUS and blank SLNs (without drug) against developing andmature biofilms was evaluated using an in vitro C. albicans biofilmmodel as described earlier (Shinde et al., 2013). Inhibition of thebiofilm growth was analysed using standard XTT-metabolic assayand the results were confirmed with scanning electron microscopy(SEM) (Shinde et al., 2013).

2.9. Challenge test

The test consisted of challenging the SLN preparation (withoutKTZ) with a prescribed inoculum of suitable microorganisms,storing the inoculated preparation at a prescribed temperature,withdrawing samples from the container at specified intervals, andcounting the organisms in the samples (BP, 1999). The preservativeproperties of the preparation are adequate if in the condition oftest, there is a predefined fall in the number of microorganisms inthe inoculated preparation at the specific times and prescribedtemperatures. The preservative used in the preparation was 0.02 %w/v benzalkoniun chloride (BAK).

2.10. In vivo safety studies

Safety assessment for ocular application was approved by theInstitutional Animals Ethics Committee, Panjab University, Chan-digarh, India (letter no. IAEC 158-169 dated 10-2-2012) andperformed as per the OECD guidelines (details described in theSupplementary section).

2.11. Cytotoxicity studies

The viability of ARPE-19 (a human retinal pigment epithelialcell line) and RCE (rabbit corneal epithelial cell lines) cells wasdetermined by cell proliferation assay using WST-1 reagent (Roche,Milano, Italy), as per the details given in Supplementary section.

, (b) �200,000, and (c) �400,000 magnification.

Table 1Characterization of KTZ-SLNs.

TDC (%) (n = 6) Entrapment efficiency (%) (n = 6) Particle size* (nm) (n = 4) Zeta potential pH Osmolarity (mOsms/l) RI

92.36 � 1.91 70.19 � 1.94 126.35 � 2.2 �3.19 7.03 � 0.15 110 1.34

Values are mean � standard deviation.* PDI = 0.28 � 0.02.

S. Kakkar et al. / International Journal of Pharmaceutics 495 (2015) 276–289 279

2.12. Ocular tolerance evaluation

To examine the effects on ocular structure and integrity, the lefteyeball was removed from rat, 10 min., 0.5 h and 2 h post ocularadministration of SLNs (without BAK, in order to avoid inherenttoxicity related to it) using corresponding right eye of the sameanimal as control. The eye balls were washed with saline, fixedwith a formalin solution 8% (w/w), dehydrated with an alcoholgradient, put in melted paraffin and solidified in block form. Cross-sections (<5 mm) were cut, stained with haematoxyline and eosine(H and E) and observed microscopically (Nikon eclipse 90i, Japan)for any pathological modifications (Baydoun et al., 2004).

2.13. Ocular pharmacokinetic studies

Bioavailability of the developed formulation (KTZ-SLN) in theaqueous and vitreous humor of rabbits was compared with freedrug suspension, at 0.5, 2, 6, 12 and 24 h after topical administra-tion. The presence of nanoparticles in the aqueous and vitreoushumor was verified by observing the withdrawn samples underTEM (details included in the Supplementary section).

Fig. 2. FTIR spectra of drug, KTZ-SLNs, Compritol1 888 ATO, physical mixture, PEG 600,

2.14. Statistical analysis

All results are expressed as the mean � standard deviation. Theresults were analyzed for statistical significance by a one-wayanalysis of variance (ANOVA) test followed by the Tukey’s test. Theraw data obtained from ex vivo studies were analyzed by applyingthe correction factor for volume and drug losses during sampling.

3. Results

3.1. Characterization of SLNs

Transmission electron microscopy indicated SLNs to be small insize (between 70 and 135 nm) and spherical in shape, with noaggregation/irregularities in the system (Fig. 1). Particle size, totaldrug content, EE, zeta potential, pH, osmolarity and RI of thedeveloped KTZ-SLNs is included in Table 1.

The PDI of <0.3 (presently 0.28 � 0.02) indicates narrowparticle size distribution (Madheswaran et al., 2013). No micron-ized particles were observed in the entire population and majority(90%) of the particles were �282.2 nm in size. Highest observedsize was 538.9 nm (results shown in Supplementary data; Fig. 1).

Compritol1 888 ATO along with PEG 600, blank SLNs, phospholipid and Tween 80.

Fig. 3. Thermograms of (A) ketoconazole (KTZ), (B) ketoconazole loaded solid lipid nanoparticles (KTZ-SLNs), (C) Compritol1 888 ATO.

280 S. Kakkar et al. / International Journal of Pharmaceutics 495 (2015) 276–289

The developed formulation was found to be safe for ocular usein terms of pH (7.03 � 0.15), osmolarity (110 mOsms/l) and RI (closeto that of water at 1.33).

The FTIR spectra for KTZ, KTZ-SLNs,Compritol1888 ATO, physicalmixture, molten Compritol1888 ATO along with PEG 600 are shownin Fig. 2. Pure KTZ showed intense absorption bands at 1648 cm�1

(C¼O stretch), 1514 cm�1 (C¼C aromatic stretch), 1249 cm�1 (C��Ostretching of cyclic ether), 1037 cm�1 (C��O stretching of aliphaticether), and 817 cm�1 (C��Cl stretch), respectively (Karolewicz et al.,2014). Compritol1 888 ATO exhibited characteristic peaks at2918 cm�1 and 2851 cm�1 (alkanes stretch) and 1741 cm�1 (C¼Oester stretch). PEG 600 displayed strong peaks at 2869 cm�1 (alkanesstretch) and 1097 cm�1 (ether stretch). All characteristic absorptionbands of KTZ, Compritol1888 ATO and PEG 600 were observed in thephysical mixture’s spectra, however a reduction in the sharpness ofsome peaks was observed when compared to the spectra of purecomponents. The retention of characteristic peaks suggests absenceof interaction of the components. The spectra of molten Compritol1

888 ATO with PEG 600 also showed characteristic peaks ofCompritol1 888 ATO and PEG 600, indicating absence of anyinteraction between the two even on heating (PEG 600 does notdissolve in molten Compritol1 888 ATO). It may be said that both ofthem retain their properties when incorporated into nanoparticles.In the FTIR spectra of KTZ-SLNs, peaks corresponding to KTZdisappeared indicating its efficient entrapment in the lipid matrixexcept for a peak at 1640 cm�1. It may however be noted that thispeak was also present in blank SLNs (1640 cm�1) and matches with apeak exhibited by Tween 80 at 1644 cm�1 attributed to the alkenestructure present in it. This indicates that Tween 80 is a majorsurfactant present on the surface of developed particles. While thecharacterstic peak exhibited by PEG at 2869 cm�1 is absent in thespectra of blank and drug loaded SLNs confirming its incorporationintotheirlipid core.Similar resultshavebeenobservedbyothers(Fanet al., 2014). Based on the drug-enriched core model (Muller et al.,2002), it may be presumed that orientation of KTZ towards thecentre/ lipid core rather than its presence on the surface will protectit against chemical degradation (Helgason et al., 2009). Hothomogenization technique usually results in SLNs with a drugenriched core (Mao et al., 2005) and an outer protective shell. Thesimilarity of the FTIR spectra obtained for drug loaded SLNs to thatexhibited by blank SLNs, indirectly confirms efficient encapsulationof drug within the lipidic core of developed SLNs (i.e. no/insignificantfree drug was present in the SLN dispersion oron the surface of SLNs).Although entrapment efficiency obtained by the analytical proce-dure is 70% w/v (Table 1), it may be said that the free drug is alsoassociated with the surface of these particles (Bhandari and Kaur,2013), so that it is not identifiable as a separate unit by FTIR. Thisphenomenon may help in a very efficient transport of both the freeand the entrapped drug.

In case of pure KTZ, melting endotherm appeared at 152.37 �C(Fig. 3A) corresponding to its melting point, while Compritol1

888 ATO showed a sharp peak at 73.6 �C and an enthalpy of 122.6 J/g (Fig. 3C). KTZ-SLNs showed an endothermic shift to 89.04 �C anda significantly lowered heat flow of 12.05 J/g (Fig. 3B) indicatingincorporation of drug into the lipid matrix. Lower enthalpy forKTZ-SLNs versus pure lipid indicates a reduction in particle sizeand the change in polymorphic state of the lipid from thecrystalline b form to amorphous forms (a, b’) (Muller et al., 2006)with more imperfections in the crystal lattice. Latter results incomfortable incorporation of more amount of drug within the lipidmolecules. It could be similar to nanostructured lipid carrier (NLC)type of a structure due to liquid PEG 600 within the lipid core(Sachdeva and Shah, 2014).

Pure KTZ exhibited characteristic peaks between 2u of 7.3 and48.9 (Fig. 4), while these peaks disappeared in the XRD pattern oflyophilized KTZ-SLN formulation. However, broad, and diffusepeaks with low intensities were observed and indicated amor-phousness of the formulation. PXRD pattern of Compritol1

888 ATO shows sharp peaks at 2u scattered angles 21.3 and23.4; indicating its crystalline state. Compritol1 888 ATO meltedwith PEG 600 (2u of 21.5) showed a pattern of peaks similar to thatexhibited by Compritol1 888 ATO (2u of 21.6) alone, confirmingthat PEG 600 does not interact with Compritol1888 ATO, as alsoindicated by the FTIR studies.

1H NMR spectra of KTZ, Compritol1 888 ATO, Tween 80, PEG600, Phospholipon 90G, KTZ-SLNs and blank SLNs are shown inFig. 2 of Supplementary data. Purity of the drug was established byappearance of a sharp peak and absence of any impurity peaks inNMR signals. NMR signals of lipid–water mixture did not show anysignal corresponding to ��CH2 (1.3 ppm) and ��CH3 (0.9 ppm) asexpected, because solid ingredients are not detected in the NMRspectra, under the experimental conditions, because of very shortrelaxation times. Furthermore, as Compritol1 888 ATO (glycerylbehenate) does not contain any lipid fractions which are liquid atroom temperature, no signals corresponding to the lipid weredetected in lipid-water mixture. Similar observations were madefor blank and drug loaded SLN dispersion where only Tween80 derived signals are observed. This finding confirms that themoieties i.e. the particles present in these dispersions are solid i.e.are SLNs (Jores et al., 2003). Presence of supercooled melts in NMRobserved for SLN samples by other workers (Bunjes et al., 1996),may therefore be excluded and the highly restricted mobility of theglyceride molecules is attributed to their solid state. Latter isachieved by cooling the SLN dispersions after preparation (West-esen et al., 1997).

Broad and weak NMR signals are characteristic for molecules ofrestricted mobility. Decreased mobility of the molecules leads to adecrease in proton relaxation times, which results in considerableline broadening. Sharp and intense signals are derived frommolecules with high mobility (Jenning et al., 2000). Phospholipon90G shows sharp and intense peaks in SLN dispersion, hence it can beconcluded that it does not lose mobility, and is present on the surface

Fig. 4. Powder X-ray diffraction patterns of Compritol1 888 ATO (A), ketoconazole(KTZ; (B), ketoconazole loaded solid lipid nanoparticles (KTZ-SLN; (C), Compritol1

888 ATO melted with PEG 600 (D) and Compritol1 888 ATO melted alone (E).

S. Kakkar et al. / International Journal of Pharmaceutics 495 (2015) 276–289 281

rather than being entrapped within the lipid core of the preparedSLNs (Mao et al., 2005). It may be noted that solidification of lipid notonly resulted in a loss of the strong triglyceride signals but it also ledto a considerable broadening or loss of the drug signals. Theconsiderable line widths of the drug signals confirm that thesesignals are generated from molecules which are not dissolved in theaqueous phase, rather are in a viscous immobile phase, i.e. mainlydispersed within the lipid core. The reduced mobility of the drugmolecules of the SLN dispersion also indicate that drug is associatedwith the lipid as expected due to its lipophilic nature. Obtained NMRdata indicate that the SLNdispersionmayserve asa sustained releasesystem from which the drug will be released slowly. Furthermore,the results show broad and very low intensity signals of PEG 600 inthe SLN dispersion indicating its probable incorporation in the solidlipid core. A small amount of PEG 600 may however be in a free formbut associated with the lipid, signifying its low mobility and weaksignals.

3.2. Ex vivo/corneal permeability studies

The lmax for standard plot in chloroform:methanol (1:1) andethanol as solvents was different (242 nm and 244 nm, respective-ly) than that obtained for GBR + 2% SLS (291 nm). This may beattributed to a bathochromic shift in UV absorption maxima. Thechange of absorption wavelength due to changing solvent polarityis a phenomena called solvatochromism. Positive solvatochromismis an increase in absorption wavelength due to increase in solventpolarity (Reichardt, 2004).

Histological section of the cornea (Fig. 5A) confirmed thatobtaining the whole eye bulbus from the local slaughter house andisolation of cornea from the former in our laboratory, in no waycompromised its integrity and suitability for use in the permeationstudies (Thiel et al., 2002). KTZ-SLNs showed a significantimprovement in apparent permeation coefficient (Papp) (i.e.14.31 �10�6� 1.25 �10�6 cm/s) as compared to aqueous suspen-sion of free drug (8.68 � 10�6� 0.84 �10�6 cm/s) taken as control(Table 2); amount permeated and percentage permeated after15 min; and steady state flux (Fig. 5B). It may be noted that thelatter comprised of 0.5% w/v Tween 80 which though incorporatedpresently, as a dispersing agent, may also act as a penetrationenhancer (Kaur and Smitha, 2002).

3.3. Stability studies

There was no significant change in entrapment efficiency, totaldrug content and particle size of KTZ loaded SLNs underrefrigeration for 12 months (p < 0.001) and upon autoclaving(p < 0.05) (Table 3).

In case of photostability studies, the total drug content of KTZ-SLNs did not change significantly (p < 0.05; Table 3, Supplementarydata) before and after exposure, while KTZ-SUS showed asignificant (p < 0.05) drug loss (�20%). Latter turned slightly pinkupon storage, whereas KTZ-SLNs retained their white color.

3.4. Antifungal activity

Mean values for zones of inhibition obtained with KTZ-SLNswere significantly (p < 0.05%) high than those observed for KTZ-SUS at corresponding concentrations (Table 4). Vehicle controls(Tween 80 and PEG 600) and blank SLNs included in the study didnot show any antifungal activity.

3.5. Anti-biofilm efficacy

KTZ-SLNs exhibited inhibitory activity similar (p < 0.05) to thatof KTZ-SUS (Fig. 6), against planktonic growth, developing biofilm

and mature biofilms (Fig. 3, Supplementary data). The blank SLNsdid not show any inhibition of C. albicans.

3.6. Challenge test

The formulation containing 0.02% w/v benzalkonium chloridepassed the pharmacopoeial (BP, 1999) challenge test (Table 5),

Fig. 5. (A) Porcine corneal section at 100� (Nikon eclipse i80, Japan). (B) Percentage amount of KTZ-SLNs and KTZ-SUS permeated through porcine cornea at various timeintervals (n = 6).Values are mean � standard deviation. The results were analyzed for statistical significance by a 1-way analysis of variance (ANOVA) test followed by the Tukey’s test. All thevalues of KTZ-SLNs were significantly different from KTZ-SUS except those marked p < 0.05.

Table 2Comparison of KTZ-SLNs with KTZ-SUS, in terms of total amount permeated, percentage permeation, steady state flux and apparent permeability coefficient (Papp) obtainedduring ex-vivo permeation studies using porcine cornea (n = 6).

Formulation Total amount permeated in 4 h (mg) % Permeation Steady state flux (mg/min/cm2) Apparent permeability coefficient Papp (cm/s)

KTZ-SLN 145.69 � 8.14 29.1 � 1.63 0.43 � 0.03 14.31 �10�6� 1.25 �10�6

KTZ-SUS 85.07 � 9.03 17.01 � 1.81 0.26 � 0.025 8.68 � 10�6� 0.84 �10�6

Values are mean � standard deviation. All the values were significantly different from one another, at p < 0.05, as per one-way ANOVA followed by the Turkey’s test.

Table 3Stability study under refrigerated condition (2–8 �C) and upon autoclaving (n = 3).

Time points Total drug content (%) Entrapment efficiency (%) Average particle size (nm)

Samples stored at 2–8 �C0 time** 92.9 � 0.73 71.99 � 2.84 129.03 � 2.996 months** 91.64 � 2.47 68.71 � 4.35 130.07 � 8.6712 months** 89.11 � 4.40 63.67 � 2.13 151.27 � 1.98

Stability upon autoclavingBefore* 92.12 � 1.81 72.31 � 0.84 123.8 � 8.47After* 90.47 � 0.93 70.26 � 0.97 145.4 � 10.49

Values are mean � standard deviation. The results were analyzed for statistical significance by a one-way analysis of variance (ANOVA) test followed by the Tukey’s test.* Values were not significantly different from one another, at p < 0.05.** Values were not significantly different from one another, at p < 0.001.

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Table 4Antifungal activity expressed as zone of inhibition produced by KTZ-SLNs and KTZ-SUS.

S. No. Conc. (mg/ml) Zone of inhibition (cm) (n = 6)

KTZ-SLN KTZ-SUS

1 500 1.26 � 0.08 0.91 � 0.112 800 1.47 � 0.16 1.1 � 0.083 1000 1.62 � 0.08 1.22 � 0.14 1200 1.73 � 0.12 1.33 � 0.16

Values are mean � standard deviation. Values of KTZ-SLNs were significantlygreater than those obtained for KTZ-SUS, at p < 0.05 when one-way ANOVAfollowed by the Tukey’s test was applied.

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confirming suitability of BAK as a preservative for the developedformulations. Since nanoparticles and their employed componentsmay adsorb, degrade or inactivate the incorporated preservative,resulting in its decreased efficacy, hence it is important to confirmthe latter by challenging the formulation with standard bacterialand fungal strains. Study was performed in blank SLNs consideringthat inclusion of KTZ may affect the evaluation especially whenfungal strains are employed.

Fig. 6. Activity of KTZ-SLNs, KTZ-SUS and blank SLNs against C. albicans (ATCC 90028). (produced no inhibitory effect at any concentration.Values are mean � standard deviation. The results were analyzed for statistical significValues of KTZ-SLN were similar (p < 0.05) to that of KTZ-SUS except at points marked

3.7. In vivo safety studies

3.7.1. Dermal irritation/corrosion testSLNs were nonirritant to dermal tissues as indicated by 0/

40 score for erythema and oedema each. This evaluation precededthe acute eye-irritation test.

3.7.2. Eye irritation/corrosion testThe score of 0/195 for single and 0/195 for repeat instillation

establishes that developed SLNs are completely safe for ocular use.The treatment of ocular fungal infection involves long term therapy,so we evaluated the developed formulation for chronic repeatinstillation and the formulation again exhibited a score of 0/312.

3.8. Cytotoxicity studies

KTZ-SLNs did not show any cytotoxicity when (Fig. 7)administered to ARPE-19 and RCE cell lines, at different concen-trations and for times upto 96 h (p < 0.001).

3.9. Ocular tolerance evaluation

Corneal, retinal and conjunctival cross-sections (Fig. 4, Supple-mentary data) of the rat eye exposed to the formulation for

A) planktonic growth; (B) developing biofilms, and (C) mature biofilms. Blank SLNs

ance by a one-way analysis of variance (ANOVA) test followed by the Tukey’s test.as (n = 3).

Table 5Results of challenge test of blank SLN formulation.

Strain Time Log reduction

Forformulation

Pharmacopoeialrequirement

Candida albicans 7 days 2.43 228 days 5.66 No increase in bacterial

populationAspergillus niger 7 days 2.99 2

28 days 5.28 No increase in bacterialpopulation

Staphylococcusaureus

0 h 0.016 h 2.03 224 h 3.66 328 days 5.69 No recovery in bacterial

populationPseudomonasaeruginosa

0 h 0.026 h 2.09 224 h 3.84 328 days 5.62 No recovery in bacterial

population

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different times, confirmed the intactness of tissue integrity andabsence of any adverse events. No inflammation or damage wasobserved in the retina and sclera, confirming ocular biocompati-bility of the developed formulation.

3.10. Ocular pharmacokinetics

The concentration-time profiles and corresponding pharmaco-kinetic parameters of KTZ-SLN and KTZ-SUS after topicaladministration were monitored and determined in the aqueousand vitreous humor as shown in Fig. 8 and Table 6, respectively. Asignificantly (p < 0.05) higher Cmax (1.6-fold) and AUC (a 2.5 foldincrease) for KTZ-SLN with respect to the KTZ-SUS was noted inaqueous humor. Similarly, a 1.7 fold and 1.6 fold higher effects invitreous humor were observed for Cmax and AUC, respectively. Theconcentration of KTZ in the aqueous and vitreous humor in the SLNtreated group was found to lie in the range of 1.37–0.1 mg/ml and0.83–0.05 mg/ml, respectively, which were several times higherthan those observed for KTZ-SUS group (0.87–0.01; 0.49–0.01 mg/ml). As over 70% of fluid in vitreous humor moves toward and exitsthrough the retina, higher concentrations of KTZ in the vitreoushumor may indirectly establish its effectiveness against fungalretinitis (Shen and Tu, 2007).

An early Tmax (0.5 h) observed for KTZ-SLNs indicates its highpermeation efficiency. Latter may be due to the nano nature andsurfactant layer around these nanoparticles. A Tmax of 20 min. isreported for a cyclodextrin complex of KTZ; presentation of KTZ inpolyethoxylated castor oil resulted in a Tmax of 1 h.

Cmax of 1.37 mg/ml of aqueous humor progressively reduced to0.82 mg/ml at 2 h followed by 0.55 mg/ml and 0.37 mg/ml at 6 and12 h, respectively, indicating a sustained and prolonged stay ofKTZ-SLNs in the aqueous humor.

Presence of intact SLNs in the aqueous and vitreous humorsamples, collected at various times upto 24 h (Fig. 5A and B ofsupplementary section) further confirm the above claims.

4. Discussion

Majority of fungal pathogens including Aspergillus sp., Candidasp., and Fusarium sp. and their clinical isolates are susceptible andsensitive to KTZ (Therese et al., 2006). Inspite of showingeffectiveness against the commonly occurring infections of theeye (Therese et al., 2006; Grossman and Lee, 1989), no ocularformulation of KTZ is in the market. Effective concentrations of KTZhave been established, but only in the debrided cornea (Hemady

et al., 1992), indicating that permeation through intact cornea maybe an issue. Furthermore, poor aqueous solubility (0.04 mg/ml)may also pose a problem of presenting it in a soluble form on thecorneal surface.

Presently we encapsulated KTZ into SLNs (i) to present it in anaqueous dispersion, (ii) provide benefits of applicability as oculardrops, and (iii) treat both endophthalmitis (intact particles wereobserved in the vitreous) and keratitis following topical applica-tion. KTZ-SLNs were prepared without the use of any organicsolvent as the latter can be a source of irritation and ocular toxicity.

Cyclodextrin complexes of KTZ have been prepared andreported (Zhang et al., 2008). Although such a system will caterto solubility issue of KTZ, however it cannot effectively improvepenetration across the cornea and transport the drug to thevitreous. Cyclodextrins are true carriers in the sense that theydeliver their cargo (drug molecule) in a solution form, to thebiological membrane but cannot transport across it, due to theirlarge size. Further to this high cost and crusting of cyclodextrins oneyelid, at times, result in poor compliance (Kaur et al., 2004, 2011).Souto and Muller (2005) have reported preparation of KTZ loadedSLNs and nanostructured lipid carrier (NLCs) for application toskin. Their composition includes Compritol1 888 ATO, poloxamerand sodium deoxycholate; and a-tocopherol for NLC. However,sodium deoxycholate is an ocular irritant and not used in eye drops(Dai et al., 2013). The size of SLNs prepared by them wasapproximately 200 nm; NLCs were bigger than SLNs. Storagestability for 90 days, under refrigeration indicated an increase insize for NLCs while KTZ loaded in SLNs underwent photo-degradation and a change in color to purple. Authors howeverdo not specify entrapment efficiency while the loading of KTZ was5.26% with respect to the lipid used. Howsoever, in the presentstudy in addition to a drug loading of 10%, the prepared SLNsprovided protection against photodegradation with no colorchange. Free KTZ (KTZ-SUS) however did show pink discoloration.

Ramasamy et al. (2012) prepared KTZ loaded SLNs of sizeranging between 1–3 m, for application on skin, using chloroform,Tween 80 and Dynasan. Size could be reduced to 172 nm followingsonication for 20 min, howsoever the drug loading again variedbetween 3.38–5.12% with an entrapment efficiency of 82.15–92.4%.

Nano-sized particles (presently 126 nm) provide better bio-adhesion (Yoncheva et al., 2005) and greater surface for associationwith the cornea and conjunctiva. They can also pass across theanatomical constraints of the eye and provide enhanced penetra-tion through the cornea (Shen and Tu, 2007; Nagarwal et al., 2009).Nanocarriers are better retained in the cul-de-sac of the eyebecause of their size and ability to adhere to the tissue surface(Kassem et al., 2007). Furthermore, their interaction with theglycoproteins of the cornea and conjunctiva can form a precornealdepot resulting in prolonged release of the encapsulated drug. Thiscan have a special application in the treatment of superficial fungalinfections.

Usually a zeta potential of ��25 mV is recommended forachieving stable dispersions. High potential indicates existence ofrepulsive forces between particles, which prevents their contactwith one another resulting in agglomeration. However, gelation ofSLN dispersions has been observed at zeta potential as high as30 mV (Heurtault et al., 2003). The neutral particles obtainedpresently were intrinsically stable (upto 1 year) with no significantchange (p < 0.001) in their size. This may be attributed to theabsence of inter particulate molecular interactions (both, attractiveand repulsive) (Monem et al., 2000; Bhandari and Kaur, 2013).

Charge on the nanoparticles is also reported to influence their invivo behavior. The positively charged nanoparticles are taken upmore rapidly by the cell membranes which have a slightly negativecharge on them, but their binding to the biological membranes isreported to induce their fluidity. Similarly negatively charged

Fig. 7. Cell viability of corneal (A) and retinal (B) cell lines after exposure (for 24, 48 and 96 h) to KTZ-SLNs and blank SLNs at three different concentrations (1.5, 3 and 6 mg/ml). Values are mean � standard deviation. The results were analyzed for statistical significance by a 1-way analysis of variance (ANOVA) test followed by the Tukey’s test. Allthe values were not significantly different from their corresponding control at a particular time and concentration (p < 0.001).

S. Kakkar et al. / International Journal of Pharmaceutics 495 (2015) 276–289 285

nanoparticles induce gelation of the lipid bilayers. However, nosuch adverse events are reported for neutral nanoparticlesindicating them to be safe (Bhandari and Kaur, 2013).

The ideal pH of an ophthalmic preparation for maximumcomfort in the eye is 7.2 � 0.2 (Mathis, 1999). Instillation of asolution with a different pH is irritating and may cause pain andalso initiate lacrimation. Latter dilutes the instilled drug solution ormay even wash it off from the eye fornix, resulting in a poorbioavailability. pH of the presently developed formulation wasnear neutral and was used as such. Osmolarity of tears is297 mOsm/l (Versura et al., 2010). However, evaporation of thetear film can lead to an increased osmolarity and consequentdamage to the ocular surface epithelia (Iester et al., 2000). Hence,use of hypotonic solutions, as obtained presently for KTZ-SLNs, isadvocated for ocular delivery (Iester et al., 2000; Aragona et al.,

2002). Since RI of the formulation is close to water, hence it may besaid that KTZ-SLN will not interfere with vision on application andis appropriate for ocular use. For minimum interference withvision, a refractive index close to that of ocular tissues (1.34–1.41) isdesirable (Anjana et al., 2012).

The loss of crystallinity of the lipid and its shift towards theamorphous state in XRD studies, indicate successful incorporationof KTZ into the lipid matrix of the SLNs. The thermogram of KTZ-SLNs also confirms loss of crystallinity of Compritol1 888 ATOwhen present in the SLN formulation (Jannin et al., 2008),indicating loss of its highly ordered structure (Attama et al.,2007). Crystallinity/amorphousness of a lipid matrix has an effecton the functional properties such as drug incorporation and releaserates (Gill et al., 1993). XRD and FTIR data confirmed incorporationof KTZ into SLNs.

Fig. 8. Concentration profile of KTZ in aqueous and vitreous humor upon topical administration of KTZ-SLN or KTZ-SUS.

Table 6Pharmacokinetic parameters of KTZ in aqueous and vitreous humor, after topical administration of KTZ-SLN and KTZ-SUS.

Formulation Aqueous humor Vitreous humor

Cmax (mg/ml) AUC0–t(mg/ml h) MRT (h) Kel (/h) Cmax (mg/ml) AUC0–t (mg/ml h) MRT (h) Kel (/h)

KTZ-SLN 1.37 � 0.18 10.31 � 0.79 9.74 0.094 0.83 � 0.09 4.05 � 0.27 9.58 0.092KTZ-SUS 0.87 � 0.11 4.16 � 0.31 4.99 0.17 0.49 � 0.13 2.48 � 0.35 6.96 0.13

Values are mean � standard deviation. The results were analyzed for statistical significance by a one-way ANOVA followed by the Tukey’s test. All values observed for KTZ-SLNs were significantly different from those for KTZ-SUS, at p < 0.05.

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Procuring DSC and XRD data for a colloidal dispersion confirmsits solid nature (Westesen et al., 1997). XRD and FTIR scans of themolten lipid-PEG mixture indicate presence of peak correspondingto Compritol1 888 ATO indicating that PEG does not in any wayinfluence properties of the former. DSC, FTIR and NMR studiesindicate presence of PEG 600 within the lipid matrix asrepresented in Fig. 9. NMR spectra confirm that KTZ is majorlydissolved or dispersed in the lipid core while upon cooling some ofit may be thrown out on the surface of nanoparticles as indicatedby the broad signals observed for it. Presence of PEG is expected toinduce imperfections in the lipid lattice similar to those introducedby low melting point lipids/oils in NLCs, allowing entrapment ofKTZ which could not be entrapped in SLNs prepared without PEG. Itmay be concluded that KTZ is either dispersed in the lipid-PEG coreor is solubilised in the PEG core which is mixed and surrounded bythe lipid matrix (Fig. 9). Although, some unentrapped (approx.30%) drug is also present in the dispersion yet it is in a solubilised/dispersed form with no separation/settling being observed even onlong term storage (1 year) under refrigerated conditions.

Fig. 9. Schematic representation of the lipid nanoparticles with PEG core dispersedin the aqueous phase.

The usually reported lipo-polymeric systems involve a polymerand a phospholipid; both these components are costly and thenatural origin of phospholipid also makes it unstable (Meenachet al., 2013; Nag and Awasthi, 2013). Furthermore, polymersemployed are complex and even if they are biocompatible, it maynot be essential that their monomers are also safe and do notaccumulate in the body. Moreover, preparation of polymericsystems always involves an organic solvent (for dissolving thepolymer) while presently described system is an aqueousdispersion and PEG (with KTZ dissolved in it) is directly mixed/dispersed with the lipid component collectively forming the non-aqueous phase.

Since one aim of the study was to deliver KTZ across the corneainto the aqueous and vitreous chambers, hence we conducted exvivo permeability studies using porcine cornea. Further, since verysmall volume of fluids are present on the precorneal surface andthe volume of both aqueous and vitreous humor is also limited,hence, performance of in vitro release studies may not be a fairmeasure of drug release from SLNs. In this regard, presentlyconducted corneal permeation study is considered to be acombination of release cum permeation studies (Kaur et al.,2010). For the corneal permeation studies, GBR with 2% SLS wasused in order to provide sink conditions. The solubility of KTZ inGBR with 2% SLS was 4.67 mg/ml. Corneal permeation dataindicates a significant improvement in flux and total amount ofKTZ permeated (0.43 mg/min/cm2; 145.69 mg) when applied asKTZ-SLNs versus KTZ-SUS (0.26 mg/min/cm2; 85.07 mg).

Developed system was found to be stable on storage. SLNsgenerally undergo gelation on storage, and factors like increasedlight, temperature, and shear stress increase kinetic energy of theparticles to favour their collision. Most nanostructured systemsneed to be dialysed, or lyophilized to retain their stability onstorage. Such methods are (i) costly, (ii) time consuming (iii) mayresult in aggregates and microsized particles on reconstitution, and(iv) change the native milieu of nanoparticles, which may

S. Kakkar et al. / International Journal of Pharmaceutics 495 (2015) 276–289 287

destabilize the system. Presently prepared aqueous SLN dispersionwas stable for 1 year at 4 �C. This may be attributed to themaintenance of SLNs in their native state in which they wereproduced (Kumar et al., 2014) comprising suitable concentration ofsurfactants in the aqueous phase in which they remain dispersedeven on long term storage. Another reason for good stability is thesuitable composition of SLNs, like a suitable surfactant, surfactant:co-surfactant ratio and incorporation of PEG in the formulation.The surfactant to co-surfactant ratio was kept suitably high toachieve not only a small size but also a significant EE with a betterocular distribution (Kumar et al., 2014; Bhandari and Kaur, 2013).PEG being a plasticizer also increases the rigidity of the system,thus facilitating its stability (Heinämäki et al.,1994; Laboulfie et al.,2013). KTZ loaded SLNs were photostable while free drugsuspension showed >20% drug loss on exposure to UV-light. KTZis reported to undergo photodegradation (Staub et al., 2010).

The developed SLN formulation was found to resist any changein size or entrapment efficiency upon steam sterilization. Thisstability is attributed to the choice of emulsifiers and use of PEG inthe preparation of SLNs (Mehnert and Mäder, 2001). The surfactantfilm comprising PEG (Mehnert and Mäder, 2001) can alter itsperformance, with the change in temperature (Bunjes et al., 1996).Use of lecithin is harmonious to steam sterilization andformulations containing lecithin produce less significant increasein particle size post steam sterilization. Absence of any significantchange in particle size after autoclaving is reported for trilaurinebased SLNs loaded with azidothymidine palmitate (Heiati et al.,1998). SLNs are anticipated to melt during autoclaving followed bytheir recrystallization upon cooling (Muller et al., 2000).

The developed SLNs were established to be safe for ocular use inhistological and in vivo experiments in rabbits. Studies have shownthat the rabbit eye is more sensitive than the human eye and has alonger epithelial repair time. Thus, it is reasonable to expect bettertolerance to SLN formulation in the human eye (Gokce et al., 2009),once its safety is established in the rabbit eye. Cytotoxicity studieson corneal and retinal cell lines also confirmed biocompatibilityeven at concentrations as high as 6 mg/ml and for exposures as longas 96 h. The tested concentration of 6 mg/ml is more than themaximum concentration of KTZ (1.37 � 0.18 mg/ml) achieved in theaqueous humor following topical administration. These resultsprovide a clear evidence of wide safety of the formulation.However, the safety was envisaged as all the ingredients used informulation are biodegradable, biocompatible and safe for ocularuse (Seyfoddin et al., 2010) at the employed concentrations (http://www.accessdata.fda.gov/scripts/cder/iig/index.Cfm).

The larger zones of inhibition obtained with KTZ-SLNs against C.albicans shows that SLNs or the free drug released from SLNsdiffused to a higher extent (both in terms of distance andconcentration) than the free drug suspension (KTZ-SUS). Similarly,in the case of mature biofilms concentration >8 mg/ml showedsignificantly (p < 0.05) better activity. It may be appreciated thatSLNs invariably showed better effect than free drug, inspite of thefact that KTZ is released slowly from SLNs and at any given time theamount of free drug in KTZ-SLNs will be less than free drugsuspension. The effect may either be due to presentation of KTZ in asolubilized and permeable form in SLN formulation or due tosignificant uptake of intact KTZ-SLNs by the fungal cells. Onceinside, release of entrapped KTZ, even in small quantity, will ensuremore efficient antifungal activity. Formation of biofilms confersmicroorganisms an ability of drug resistance and capacity towithstand high concentrations of antimicrobial drugs/antibioticswhich is not seen in their planktonic counterparts (Donlan andCosterton, 2002). Candida biofilms are 30 to 2000 times moreresistant to antifungal agents such as, KTZ, itraconazole, ampho-tericin B, and fluconazole than the corresponding planktonic cells(Martinez and Fries, 2010). Hence, significantly better efficiency

exhibited by KTZ-SLNs against the mature biofilms is a highlyvalued result.

Hironaka et al. (2009, 2011) have discussed the delivery of drugsto the posterior segment of the eye via topical administration. Theyproposed that the liposomes travelled majorly through the cornealand conjunctival route involving the iris and the ciliary body. Sucha transport mechanism was attributed to the rigidity and nano sizeof the liposomes. Rigidity is expected to maintain the stability ofthe carrier system in the biological environments such as tear filmand ocular mucosa. Solid nature (as indicated from the characteri-zation data) of presently prepared SLNs could thus be responsiblefor the efficient posterior eye targeting achieved by the former.

Pharmacokinetic studies showed higher bioavailability of KTZwhen administered as SLNs versus the free drug suspension, bothin the aqueous and the vitreous humor. Former was ascribed tolong preocular retention, small size of KTZ-SLNs, their presumableentrapment and retention in the mucin layer covering the cornealepithelium (Cavalli et al., 2002). Further, cellular uptake ofnanoparticles by corneal epithelial cells and different tissues ofthe eye (Gokce et al., 2008) is also expected to enhancebioavailability of KTZ-SLNs. It may however be noted thatsignificant permeation exhibited by KTZ-SLNs is also attributableto the presence of surfactant (i.e. Tween 80) and the intrinsiclipophilic nature of KTZ. Tween 80 increases the permeability ofcell membranes and/or loosens the tight junctions (Sultana et al.,2006). In addition to this, both KTZ (Ogasawara et al., 2007) andTween 80 (Al-Mohizea, 2010), inhibit the P-gp efflux existing in thecornea (Kawazu et al., 1999), conjunctiva (Saha et al., 1998), andretina (Constable et al., 2006). However, this nature of KTZ andTween 80 to inhibit P-gp efflux transporters is relevant to KTZ-SUSalso. Additionally, encapsulating these agents in the protectivelipid matrix of the SLNs may also provide a reduced exposure to theenzymatic and metabolic milieu of the eye, post administration,thus minimizing their clearance.

Cmax of 2.67 mg/ml at 20 min, was achieved for KTZ complexedwith hydroxypropyl-b-cyclodextrin versus 0.44 mg/ml, at 30 min,for the corresponding free drug suspension in aqueous humor(Zhang et al., 2008). The researchers did not determine drugconcentration in the vitreous. This Cmax was higher than the Cmax

obtained with the present formulation, which may be due to thedifference in amount of drug administered. These workersadministered 750 mg of drug while we instilled 345 mg of KTZ.Furthermore, a concentration of 0.64 mg/ml of KTZ was present at2 h which reduced to 0.38 mg/ml at 3 h (no values reportedthereafter) in the former case while we could attain a concentra-tion of 0.82 mg/ml at 2 h which reduced to 0.55 mg/ml at 6 h and0.37 mg/ml at 12 h even though almost half the amount of KTZ wasinstilled presently. This confirms a prolonged residence of nano-particles and release of KTZ from the presently developed SLNs. Inanother report (Hemady et al.,1992), drug levels as high as 32.5 mg/ml were achieved on topical administration of 1% KTZ prepared inpolyethoxylated castor oil, in aqueous humor, after 1 h ofadministration which decreased to 1 mg/ml after 4 h, while, oncorneal debridement, levels were 46.3 mg/ml at 1 h and 8 mg/mlafter 4 h. No drug was detected in the vitreous except followingepithelial debridement, when 9.2 mg/ml at 15 min; 8.3 mg/ml at1 h; 0.13 mg/ml at 2 h; and no drug at 4 h was observed, afteradministration of approximately 2400 mg. However, we achieveddrug levels of 0.83 � 0.09 mg/ml at 0.5 h in vitreous humor when345 mg of KTZ-SLNs were instilled in intact eye. Howsoever,sampling times were invariably wide in the present study, majorlybecause rabbits are large animals and use of more number hadethical implications; use of only a limited number of rabbits wasallowed by the ICSEA. Furthermore, the data at each time pointrepresents values for four eyes; bigger sample size with closerspacing may give a better picture of the pharmacokinetic

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performance of KTZ-SLNs. Another point, which is noteworthy inthe study, is the fact that peak effects were obtained within 30 minafter administration of the last drop of SLNs. This indicates a fastpermeation of developed system across various barriers to reachthe aqueous and especially so the vitreous humor. Further to this,bioavailability of KTZ is also expected to improve in situation ofactual use because fungal infections are at times associated withcorneal debridement. The presence of intact SLNs in vitreousprovides significant and lucid evidence that SLNs cross, differentocular barriers, to successfully reach the vitreous humor and thuscan be especially useful for treatment of endophthalmitis.

5. Conclusion

Ketoconazole was successfully entrapped in lipidic coreproviding enhanced permeation through cornea and higherbioavailability both in the aqueous and vitreous humor. Thedeveloped nanoparticles were able to cross ocular barrier, reachposterior segment of the eye and had significant antifungalpotential. Thus, the developed SLNs can be used both for thetreatment of keratitis and endophthalmitis. Corresponding safetyin corneal and retinal cell lines followed by in vivo acute andsubchronic toxicity studies in rabbits coupled with their autoclav-able nature, establish suitability of developed SLN formulation forocular use. Furthermore, it is a lipo-polymeric (PEG) system whichhas the capacity for both, high loading and entrapment efficiency,due to the intelligent incorporation of lipid and PEG in the core ofparticles. Reported formulation is clearly different from aPEGylated system, which has a limited advantage of being stealthin comparison to the parent nanoparticles. The present systemprovides advantage of incorporating drug in a soluble form withinthe nanoparticle core, endows amorphousness and imperfectionsto the lipid core, in addition to being rigid and stable.

Declaration of interest

The authors report no declarations of interest.

Acknowledgments

The funding provided by DST, New Delhi, India, is highlyacknowledged. Dr Jayant Raut is thankful for the UGC-D S Kotharipost doctorate fellowship.

Characterization studies conducted at PU-SAIF centre are highlyacknowledged. Special thanks are due to Mr. Avtar Singh and Mr.Manish Kumar for interpretation of NMR data and to Mr. DineshSharma for TEM pictures.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijpharm.2015.08.088.

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