International Journal of Pharmaceutics of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, Egypt A R T I C L E I N F O Article

International Journal of Pharmaceutics 492 (2015) 28–39

Pharmaceutical nanotechnology

Tri/tetra-block co-polymeric nanocarriers as a potential ocular deliverysystem of lornoxicam: in-vitro characterization, and in-vivo estimationof corneal permeation

Alaa Hamed Salamaa, Rehab Nabil Shammab,*aDepartment of Pharmaceutical Technology, National Research Center, Cairo, EgyptbDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, Egypt

A R T I C L E I N F O

Article history:Received 24 April 2015Received in revised form 25 June 2015Accepted 2 July 2015Available online 4 July 2015

Keywords:Ocular drug deliverySingle and mixed nanomicellar systemsLornoxicamConfocal laser scanning microscopyHistopathological studies

A B S T R A C T

Polymeric micelles that can deliver drug to intended sites of the eye have attracted much scientificattention recently. The aim of this study was to evaluate the aqueous-based formulation of drug-loadedpolymeric micelles that hold significant promise for ophthalmic drug delivery. This study investigatedthe synergistic performance of mixed polymeric micelles made of linear and branched poly(ethyleneoxide)-poly(propylene oxide) for the more effective encapsulation of lornoxicam (LX) as a hydrophobicmodel drug. The co-micellization process of 10% binary systems combining different weight ratios of thehighly hydrophilic poloxamers; Synperonic1 PE/P84, and Synperonic1 PE/F127 and the hydrophobicpoloxamine counterpart (Tetronic1 T701) was investigated by means of photon correlation spectroscopyand cloud point. The drug-loaded micelles were tested for their solubilizing capacity towards LX. Resultsshowed a sharp solubility increase from 0.0318 mg/mL up to more than 2.34 mg/mL, representing about73-fold increase. Optimized formulation was selected to achieve maximum drug solubilizing power andclarity with lowest possible particle size, and was characterized by 1HNMR analysis which revealedcomplete encapsulation of the drug within the micelles. Further investigations by histopathological andconfocal laser studies revealed the non-irritant nature and good corneal penetrating power of theproposed nano-formulation.

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1. Introduction

Drug delivery to the eye is one of the most challenging problemsfacing pharmaceutical researchers. The eye specific anatomy,physiology and biochemistry of make it practically inaccessible todrugs. It is claimed that less than 5% of the applied dose reachesintraocular tissues (Zhang et al., 2004). Moreover, there is a majorobstacle for successful and effective therapy of hydrophobic drugabsorption, because of their poor water solubility (Aliabadi et al.,2007). Aqueous formulations of drug-loaded polymeric micellescan overcome the ocular barriers through improving the perme-ation and enhancing the residence time on the ocular surface(Pepic et al., 2010).

Polymeric micelles with core–shell architecture have attractedmuch attention as a nano-sized drug carrier in drug deliverysystems. Hydrophobic drugs are encapsulated and solubilized intotheir hydrophobic cores through hydrophobic interactions (Harada

* Corresponding author.E-mail address: [email protected] (R.N. Shamma).

http://dx.doi.org/10.1016/j.ijpharm.2015.07.0100378-5173/ã 2015 Elsevier B.V. All rights reserved.

et al., 2011). In addition, they can be formed spontaneously,allowing large quantities to be prepared in fast and industriallyscalable technology (Ribeiro et al., 2013). Polymeric nanomicelleshave gained much attention in solubilizing hydrophobic drugs forthe different reasons. First, they offer large drug loading capacityand ensure water solubility of the nanocarrier system. Second, theyhave low critical micellar concentration (CMC), compared to othercommon low molecular weight surfactants. This offers greaterthermodynamic stability to withstand dilution, and enhance drugsolubilizing capability (Torchilin, 2001; Adams et al., 2003) toensure successful in-vivo targeting (Yokoyama, 2005).

Previous studies have shown that to prepare mixed polox-amers/poloxamine nanomicelles, the copolymers should behydrophobic blocks of similar molecular weight and differentHLB balance (Li and Tan, 2008; Wei et al., 2009). Thus, the aim ofthis work was to elucidate the possibilities of developing apolymeric nanomicellar system for the ocular administration oflornoxicam (LX). LX is an NSAID drug which is used topically todayfor the treatment of ophthalmic surface inflammation, and toreduce inflammation after cataract surgery; further, it is used astreatment for post cataract macular edema (Diakonis et al., 2013).

A.H. Salama, R.N. Shamma / International Journal of Pharmaceutics 492 (2015) 28–39 29

LX was also reported to decrease the severity of cornealultrastructural damage induced by UVB irradiation (Yin et al.,2008). However, its poor aqueous solubility constitutes a hurdle inthe development of ocular formulations. Thus, amphiphiliccopolymers are particularly suitable to increase drug solubility,prolong the residence time on the ocular surface, and improvecornea penetration, to ensure better ocular bioavailability com-pared to other ophthalmic liquid preparations (Jiao, 2008).

Specifically, the work was carried with Tetronic1 701, a non-ionic tetra-functional block copolymer surfactant that is 100%active and relatively non-toxic. Tetronic1 701 is formed by fourpoly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO) chainsconnected through a central ethylene diamine group (Alvarez-Lorenzo et al., 2010).

To the best of our knowledge, incorporation of LX in polymericmicelles has not been reported yet. Thus, as a first step, the effect ofcopolymer concentration on the formation of LX-loaded nano-micellar systems was studied. The prepared LX-loaded nano-micelles were characterized, by means of photon correlationspectroscopy and transmission electronic microscopy. LX releaserate was also evaluated. Finally, physical stability of LX-loadedmicelles throughout three months storage, and their oculartolerance in healthy rabbits were evaluated in order to furtherelucidate the feasibility of poloxamine-based systems as ocularformulations.

2. Experimental

2.1. Materials

LX was kindly provided by Delta Pharma, 10th of Ramadan City,Egypt. Tetronic1 701 (T701), Synperonic1 PE/F127 (F127),Synperonic1 PE/P84 (P84), and Rhodamine B (RhB), dialysistubing cellulose membrane (molecular weight cut-off 12,000 g/mole) was obtained from Sigma Chemical Company, USA. Absoluteethanol was purchased from El-Nasr Chemical Co. (Cairo, Egypt).

2.2. Methods

2.2.1. Preparation of polymeric nanomicellar systemsSingle and mixed polymeric nanomicellar systems were

prepared by direct equilibrium technique (Chen et al., 2010). Toprepare mixed T701:F127 and T701:P84 polymeric micelles (10%w/v), the required amount of each copolymer was first dissolved indistilled water, and the system was left to equilibrate at 25�/37 �Cfor 24 h. Pure T701, P84 and F127 (10%) single nanomicellarsystems were also prepared for comparison purposes, and aredenoted SM-1, SM-2 and SM-3, respectively.

2.2.2. Characterization of the prepared polymeric nanomicellarsystems

2.2.2.1. Cloud point (CP). CP measurements were conducted byplacing glass vials containing single and mixed nanomicellarsystem in a water bath at 25 �C. Then, the temperature wasincreased gradually from 25 �C (1 �C/min) until the point of suddenvisual appearance of turbidity. Assays were carried out in triplicate(Parmara et al., 2014).

2.2.2.2. Determination of the size of single and mixed polymericnanomicellar systems. The hydrodynamic diameter (Dh) and thepolydispersity index (PDI) of LX-free and LX-loaded single andmixed polymeric nanomicellar systems were determined byphoton correlation spectroscopy (PCS) that analyzes thefluctuations in light scattering owing to the brownianmovement of particles. Each nanomicellar system was diluted

(10 times) with bi—distilled water and was placed into a quartzcuvette at 25 � 0.5 �C, at 90� to the incident beam using a ZetasizerNano ZS (Malvern Instruments Ltd., Worcester-shire, UK). Allmeasurements were performed, in triplicate.

2.2.2.3. Measurement of % transmittance (%T). The turbidity ofsingle and mixed nanomicellar systems was monitored byspectrophotometric (lmax 520 nm) measurement of the % T ofeach formula using distilled water as a blank. The transmittancepercentages of the dispersions were measured 24 h afterpreparation (Sakai et al., 2011).

2.2.2.4. Encapsulation of LX. LX (in excess) was added to theprepared single and mixed polymeric nanomicellar systems (3 mL)and samples were shaken for 48 h at 37 �C. LX-loaded micelleswere separated by centrifugation of the micellar dispersions at5000 rpm for 15 min at 25 �C (Beckman centrifuge, Fullerton,Canada). The LX concentration was determined byspectrophotometric measurement at 378 nm. Solubility factors(fs) were calculated according to the following equation:

f s ¼Sa

Swater

where Sa and Swater are LX apparent solubility in micelles and indistilled water, respectively.

2.2.2.5. In-vitro release studies. LX release from the loadedmicellar systems was performed using dialysis bag technique(Das et al., 2011; Kumbhar and Pokharkar, 2013). Before theexperiment, the cellulose dialysis tubes were soaked in the releasemedia overnight. Two milliliters of LX-loaded micellar dispersionwere placed in a cellulose membrane dialysis bag (7 cm length,1 cm width, molecular weight cut-off 12,000 g/mole), then tied atboth ends. The dialysis tube was immersed in a beaker containing50 mL of phosphate buffer (pH 7.4) and shaken using athermostatically controlled shaker (GLF Corp., Burgwedel,Germany) adjusted at 100 strokes per minute maintained at32 � 0.5 �C. At pre-set time intervals, samples from the releasemedium were withdrawn and assayed spectrophotometrically at378 nm. Each withdrawn sample was replaced by an equal volumeof fresh release medium. To ascertain the kinetic modeling of drugrelease, the release data of LX from LX-loaded micellar dispersionswere fitted to zero-order, first-order and Higuchi diffusion models(Higuchi, 1962).

2.2.3. Optimization of LX-loaded micelles using a 21.31 full factorialdesign

LX-loaded micellar systems were optimized using a 21.31 fullfactorial experimental design in order to elucidate the influence ofdifferent formulation variables using Design-Expert1-8 Software.Two factors were evaluated, one factor at 2 levels, and the other at3 levels. The independent variables were the type of Synperonic1

(X1) and the concentration of the Tetronic1 in the nanomicellarmixture (X2) (Table 1). The percentage transmittance (Y1), themajor peak particle size (Y2), the LX encapsulation (Sa) (Y3), and the% drug released after 6 h (Y4) were selected as the dependentvariables. Table 2 depicts the composition of the preparedformulations.

2.2.4. Morphological examinationThe morphological appearance of a selected LX-loaded micellar

system was evaluated using TEM. One drop of the diluted vesiculardispersion was deposited on the surface of a carbon coated coppergrid, negatively stained with 2% phosphotungstic acid thenallowed to dry at room temperature for 10 min for investigationby TEM.

Table 1Full factorial design used to optimize the LX-loaded nanomicellar systems.

Level

Low (�1) Medium (0) High (1)

Factors (independent variables)X1: Type of Synperonic P84 F127X2: Concentration of T701in the total polymer (%) 75 50 25

Response (dependent variables) ConstraintsY1: % T MaximizeY2: PS MinimizeY3: Sa MaximizeY4: % Released after 6 h Maximize

30 A.H. Salama, R.N. Shamma / International Journal of Pharmaceutics 492 (2015) 28–39

2.2.5. 1NMR characterizationThe optimized formula was frozen at �20 �C, and then

lyophilized at �45 �C and pressure of 7 � 10�2mbar for 24 h(Novalyphe-NL 500; Savant Instruments Corp., USA). Drug-loadingcharacteristics of the lyophilized LX-loaded nanomicelles wasassessed by recording the 1H NMR spectra of LX, plain nano-micelles and LX-loaded nanomicelles in deuterated chloroform(CDCl3) (Varian 300 MHz NMR spectrometer, Varian Mercury VX-300, CA, USA) at room temperature. For comparison purpose, 1HNMR spectra in deuterated water (D2O) were also assessed (Leeet al., 2003). Chemical shifts were quoted in d, related to that of thesolvents and measured from residual protons in CDCl3 or D2O(Causse et al., 2006).

2.2.6. Physicochemical stability of LX-loaded nanomicellar systemsSelected LX-loaded nanomicellar formulation was stored at

25 �C at ambient conditions in a glass stoppered container for aperiod of 3 months. LX remaining in solution, samples weremeasured as described before, and the % LX was calculated(n = 3 � SD). In addition, the size and size distribution of the drug-loaded nanomicelles were also measured (see above).

2.2.7. Evaluation of micellar stability during membrane sterilizationSelected formulation was filtered through a sterile 0.22 mm

pore size syringe membrane filter. Particle size was measuredbefore and after filtration to check integrity of the micellar system.

2.2.8. In-vivo tolerance assay and histopathological studiesAnimal ethics clearance was obtained from the Ethical

Committee of Faculty of Pharmacy, Cairo University (approvalnumber PI 1381). All animals and biological tissues were handledaccording to standard operating procedures (SOP) of the Univer-sity’s Central Animal Services (CAS) unit. Furthermore, guidelinesof Association for Research in Vision and Ophthalmology (ARVO)resolution on the use of animals in Ophthalmic Research andVision Research (Rockville, MD, USA) were followed. An acutetolerance test was performed on three New Zealand white albino

Table 2Composition of single micellar systems and factorial deign—based mixed micellarsystems.

Formula % of copolymer solution

Tetronic1 701 Synperonic1 PE/P84 Synperonic1 PE/F127

SM-1 100 – –

SM-2 – 100 –

SM-3 – – 100MM-1 75 25 –

MM-2 50 50 –

MM-3 25 75 –

MM-4 75 – 25MM-5 50 – 50MM-6 25 – 75

rabbits of 2.5 kg weight in order to determine the ocular toleranceof the prepared nanomicellar systems. Healthy animals free ofclinically observable abnormalities, housed singly in standardcages, in a light-controlled room (12 h light and 12 h dark cycles) at20–24 �C and 30–75% relative humidity, with no restriction to foodor water.

One drop (0.1 mL) of the selected formulation was instilled inthe one eye, and the other eye was kept as control, receiving onlyphysiological saline. The tested formula onto the rabbit’s corneawas repeatedly applied every hour through a period of 6 h. Botheyes of the rabbits under test were examined for any irritationsigns, such as conjunctival, corneal edema and/or hyperemia onthe basis of direct visual observation using a slit lamp. Evaluation ofirritation was conducted according to a scoring system of 0(absence) to 3 (highest) (Moosa et al., 2014).

Thirty minutes after the last instillation, rabbits were eutha-nized by an intravenous injection of an overdose of sodiumpentobarbital given via a marginal ear vein. The corneas wereisolated, rinsed in physiological saline and subsequently fixed in10% formalin for 24 h. The cornea of one eye receiving of the drugloaded nano-formulation, and the cornea of the control eyereceiving physiological saline were examined. The washing wasdone in tap water then serial dilutions of alcohol (methanol,ethanol and absolute ethanol) were used for dehydration. Speci-mens were cleared in xylene and embedded in paraffin at 56 �C inhot air oven for 24 h. Paraffin beeswax tissue blocks were preparedfor sectioning at 4 mm thickness by sledge microtome. Theobtained tissue sections were collected on glass slides, deparaffi-nized, stained by hematoxylin and eosin stain for examinationthrough the light electric microscope (Banchroft et al., 1996).

2.2.9. Corneal visualization using confocal laser scanning microscopy(CLSM)

Observing the penetration of fluorescently-labeled nanomicel-lar systems within the cornea was performed using Invert CLSM(LSM 510 Meta, Carl Zeiss, Jena, Germany). RhB (a lipophilic dye)was selected to simulate a lipophilic drug incorporated. RhB-loaded nanomicellar systems were fabricated using the samemethod employed for preparing LX-loaded nanomicellar systemswhere RhB, at a concentration of 0.1% (w/w), replaced LX in theselected mixed polymeric nanomicellar formulation. RhB aqueoussolution, at the same concentration was used for comparison.

One drop of RhB-loaded micellar system (or aqueous solution)was applied as mentioned earlier (every hour, 6 times in all). Therabbit was scarified using the same procedure mentioned underthe histopathological section, and then the corneas were isolatedand rinsed in physiological saline. Subsequently, the cornealspecimens were directly mounted on glass slides and intracellularfluorescence was observed by CLSM without additional tissueprocessing. A CLSM system (TCS SP2/AOBS, Leica, Germany), linkedto an inverted microscope with a HCX PL APO 100�, 1.40–0.70 oil


CS objective (DM IRE2, Leica, Germany) was used for confocalimaging. An Ar excitation laser (excitation 485 nm) and a HeNeexcitation laser (excitation 595 nm) were selected for RhB. Opticalcross-sections in the vertical plane were made by scanning in the Xand Z direction, and cross-sections in the horizontal plane weretaken by scanning at different depths in the tissue in the X and Ydirection. Image acquisition and analysis were processed usingLEICA Confocal Software, release 4.2 (Carl Zeiss microimaging,Gottingen, GMBH) (Shamma and Aburahma, 2014).

3. Results and discussion

In order to prepare poloxamer/poloxamine single, and mixedpolymeric micelles, 3 amphiphiles were employed; two hydro-philic amphiphiles: F127 and P84 and a lipophilic poloxaminecomponent (T701 with HLB 1–7). Presence of copolymers withdifferent HLB values could help achieving the maximum thermo-dynamic and kinetic stabilities for the prepared nanomicelles. Itwas reported that low HLB poloxamers would increase thethermodynamic stability of the micelles due to the tighthydrophobic interactions with hydrophobic PO blocks, while highHLB poloxamers would increase the kinetic stability of thenanomicelles (Lee et al., 2011; Abdelbary and Tadros, 2013). Theseamphiphilic carriers were evaluated for their ability to form singleand mixed polymeric nanomicellar systems as nanotechnologyplatform suitable for solubilization, and encapsulation of LX for apotential topical treatment of ocular inflammations. Directequilibrium method was selected for micelle formulation, sinceit is a simple and cost effective technique with improved physicalstability and fulfils the requirements for industrial acceptance.According to Chen et al., direct equilibrium technique was found tobe superior to thin film hydration and solvent hydrationtechniques in the preparation of Triclosan-loaded micellarsystems, where the prepared micellar systems showed higherdrug encapsulation and smaller particle size compared to the othertwo techniques (Chen et al., 2010).

T701 (EO2.1–PO14–EO2.1) is a lipophilic polymer with a very lowability to form stable micelles in aqueous media. The longhydrophobic PO block of T701 enhance its aggregation in anaqueous environment due to the hydrophobic interactions, whilethe short hydrophilic ethylene oxide (EO) block of T701 cannotprovide sufficient steric hindrance (Lee et al., 2011). Therefore, theaddition of a more hydrophilic derivative is essential. Addition ofthe highly hydrophilic F127 (EO100–PO65–EO100, HLB 22), havinglong EO chains, to the more hydrophobic Pluronic1 L121 wasreported previously. This mixture enabled the production of small,spherical and highly stable mixed nanomicellar dispersion(Alexandridis et al., 1994). Poloxamers were also previously mixedwith poloxamines to improve the physical stability of efavirenz-loaded micelles (Chiappetta et al., 2010a,b). Therefore, in our studywe included F127 and P84 as representatives of high HLB

Table 3Micellar size (Dh), size distribution and polydispersity index (PDI) of single and mixed coconcentration was 10% w/v).

100% T701 100% P84 100% F127 T 701: P84

75% T701 5

25 �C 37 �C 25 �C 37 �C 25 �C 37 �C 25 �C 37 �C 2

Peak 1 Dh (nm) 1383.0 695.7 20.6 21.6 6.9 31.4 458.9 336.5

% 81.7 66.3 73.4 75.4 49.4 49.8 73.7 99.4

Peak 2 Dh (nm) 109.2 149.1 190.3 4.0 36.8 7.1 121.5 4925.0 4% 18.3 33.0 14.7 15.4 34.3 40.0 19.0 1.3

Peak 3 Dh (nm) – 5371.0 396.3 5059.0 391.4 350.7 4957.5 – –

% – 1.4 8.8 4.6 13.2 7.2 7.3 – –

PDI 1.0 0.5 0.4 0.3 0.3 0.3 0.5 0.2

amphiphilic carriers in order to elucidate the role of this parameteron the formation and stabilization of a hydrophobic drug as LX innanomicellar systems.

3.1. Studies on drug free micelles

3.1.1. Effect of preparation temperature on the size of polymericnanomicellar systems

At 25 �C, T701 displayed a bimodal appearance (Table 3). Themajor peak (Dh = 1383 nm, 81.7%) corresponds to polymericmicelles, while the minor peak fraction (Dh = 109.2 nm, 18.3%)represents unimers or incomplete aggregates.

Addition of the hydrophilic amphiphile (P84/F127) resulted in asignificantly smaller Dh as compared to pure T701 nanomicelles,with sizes gradually shrinking for increasing the hydrophiliccomponent concentration (T701:P84 and T701: F127 mixed nano-micelles showed major peak Dh between 20.62–458.9, and 205–344 nm, respectively). This could be attributed to the formation ofkinetically stable spherical micelles produced by the presence ofthe hydrophilic amphiphiles that stabilize and regulate thehydrophobic interactions of PO blocks of T701.

At 37 �C, the minor peak fraction was greatly minimized(Table 3). This observation ensures that the micelles arepredominantly formed with the temperature rise, due to thedehydration of PO and EO blocks (Chiappetta et al., 2010a,b). Thisprocess promotes the formation of more micelles (Perreur et al.,2001). The PDI values were found to decrease with the tempera-ture increase. The major peak corresponding to micelles indicatesthat the unimers closely associate with micelles, as reported forother copolymers (Barbosa et al., 2007; Chiappetta et al., 2010a,b).Table 3 shows that at 37 �C, all the mixed polymeric micellarsystems (with the exception of 25:75 ratio for both T701:P84, andT701:F127) displayed monomodal size distributions with smallPDI-values between 0.10 and 0.54. This was supported by theabsence of small sized unimers.

It could be noticed that the single nanomicellar systems(F127 or P84) were having the lowest particle size-values. Thismight be attributed to the polar and semi-polar nature of F127 andP84, respectively, which could enable them to form relatively smallmicelles in water. Similar results were obtained in previous studiesutilizing Pluronic1 P123 alone or in a mixture with Pluronic1

F127 for the micellar solubilization of paclitaxel, whereas theparticle size was nearly 20 nm (Han et al., 2006; Wei et al., 2009).On the other hand, presence of hydrophobic component, T701, inthe mixed nanomicellar systems led to a significant increase in theparticle size (Table 3). These results are in accordance with thepreviously noticed findings by AbdElbary and Tadros, whoprepared mixed micelles containing different concentrations ofPluronic1 L121 and Pluronic1 P123 for brain targeting ofolanzapine (Abdelbary and Tadros, 2013).

polymeric nanomicellar carriers prepared at 25 �C, and 37 �C. (The final copolymer

T 701: F127

0% T701 25% T701 75% T701 50% T701 25% T701

5 �C 37 �C 25 �C 37 �C 25 �C 37 �C 25 �C 37 �C 25 �C 37 �C

89.4 169.5 20.6 29.9 205.4 419.9 344.6 275.1 297.3 236.097.5 100.0 68.5 89.8 99.3 94.0 92.2 98.8 86.5 89.2

633.0 – 586.5 214.0 5163.0 4588.0 4582.5 4758.0 29.0 31.32.5 – 18.6 9.2 1.4 6.0 7.9 2.4 7.8 10.9

– 3.8 5368.0 – – – – 4659.5 –

– 10.0 2.2 – – – – 5.8 –

0.3 0.1 0.5 0.3 0.2 0.3 0.4 0.2 0.5 0.4

Table 4Measured parameters for blank and LX-loaded micelles.

Formula Blank micelles LX-loaded micelles R2 results of fitting the release data to kinetic model

CP (�C) % T Sa (mg/mL) Fs % released after 6 h Zero First Diffusion Release order

SM-1 Less than RT* 0.18 0.41 14.76 55.16 0.994057 0.979562 0.966644 ZeroSM-2 80 97.01 0.33 136.43 19.46 0.965702 0.987279 0.912183 FirstSM-3 More than RT 97.79 0.28 73.57 42.23 0.991672 0.983468 0.953825 ZeroMM-1 Less than RT 31.64 4.49 46.31 23.57 0.992201 0.951813 0.936634 ZeroMM-2 48 95.83 2.34 12.64 38.91 0.960777 0.906757 0.975491 DiffusionMM-3 64 96.69 1.62 14.76 31.36 0.973847 0.896373 0.986573 DiffusionMM-4 Less than RT 13.88 2.75 77.29 13.29 0.995251 0.929493 0.968494 ZeroMM-5 Less than RT 23.35 2.30 72.50 33.04 0.980353 0.913518 0.985573 DiffusionMM-6 75 98.02 1.25 49.57 32.25 0.967904 0.892019 0.976216 Diffusion

*RT: Room temperature


3.1.2. Cloud pointCP of mixed polymeric nanomicellar dispersion depends on the

inter-micellar interactions. The clouding behavior of mixedpolymeric nanomicelles will be totally different from the singlenanomicelles. Cardoso da Silva and Loh studied the CP behavior ofdifferent co-polymeric nanocarriers and have reported theformation of mixed micelles based on single value of CP observedin these systems(da Silva and Loh, 1998).

Table 4 shows values of CP of single and mixed micellarformulations. The turbidity of 10% micellar solutions was moni-tored with temperature rise in order to determine the CP (Xiuliet al., 2004). T701 exhibited phase separation at a very lowtemperature (<25 �C) compared to the more hydrophilic polox-amers (F127 or P84) (�70 �C; Table 4). This is in accordance withthe polymer monograph, where CP-values for T701, P84, andF127 were 16–19�, 71�, and 105 �C, respectively. In addition, CPvalues of mixed polymeric nanomicelles were always smallercompared to those of the single hydrophilic poloxamers (Table 4).This may be attributed to the formation of a more hydrophobicsystem on addition of T701. Previous researchers reported thesame findings elsewhere in the literature (Chiappetta et al., 2011;Ribeiro et al., 2012; Ribeiro et al., 2013).

3.2. Analysis of factorial design

A full 21.31 factorial design selected for planning andoptimization of experimental series. Factorial designs are com-monly used to study the effect of different variables on theproperties of a drug loaded formulations (Araujo et al., 2010).Comparing the value of predicted R2 with the adjusted R2 canmeasure how good the model could predict a response (Annaduraiet al., 2008). As shown in Table 5, the predicted R2 values werefound to be in good agreement with the adjusted R2 in all responses(approximately 0.2 difference between them) (Quinten et al.,2009; Basalious et al., 2011). Adequate precision measured thesignal-to-noise ratio to ensure that the model can be used toevaluate the design space (Lima et al., 2011). A ratio greater than 4(the desirable value) was observed in all responses.

ANOVA test was performed to evaluate the level of significanceof the tested factors on the peak % transmittance (Y1), particle size

Table 5Output data of the 21.31factorial analysis of LX – loaded nanomicellar systems.

Responses R2 Adjusted R2 Predicted R2

Y1: % T 0.8973 0.8117 0.5891

Y2: PS 0.7029 0.4553 -0.1885

Y3: Sa 0.9814 0.966 0.9257

Y4: % released after 6 h 0.9323 0.8759 0.7292

and PDI (Y2), Sa (Y3), and the % drug released after 6 h (Y4) as well asthe interactions between these factors.

3.2.1. Influence of formulation variables on % transmittance andparticle size

The appearance of turbidity (low % T) in the system is usuallyrecognized as a result of transient separation of particles (Barlowet al., 1996; Kulthe et al., 2011). Therefore, measurement ofturbidity can indicate the presence of large aggregates in thesystem(Kulthe et al., 2011). As shown in Table 4 and Fig. 1a,T701 single micelles formed a very cloudy dispersion (% T = 0.18%).This is in accordance with the particle size analysis, whereT701 single micelles had a large droplet size (1383 nm, Table 3).This turbidity was significantly reduced by increasing either P84 orF127 content in the mixed micellar system. The increase in % T andthe reduction of particle size of T701 dispersions as a result ofincreasing the hydrophilic component (P84 or F127) indicates thatthe growth of T701 large aggregates was hindered. This is in goodagreement with the results obtained by Abdelbary and Tadros,(2013), and Kulthe et al., 2011, in a study on the formation of mixedmicelles using hydrophobic and hydrophilic Pluronic copolymers.Similar results were also obtained by Lee et al. 2011 in a study onthe preparation of binary micelle systems of Pluronic1 L121 andPluronic1 P123. They reported that the decrease in turbidity of themixed micellar systems with increasing the hydrophilic compo-nent; Pluronic1 P123, in the system might have resulted from theconversion of the lamellar structure of the Pluronic1

L121 dispersion to the spherical structure of the mixed nano-micellar dispersion.

3.2.2. Influence of formulation variables on the micellar size and sizedistribution

ANOVA results show that only the type of Synperonic1 had asignificant effect on the micellar particle size (p = 0.0242). Particlesize of mixed nanomicellar systems prepared using F127 weresignificantly larger than those prepared using P84 (Fig. 1b).However, changing the % T701 had no significant impact on the PSof LX nanomicellar systems (p > 0.05).

PCS measurements revealed that the degree of polydispersity ofthe mixed micelles ranged from 0.10 to 0.54. According to Araujoet al., 2009 a PDI value of 0 indicates highly mono dispersed sample

Adequate precision Significant factors (p value)

7.148 X2 (0.0021)4.439 X1 (0.0242)

22.764 X1 (0.0009), X2 (<0.0001), X1X2 (0.0022)11.115 X2 (0.0005), X1X2 (0.0271)

Fig. 1. Line charts (a and b), and response surface plots showing the effect of different formulation variables on the properties (c and d), and on the desirability (e) of theprepared nanomicellar formulations.


Fig. 2. Release profiles of LX-loaded single (a) and mixed (b) nanomicellar carriers.

Fig. 3. Transmission electron micrographs of the optimized formulation, atdifferent magnifications.


whereas PDI � 1 indicates highly poly dispersed systems (Araujoet al., 2009).

3.2.3. Influence of formulation variables on encapsulation of LXAn important property of micelles is their ability to host poorly

water-soluble active compounds (Ribeiro et al., 2013). ANOVAresults show that both variables (type of Synperonic1 and theconcentration of the Tetronic1 in the micellar mixture) as well astheir interaction had a significant effect on LX encapsulation (Sa)within the polymeric nanomicelles (p = 0.0009, <0.0001, and0.0022, respectively) (Table 5).

All mixed nanomicellar systems led to sharp increases in drugsolubility (Table 4), at least seven-fold compared with LX aqueoussolubility (0.0318 mg/mL). T701 single micelles increased up to14.76 times the apparent solubility of the drug. Solubilizing abilityof the used polymer is ranked in the order: T701 � P84 > F127. Theaqueous drug solubility is inversely proportional with the HLBvalues of the polymeric mixed micelles (Fig. 1c). The lower the HLBvalues, the greater the increase in LX apparent solubility is. This isin accordance with the results obtained by Riberio et al., in a studyon using single and mixed poloxamine nanomicelles for ethox-ozolamide solubilization (Ribeiro et al., 2012).

T701 containing mixed micelles showed a statistically signifi-cant higher (p < 0.05) encapsulation capacity as compared to theirsingle micellar systems. For example, the ratio 75:25 of T701:P84 increased the Sa136.43 times, while the pure T701 andP84 micellar systems increased the Sa 14.76, and 12.64 times,respectively. The greater the T701 content, the higher theencapsulation extent observed; e.g., the fs-values for 75:25, and50:50 ratios of T701:P84 systems were 136.43 and 73.57,respectively (Table 4).

3.2.4. Influence of formulation variables on in-vitro release of LXThe in-vitro release experiment was carried out in order to

elucidate LX release profile from the nanomicellar systems. Fig. 2shows detailed plots of LX release profiles. Values of % drugreleased after 6 h and ANOVA results are depicted in Tables 4 and 5,respectively. Although all micellar systems provided a sustaineddrug release, the highest values for both % drug released after 6 hand release efficiency were observed in the case of the singlemicelles and were in order: P84 > T701 > F127. This behaviorindicates that the greater the hydrophilicity of the copolymer,the smaller is the micellar capability of the nanomicellar system toretain the drug within their core. In other words, the higher % drugreleased at 6 h value of T701 compared to that F127 may be relatedto the unstable lamellar structures provided by the hydrophobicpolymer (T701). Interestingly, mixed micelles showed intermedi-ate release percentages, presenting an additional feature thatenables us to fine tune the release rate. Remarkable differences inboth % drug released after 6 h was observed upon changing eitherthe Synperonic1 type or its ratio to T701 (p < 0.05) (Fig. 1d). Aspreviously demonstrated in the single micellar system, the morehydrophilic nature of P84 had a significant role in enhancing LXrelease from the mixed micellar systems as compared to thosecontaining F127. Moreover, the ratio of T701 to either Synperonic1

had a significant effect on LX release with the highest values alwaysbeing achieved when the ratio is 1:1.

Finally, it would be suggested that presenting the suggestednanomicellar system offering sustained drug release can offer agreat advance in formulating LX for the ocular application. Servingas reservoirs with sustained drug release property, polymericmicelles can remain in the apical zone of the eye or slowlypenetrate inside the cornea, releasing the drug over time, aspreviously demonstrated by other micellar carriers (Ribeiro et al.,2012), i.e., once formulated in poloxamine micelles, LX could beabsorbed either in its free form or encapsulated in the carrier.

The release data obtained from the investigated micellarsystems were analyzed using zero-order, first-order, and Higuchidiffusion models in order to determine the mechanism of LXrelease. The in-vitro release of LX was found to follow zero-orderrelease kinetics in case of mixed micelles systems with the lowestconcentration of T701 (25%), whereas other mixed micellarsystems containing equal or higher concentration of Tetronic1

followed diffusion release kinetics.

Fig. 4. The 1H NMR spectra of LX in CDCl3 (a), LX-free mixed micells in CDCl3 (b) and LX-loaded mixed micells in CDCl3 (c) and D2O (d).



3.3. Optimization and validation of LX-loaded nanomicellar systems

Optimization of all independent variables at the same time isvery hard as the optimum condition for one response might haveopposite effect on the another (Singh et al., 2012). Therefore, thedesirability function approach is widely used in researches for theoptimization of multiple response processes. The desirabilityfunction combines all the responses into one variable to predict theoptimum levels for the studied factors (Pandya et al., 2011).Accordingly, desirability was calculated to select the optimizedformula with the least PS along with the highest drug entrapment,% T and % LX released after 6 h (as absolute value). The highestdesirability value (0.681) was depicted in the formulationencompassing 50% T701 together with P84 (Fig. 1e). Hence, thisformula was selected for further investigation.

3.4. TEM microscopy

TEM images of the optimized LX-loaded nanomicellar systemconfirmed that the particles were non-aggregated and spherical inshape with narrow size distribution (Fig. 3). It was observed from

Fig. 5. (a) Eye of rabbit in group 1 showing normal histological structure of the ciliarinflammatory cells infilteration underneath the conjuctiva and the sclera.

different frames that only a monomodal size species was presentthroughout the investigated sample. The mean size of the particlesobserved in the TEM micrographs was in good agreement with thesize obtained from particle size analyzer (Table 3).

3.5. 1HNMR

In order to further confirm the solubilization and entrapment ofLX into the micellar core, LX-loaded T701:P84 micelles wereanalyzed by 1H NMR (Fig. 4). The drug loading into the inner POcore of the mixed T701:P84 micelle was confirmed by the analysisof 1H NMR spectra. Fig. 4 shows the 1H NMR spectra of LX in CDCl3(a), free mixed nanomicelles in CDCl3 (b) loaded mixed nano-micelles in CDCl3 (c) and D2O (d). As shown in Fig. 4, the resonancepeaks corresponding to LX as well as T701:P84 were clearlyobserved in CDCl3, whereas LX characteristic peaks disappeared inD2O, while those related to T701:P84 were still detected. Thesefindings suggest successful LX solubilization and entrapment intothe hydrophobic PO inner core of the mixed polymeric micelles(Wei et al., 2009). As reported by Lee et al., 2003, these resultsindicate the development of core-shell type micellar system.

y body, sclera, and extention from the conjuctiva, (b) eye of rabbit showing local


3.6. Physicochemical stability of LX-loaded nanomicellar systems

The stability of ophthalmic dosage forms is important to detectthe shelf life and expiration date of the product (Ali andLehmussaari, 2006). The optimized LX—loaded nanomicellarcarrier system remained clear over a period of 3 months. Theparticle size was not significantly changed after this storage period(p > 0.05).

It has been clarified that polymeric micelles possess highstructural stability provided by the entanglement of copolymerchains in the inner core. Hence, polymeric micelles can befabricated by simple techniques with better physical stability incomparison to other nanomicellar carrier systems.

3.7. Evaluation of micellar stability during membrane sterilization

An essential requirement for the eye drops is being sterile. Themajor benefit of polymeric nanomicelles is the ease of theirsterilization in pharmaceutical productions. Polymeric micellescan be simply and inexpensively sterilized by filtration usingtypical sterilization membrane filters with 0.45 mm or 0.22 mmpores. In contrast, other typical pharmaceutical nanocarriersystems (niosomes, liposomes) need a removal of contaminatedmicron sized particles (Yokoyama, 2011). The optimum micellarformulation was subjected to filtration using a sterile syringemembrane filter with 0.22 mm pore size. A check of the micellarsize was done before and after filtration to ensure the efficiency ofthe method and maintenance of micelles integrity. No significantchange (p > 0.05) in particle size or PDI was observed before andafter filtration was noticed (PS = 177 and PDI = 0.456 vs 168 and0.416, respectively).

Fig. 6. CLSM z-stack of the corneal epithelia separated from the rabbit eye trated with

increments.

3.8. In-vivo ocular irritation test and histopathological studies

The unique characteristics and high sensitivity of the eye tissuesimpose distinctive safety requirements and great restrictions onthe selection of the components that can be used in the topicalocular preparations. The clinical acceptability of topically appliedocular micellar systems may be limited because of the polymericsurfactants' ability to affect the integrity of epithelial surfacespotentially causing irritation and inflammatory changes (burning,stinging, corneal opacity, conjunctival redness or chemosis, anddischarge), that may provide a reason for patients to stop theirmedication. Polymeric surfactants are known to be safer than low-molecular-weight surfactants (Yokoyama, 2011). Polymeric non-ionic surfactants are relatively harmless to the eye in comparisonto the cationic, anionic, or amphoteric counterparts. Polyoxy-ethylated nonionic surfactants have found widespread applica-tions in ophthalmics among nonionic surfactants. Some polyoxy-ethylated nonionic surfactants have a long history of being safe inophthalmic use (Pepic et al., 2012).

Accordingly, the potential ocular adverse and/or damagingeffects of the ocular nanomicellar systems under investigationwere evaluated by observing the conjunctiva and cornea of rabbits’eyes at specific time intervals after ocular insert administration.

3.8.1. Ocular irritationRabbits’ eyes were examined physically for the presence of any

irritation signs. An irritation score of 0 for drug-loaded formulationwas obtained. Remarkably, the optimized formulation wasvirtually non-irritant even with repeated instillation. The resultsshowed that the tested formulation showed no sign of redness orinflammation or increased tear production proving the safety of

RhB-labeled nonomicellar systems, and sectioned from 0 mm to 18 mm with 1 mm


the used non-ionic surfactants to be applied topically in the eye. Itis a pre-requisite for ocular systems to have no harmful effect onthe eye, and the used surfactants showed a good suitability for theintended application.

3.8.2. Histopathological studiesHistopathological investigations were performed to assess the

safety of the LX-loaded nanomicellar system on the eye surface.The corneal topographical findings from the optimized formula-tion and control specimens from the rabbits’ eyes were recorded.As depicted in Fig. 5, the cornea treated with PBS, as well as the onetreated with LX-loaded nanomicellar system showed normalhistological structures characterized by absence of defects orinflammation. The corneal epithelium and endothelium were bothintact, while the stromal compartments were similar to that ofnormal corneal tissue. However, there was minimal markedchanges and irritation following the instillation of the optimizedmicellar formulation. The presence of both heterophils andheterophils’ exocytosis into the conjunctival epithelium in mini-mal amount indicate sub-acute inflammation. These resultsindicate that the fabricated LX-loaded nanomicelles could besuitable for ocular applications.

3.9. Corneal visualization using CLSM

Thirty minutes after the last administration of the formulationinto rabbits’ eyes, corneal samples were examined using CLSM.Fig. 6 depicts the acquisition of 18 optical sections (x–y plane)taken at successive focal planes along the z axis to obtain three-dimensional information about the penetration of the RhB-labelednanomicelles within the corneal epithelia. Fig. 7 shows a plot of themean fluorescence intensities against the z-slices depth of cornealepithelia exposed in-vivo to RhB solution and RhB-labeled nano-micellar systems. It is depicted that the dye fluorescence wasmaintained until a depth of 18 mm within the corneal epithelia. Anenhanced dye penetration into the viable corneal tissue was foundafter application of either the nanomicellar system or the dyesolution. Data evaluation through picture analysis allowed for aquantitative analysis of absolute dye penetration. In both cases, thepassive transfer of RhB from the administered drops into cellsbrought about its repartition into lipophilic cell compartments(e.g., lipid membranes and subcellular vesicles). Compared to dyesolution, the penetration of RhB into the viable cornea wasincreased following the micellar application (Fig. 7). A greaterdifference in the penetration capacity was expected between themiceller solution and the plain dye solution. This can be explained

Fig. 7. Mean fluorescence intensities of Rhodamine B isothiocyanate across thecorneal depth.

by the transfer of the fluorescent marker RhB into corneal cellsinstead of particle uptake. This is in accordance with Tian et al.,2013 who found no obvious differences between eyes treated withRhB-labeled nanoparticles and the solution of pure dye alone. Aprevious report by Haynes and Cho, (1988) suggested that thetransfer of lipophilic markers from nanoparticles into cells mayoccur by different processes like phagocytosis, or by partitioning ofthe free marker into the cells after its dissociation from theparticles. Another process was also postulated, which is collisioninduced non phagocytic process through partitioning of a part ofthe entrapped material from the surface of donor particles directlyinto the cells.

4. Conclusion

Nanomicelles were fabricated by simple and cost effectivetechniques with improved physical stability which fulfills therequirements for industrial acceptance, in addition to greaterpatient compliance due to the ease of application without blurringof vision and discomfort. In this study, polymeric micellesincluding LX were fabricated using a simple method with a meandiameter of 169.45 nm. Solubility of LX was increased �73-foldafter encapsulation in the optimum formulation with about 60% ofdrug was released within 6 h. These promising values of particlesize and solubility paved the way for further investigation for theocular tolerance in rabbit eyes. Fabricated micelles showed theabsence of physical irritation on rabbit's eye, while histopatholog-ical studies revealed subacute inflammation after repetitiveapplications within short time. The normal microstructure ofcornea tissue and retina can guarantee the safety of the fabricatedmicelles for ocular application.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of the paper.

Acknowledgement

The authors would like to thank Professor Dr. Adel Bakir(Pathology Department, Faculty of Veterinary Medicine, CairoUniversity, Egypt) for his great help in the histopathologicalevaluation of the corneal tissue.

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