Polyelectrolyte stabilized multilayered liposomes for oral delivery of paclitaxel

Biomaterials 33 (2012) 6758e6768

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Polyelectrolyte stabilized multilayered liposomes for oral delivery of paclitaxelq Sanyog Jain*, Dinesh Kumar, Nitin K. Swarnakar, Kaushik Thanki Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), Punjab 160062, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2012 Accepted 13 May 2012 Available online 29 June 2012

Paclitaxel (PTX) loaded layersome formulations were prepared using layer-by-layer assembly of the polyelectrolytes over liposomes. Stearyl amine was utilized to provide positive charge to the liposomes, which were subsequently coated with anionic polymer polyacrylic acid (PAA) followed by coating of cationic polymer polyallylamine hydrochloride (PAH). Optimization of various process variables were carried out and optimized formulation was found to have particle size of 226
17.61 nm, PDI of 0.343
0.070, zeta potential of þ39.9
3.79 mV and encapsulation efficiency of 71.91
3.16%. The developed formulation was further subjected to lyophilization using a universal stepwise freeze drying cycle. The lyophilized formulation was found to be stable in simulated gastrointestinal fluids and at accelerated stability conditions. In vitro drug release studies revealed that layersome formulation was able to sustain the drug release for 24 h; release pattern being Higuchi kinetics. Furthermore, cell culture experiments showed higher uptake of layersomes from lung adenocarcinoma (A549) cell lines as compared to free drug. This was subsequently corroborated by MTT assay, which revealed IC50 value of 29.37 mg/ml for developed layersome formulation in contrast to 35.42 mg/ml for free drug. The in vivo pharmacokinetics studies revealed about 4.07 fold increase in the overall oral bioavailability of PTX as compared to that of free drug. In vivo antitumor efficacy in DMBA induced breast tumor model showed significant reduction in the tumor growth as compared to the control and comparable to that of i.v. TaxolÒ. In addition, the toxicity studies were carried out to confirm the safety profile of the developed formulation and it was found to be significantly higher as compared to TaxolÒ. Therefore, the developed formulation strategy can be fruitfully exploited to improve the oral deliverability of difficult-to deliver drugs. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Layersomes Paclitaxel Pharmacokinetics Antitumor efficacy Polyelectrolytes Nephrotoxicity

1. Introduction Paclitaxel is one of the major anticancer drugs with broad spectrum of efficacy in advanced and refractory lung, ovarian and breast cancers and Kaposi’s sarcoma. However, its use is limited due to the associated toxicity contributed equally by the drug and vehicle [1]. Furthermore, the paclitaxel shows very low oral bioavailability (<10%) due to limited aqueous solubility, poor intestinal permeability, P-gp efflux and first pass hepatic metabolism [2]. The advances in the technologies have provided a vast platform for the development of such difficult-to-deliver drugs via oral route. Among lipid based delivery systems, liposomes are most commonly used formulation approaches for delivery of the anticancer drugs [3,4]. These colloidal vesicular structures are

q This work is a part of Indian Patent Application No. 3246/DEL/2011 filed on November 15th, 2011. * Corresponding author. Tel.: þ91 172 2292055; fax: þ91 172 2214692. E-mail addresses: [email protected], [email protected] (S. Jain). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.05.026

biodegradable in nature and are capable of delivering both hydrophilic and lipophilic drugs for variety of diseases including cancer, gene therapy, vaccines, proteins and peptides. With their superior encapsulation efficiency, they are also eligible for targeting capability with decreased toxic effects of the drugs [5]. Very often, the highly sensitive nature of the liposomes, limits its applicability to the variety of the situations. The phospholipids present at the surface are regarded as the culprit for the instability in the biological media and during storage [6,7]. However, meticulous efforts have been taken to stabilize the liposomes upon oral and peroral route, owing to their significant advantages over the other conventional drug delivery systems in race. Modification for its suitability for oral delivery includes alginate coated liposomes, coating with thiolated polymers [8], incorporation of the bioadhesive molecule (chitosan) [9], incorporation of glycocholate [10], using tethered lipids for formulating liposomes [11] and so on. Layer by layer coating of the polyelectrolytes over the liposomes, has recently been implemented for their stabilization [12]. It basically includes the adsorption of the polycations and polyanions on the liposome surface so as to protect the labile surface

S. Jain et al. / Biomaterials 33 (2012) 6758e6768

phospholipids from the body fluids. This coat provides adequate isolation from the outer physiological environment so as to preserve the original structure for efficient delivery of the drugs. These special tailor-made structures exploit advantages of both particulate systems (escaping P-gp efflux, increasing storage stability and robustness) and vesicular systems (effective encapsulation of drug). Further the layer by layer approach renders the process to be easy and inexpensive, thereby allowing variety of materials to be accommodated in to the multilayer structures [13]. This approach also has promising results on the stabilization of the liposomal compositions for oral delivery of the drugs. In our previous report, we had successfully shown the effectiveness of said approach in improving the therapeutic efficacy of amoxicillin and metronidazole in the treatment of Helicobacter pylori infections [14]. The present study focuses on the potential of the multiple polyelectrolyte coated liposomal composition for the oral delivery of the anticancer drug, paclitaxel (PTX). These assemblies were prepared by coating of the anionic polymer polyacrylic acid (PAA) on cationic liposomes, which acts as core. These single layer coated liposomes were further coated by cationic polymer polyallylamine hydrochloride (PAH) to provide adequate stabilization and conservation of the original quality attributes. The developed system was freeze dried using stepwise freeze drying cycle to impart long term stability to the formulation. Furthermore, therapeutic efficacy and toxicity of developed formulation were compared with marketed intravenous products TaxolÒ (Cremophor-EL based hydroalcoholic solution) and NanoxelÔ (PTX complexed albumin nanoparticles) in suitable animal models.

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Table 1 Optimization of process variables for preparation of the paclitaxel liposomes. Sr. No.

Optimization parameters

Variables

Remark

1

Phospholipid to cholesterol ratio (mole ratio)

2

Theoretical drug loading (%w/w)

Theoretical drug loading: 5% w/w Stearyl amine: 5% w/w Sonication time: 3 cycles of 30 s at amplitude 80 with 1 min gap Stearyl amine: 5% w/w Sonication time: 3 cycles of 30 s at amplitude 80 with 1 min gap

3

Sonication time

3:1 2:1 1:1 1:2 5% 10% 15% 20% 15 s 30 s 45 s 60 s

Theoretical drug loading: 5% w/w Stearyl amine: 5% w/w Sonication time: 3 cycles at amplitude 80 with 1 min gap

The optimized values are represented in bold. The polydispersity index (PDI) which is a dimensionless number indicating the width of the size distribution, having a value between 0 and 1 (being 0 for monodispersed particles), was also obtained. 2.4.2. Encapsulation efficiency The percentage of drug incorporated in liposome was determined using direct method [17]. Liposomes were pelletized using centrifugation at 40,000 g for 1 h at  4 C. Methanol was added to dissolve obtained pellet and solubilize the drug. The concentration of PTX was then estimated using validated HPLC method [18] and encapsulation efficiency was calculated as follows. Encapsulation efficiency ¼

Amount of drug in pellet  100 Amount of drug initially taken for preparation of liposomes

2. Materials and methods 2.1. Materials Soya Lecithin (92% phosphatidyl choline) was generously gifted by Cargill, Germany. Paclitaxel was availed as gift sample by Fresenius Kabi, India. Polyallylamine HCl, polyacrylic acid, stearyl amine, trehalose trihydrate, 7,12-dimethylbenz[a] anthracene (DMBA), Trypsin-EDTA, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide), coumarin-6, tween 80 and triton X-100 were procured from Sigma Aldrich, USA. Cholesterol and mannitol were obtained from Himedia labs, India. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (Antibiotic-antimycotic solution) and Hank’s balanced salt solution (HBSS) were purchased from PAA, Austria. Tissue culture plates and 8-well culture slides were procured from Tarsons and BD Falcon, respectively. Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Ranchem Fine Chemicals, India. Ultra-pure water (SG water purification system, Barsbuttel, Germany) was used for all the experiments. All other reagents used were of analytical grade. 2.2. Preparation of PTX loaded liposomes Paclitaxel liposomes were prepared by thin film hydration method with slight modification as per laboratory conditions [15]. Briefly soya lecithin (24.5 mg), cholesterol (11.5 mg), stearyl amine (2 mg) and paclitaxel (2 mg) were dissolved in 20 ml mixture of chloroform and methanol (9:1 v/v) in a round bottom flask and thin film was prepared by evaporating the organic solvents under vacuum. The thin film was further dried in dried in vacuum oven for 4 h to completely remove the traces of organic solvents. It was then hydrated at 40  C for 2 h at 100 rpm. The mixture was then probe sonicated (Mi Sonix, USA) for optimized time in ice bath to get smaller sized liposomes [16]. 2.3. Optimization of process variables Various process variables such as phospholipid to cholesterol ratio, drug loading and sonication time, as mentioned in Table 1 were optimized to get liposomes of desired quality attributes. The characterization parameters included size, polydispersity index (PDI), zeta potential and encapsulation efficiency. 2.4. Characterization of PTX-liposomes 2.4.1. Size, size distribution and zeta potential The size and size distribution of liposomes and layersomes were determined by DLS (dynamic light scattering) (Nano ZS, Malvern Instruments, UK), taking the average of 5 measurements, whereas zeta potential was estimated on the basis of electrophoretic mobility under an electric field, as an average of 20 measurements.

2.5. Polyelectrolyte coating of PTX-liposomes Paclitaxel liposomes were coated with series of polyelectrolytes using layer by layer technique, mediated by ionic interactions. Briefly the cationic liposomes were coated by anionic polyelctrolyte in the first step followed by coating with cationic polyelectrolyte in the second step. Polyacrylic acid (molecular weight w5200) was implemented as the first layering agent on the cationic paclitaxel liposomes (charge imparted by steary lamine). Its concentration and volume was optimized for achieving best possible physicochemical parameters. Optimization studies at different concentrations of PAA (0.01%e1% w/v) in water having different volumes (100 mle1000 ml of each concentration) were carried out by addition to liposomal dispersion (0.5 ml) under constant stirring at 2000 rpm for 1h using magnetic stirrer. They were then subjected to centrifugation at 20,000 g using high speed centrifuge (sigma 3K30) followed by single washing with water. All these formulations were further analyzed for particle size, charge reversal and encapsulation efficiency. These single layer coated liposomes (PAA-PTX-liposomes) were then coated with a cationic polyelectrolyte, polyallylamine hydrochloride (PAH), molecular weight w56,000. Optimization studies at different concentrations of PAH (0.01%e1% w/v) in water having different volumes (100 mle1000 ml of each concentration) were carried out by addition to the PAA-PTX-liposomes (0.5 ml) under constant stirring at 2000 rpm for 1 h using magnetic stirrer. The dispersion was then subjected to centrifugation at 20,000 g using high speed centrifuge (sigma 3K30) followed by single washing using water in order to get final formulation (PAH-PAA-PTX-layersomes). 2.6. Shape and surface morphology The shape and surface morphology of the developed formulations were assessed using transmission electron microscopy, (TEM, Philips, Japan). Samples were negatively stained with 2% aqueous solution of uranyl acetate [19]. The sample was placed on the 400-mesh carbon coated grids and allowed to stand at room temperature for 90 s. Excess sample was removed using filter paper. Then 10 ml of staining solution was placed on the grid, allowed to stand for 60 s and drained. The specimens were viewed under the microscope at an accelerating voltage of 100e200.0 kV. 2.7. Lyophilization All the developed formulations were lyophilized (Vir Tis, Wizard 2.0, New York, USA freeze dryer) using a universal stepwise freeze drying cycle developed and patented by our group [20]. Trehalose, mannitol and sucrose were tried as cryoprotectants; among which mannitol was selected on the basis of optimization studies. The condenser temperature was 60  C and pressure applied in each step was 200 Torr. A volume of 5 ml of all three formulations was filled in 15 ml glass vials and subjected to freeze drying using 5% w/v of mannitol. The freeze dried

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formulations were characterized for the appearance of cake, size, PDI, ease of redispersion and encapsulation efficiency after reconstitution [21]. 2.8. Stability studies 2.8.1. Stability in simulated gastrointestinal fluids Freeze dried PTX-liposomes, PAA-PTX-liposomes and PAA-PAH-PTX-layersomes were evaluated for their stability in different simulated GIT fluids (SGF, pH 1.2 and SIF, pH 6.8) to assess the stability of formulations under various pH and enzymatic conditions. Freeze dried formulations were reconstituted prior to use. The simulated gastric fluid (SGF) comprised of 0.2% NaCl, pepsin, 0.7% and HCl with pH 1.2 while simulated intestinal fluid (SIF) comprised of 0.685 monobasic potassium phosphate, 1% NaOH and 1% pancreatin with pH 6.8. In order to simulate the effect of bile salts, 3 mM sodium taurocholate was added in the SIF [22]. Each ml of the formulations was added to 9 ml of gastric simulated GI fluids. The samples were incubated for 2 h and 6h in SGF and SIF, respectively. The formulations were then evaluated for particle size, PDI, zeta potential and % encapsulation efficiency. 2.8.2. Storage stability Storage stability of all freeze dried formulations was tested at 4  C and at 25  C for six months. The formulations were evaluated for particle size, PDI, zeta potential and % encapsulation efficiency after reconstitution. 2.9. In vitro drug release studies The in vitro drug release from the PTX-liposomes, PAA-PTX-liposomes and PAHPAA-PTX-layersomes was carried out by dialysis membrane method. Activation of dialysis membrane (Sigma, molecular cut off w12,000) was carried out prior to use by washing the membrane under running water for 6 h to remove glycerol. Subsequently, sulfur was removed by treatment with 0.3% sodium sulfide for 1 min, followed by acidification (0.2% sulfuric acid) of membrane [23]. Finally, it was washed with water repeatedly to remove the excess acid. Phosphate buffer (pH 7.4) with 0.1% Tween 80 (to solubilize the released drug) was used as release medium [24,25]. Different formulations (1 ml) were poured in dialysis bags, subsequently suspended in 15 ml of release medium at 37  C in shaking water bath at 100 rpm. The release medium of 500 ml was withdrawn at each sampling time and was replaced with fresh medium of equal quantity. Release study was carried out up to 24 h [26]. Finally, samples were analyzed using validated analytical methods and % cumulative drug release was calculated. 2.10. Cell culture experiment A549 cells (lung adenocarcinoma cell lines), grown in 25 cm2 tissue culture flasks and maintained in 5% CO2 atmosphere at 37  C, were used for cell culture experiments. The cell medium was supplemented with Dulbecco’s Modified Eagle’s culture medium (DMEM), 20% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (PAA, Austria). The cells were harvested in 0.25% trypsinEDTA solution (Sigma) once 90% confluence in the cell culture medium was attained. The cells were then cultured in a 96 well plate (Costars, Corning Incorporated) at a density 50,000 cells/well and incubated overnight for cell attachment to proceed for subsequent studies. 2.10.1. Cell uptake studies Coumarin-6 was co-encapsulated in liposomal formulations for cell uptake studies. The dye was added in the organic phase and formulations were prepared following the optimized protocol as described earlier. A549 cells were seeded in 8well culture slides (BD Falcon) and allowed to attach overnight. The cells were incubated with prepared formulations for 3 h and extracellular particles were removed by washing with HBSS (5X). The cells were observed under the confocal laser microscope (CLSM) (Olympus FV1000).

the animals were kept under standard housing conditions. The animals were randomly distributed into four groups each containing 6 animals. PAH-PAA-PTXlayersomes, PAA-PTX-liposomes, PTX-liposomes and PTX suspension were administered to the animals by oral gavage at a PTX dose of 5 mg/kg body weight of the animal [29]. The freeze dried formulations were suitably reconstituted to achieve the desired drug dose in 0.5 ml volume, which was administered to animals. 2.11.2. Extraction and quantification of PTX in plasma samples The blood samples (w500 ml) were collected from the retro-orbital plexus under mild anesthesia into heparinized micro-centrifuge tubes (containing 30 ml of 1000 U of heparin). Plasma was separated by centrifuging the blood samples at 3000 rcf for 5 min at 15  C. To 125 ml of plasma, 25 ml of internal standard (1-amino 4-nitro naphthalene) was added and then vortexed for 60 s. Afterwards 500 ml of methanol was added to precipitate proteins and vortexed for 5 min and centrifuged at 5000 g for 10 min. Supernatant was taken and dried in vaccum oven at 40  C. Dried samples were then redispersed in 100 ml methanol and vortexed. The supernatants were separated and analyzed for drug content by validated RP-HPLC method. Calibration curves were designed over the concentration range of 25e1000 ng/ml (r2 0.998) and were used for the conversion of the PTX/1-amino 4-nitro naphthalene chromatographic area to the concentration of PTX. Mobile phase employed for analysis was the mixture of acetonitrile and phosphoric acid solution (0.01%) (50:50). Retention time of PTX and internal standard was found to be 7.1 and 4.5 min respectively. The detection wavelength (lmax) set for PTX was at 227 nm and the column was kept at room temperature. 2.11.3. Pharmacokinetic data analysis The plasma concentrationetime data was analyzed by one compartmental model, using Kinetica software (Thermo scientific). Required pharmacokinetic parameters like total area under the curve (AUC) 0N, terminal phase half life (t1/2), peak plasma concentration (Cmax) and time to reach the maximum plasma concentration (Tmax) were determined. The relative bioavailability of PTX after oral administration was calculated as follows: Relative bioavailability ¼

AUCðPAH  PAA  PTX-layersomesÞ  100 AUCðPTX SuspensionÞ

2.12. In vivo antitumor efficacy Female Sprague Dawley (SD) rats of 200e250 g were used for the induction of chemical induced breast cancer. 7,12-dimethylbenz anthracene (DMBA) in soya bean oil was administered orally to rats at 45 mg/kg dose at weekly interval for three consecutive weeks [25,30]. The tumor bearing animals were separated and divided randomly into six treatment groups. The tumor width (w) and length (l) were recorded with an electronic digital calliper and tumor size was calculated using the formula (l  w2/2). Drug treatment was started after 10 weeks of the last dose of DMBA introduced. Animals were treated with a single dose of PTX suspension (oral), PAA-PTX-liposomes (oral), PAH-PAA-PTX-layersomes (oral), TaxolÒ (i.v.), and NanoxelÔ (i.v.) all in a dose equivalent to 5 mg/kg body weight, of PTX. The positive control group received a same repeated oral administration of saline. The tumor size was calculated as (l  w2/2). The tumor size was measured up to 15 days (during the treatment period). Further the animals were observed for survival till 60 days and Kaplan Meier survival plot was constructed. 2.13. Toxicity studies

2.10.2. MTT assay The cultured cells, washed with Hank’s Buffered Salt (HBS) Solution (PAA, Austria) for three times, were taken for MTT assay. After removing the HBS solution from the plates, cells were incubated with 0.2 ml of fresh DMEM containing free PTX, PTX-liposomes and PAH-PAA-PTX-layersomes at concentrations 0.1, 1, 10 and 100 mg/ ml for 24 or 72 h. Cell viability was then determined by MTT assay [27]. Briefly, cells were washed with HBS solution and incubated again with 0.2 ml fresh DMEM containing 0.5 mg/ml MTT (Sigma, USA) for 3 h. The medium was then removed and MTT formazon was dissolved in 0.2 ml dimethylsulfoxide. The optical density was then determined at 550 nm using an ELISA plate reader (BioTek, USA) [28].

In order to address the toxicity issues pertaining to use of PAH-PAA-PTX-layersomes, biochemical parameters were determined in normal mice. Swiss mice (female) weighing w25 g were procured from Central Animal Facility (CAF) of NIPER, India. The mice were randomly divided into six groups (6 mice per group) e control, PTX-liposomes, PAA-PTX-liposomes, PAH-PAA-PTX-layersomes, TaxolÒ and NanoxelÔ. All formulations were administered intravenously at the dose of 5 mg/kg. The mice were kept in standard housing conditions. All animal experiments were performed in compliance with the institutional ethics committee regulations and guidelines on animal welfare. After 7 days, blood samples were collected in heparinized microcentrifuge tubes and centrifuged at 3000 rcf for 5 min to separate plasma. The plasma was then analyzed for the levels of various toxicity markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), plasma urea and creatinine. All the biochemical parameters were determined spectrophotometrically according to the manufacturer’s guidelines for the diagnostic kits obtained from Accurex (Accurex biomedical Pvt. Ltd., India).

2.11. In vivo pharmacokinetic studies

2.14. Statistical analysis

2.11.1. Dosing protocol Female Sprague Dawley (SD) rats of 200e250 g were supplied by the central animal facility (CAF), National Institute of Pharmaceutical Education & Research (NIPER), India. All the animal studies protocols were duly approved by the Institutional Animal Ethics Committee (IAEC), NIPER, India. Throughout the experiment,

All the data are expressed as mean
standard deviation (SD) for all in-vitro and mean
standard error of mean (SEM) for all in-vivo results. Statistical analysis was performed using SigmaStat (version 3.5) utilizing one-way ANOVA followed by TukeyeKramer multiple comparison test. p < 0.05 was considered as statistically significant difference.

S. Jain et al. / Biomaterials 33 (2012) 6758e6768

3. Results

6761

Table 3 Effect of drug loading on PTX-liposomes.

3.1. Preparation and optimization of PTX-liposomes and layersomes

Drug loading (%w/w)

Size (nm)

3.1.1. Effect of phospholipid to cholesterol molar ratio As evident from Table 2, the ratio of phospholipid to cholesterol has a significant role in the formulation of PTX-liposomes. Significant decrease in the particle size and PDI with decrease in the phospholipid concentration was observed whereas a concomitant increase in the encapsulation efficiency was noted. However, it was also noted that any increase in the concentration of cholesterol greater than phospholipid led to undesirable quality attributes of prepared liposomes. The encapsulation efficiency decreased drastically at higher concentration of cholesterol. Zeta potential in all the cases was maintained on the positive side owing to the charge imparted by stearyl amine.

2.5% 5% 10% 15% 20%

109 117 187 233 359


05.14 ± 06.35
08.67
23.72
21.29

PDI

Zeta potential (mV)

% Encapsulation efficiency

0.273
0.029 0.298 ± 0.032 0.417
0.020 0.361
0.079 0.576
0.076

þ47.9
4.1 D48.4 ± 4.6 þ48.8
3.2 þ50.4
5.7 þ53.6
5.2

84.07
2.91 81.03 ± 3.34 89.91
1.67 65.37
3.04 30.82
7.65

Values are expressed as Mean
SD (n ¼ 6). The optimized values are represented in bold.

concentration at volumes of 100 mle500 ml. However, at the concentration above 0.1%, satisfactory charge reversal (w30 mV) was observed at volumes ranging from 100 to 1000 ml. Furthermore, the particle size was also in the acceptable order of about 200 nm. However, PDI was rate limiting in this case and the acceptable (w0.2) only in the range of 400e500 ml at concentration of 0.1% PAA. Upon increasing the concentration of PAA above 0.6%, satisfactory charge reversal was found and both resultant size and PDI were in unacceptable range, hence were omitted from the selection criteria. Therefore, the acceptable range was found from 0.08% to 0.12% in the volume range of 400e500 ml and finally 0.1% (w/v) at volume of 400 ml was selected for subsequent coating. The final parameters of PAA-PTX-liposomes are shown in Table 5.

3.1.2. Effect of drug loading Table 3 reveals the effect of drug loading on the various quality attributes of the prepared liposomes. Significant increases in the particle size and PDI, whereas proportionate decrease in encapsulation efficiency was observed with increase in the drug loading. Although higher encapsulation efficiency was observed in case of 10% w/w drug loading, the formulation was found to be unstable and precipitated within 2 h of its preparation. Similar results were also obtained in case of 7.5% w/w drug loading (data not shown). A steep fall in the encapsulation efficiency was observed at higher concentration of drug loading which was indicative of inability to withhold drug molecules within the formed system.

3.2.2. PAH coating PAH was implemented as the cationic polyelectrolyte polymer for coating of anionically charged PAA-PTX-liposomes. Fig. 2 reveals the effect of strength of PAH solution on the zeta potential, particle size and PDI of PAA-PTX-liposomes. At lower and volume of PAH, partial charge reversal was obtained with precipitation upon storage. The frequency of precipitation was much higher in this case as obtained previously. This phenomenon was found in the range of 0.01%e0.08% (w/v) concentration at volumes of 100 mle400 ml. The particle size and PDI were also unacceptable in this range. However, increments in the PAH strength led to complete charge reversal (w30 mV) with satisfactory particle size (w200 nm) and PDI (w0.3). This phenomenon was observed in the range of 0.1%e0.2% (w/v) at volume of 500e800 ml. Further increments in the PAH led to much detrimental effects on PDI while zeta potential and particle size were marginally affected. The region with acceptable quality attributes was re-evaluated and finally 500 ml of 0.1% (w/v) PAH was selected for coating purpose. This formulation was then characterized and used for further studies. The final parameters of PAHPAA-PTX-layersomes are shown in Table 5.

3.1.3. Effect of sonication time Sonication time was considered as an important parameter for optimization of the liposomes and it was found to have significant impact on the quality attributes of prepared formulations. As evident from Table 4, a significant decrease (p < 0.05) in the particle size was observed with increase in the sonication time but concomitant decrease in the encapsulation efficiency was also seen simultaneously. Furthermore, a complementary higher PDI at extremities of the tested sonication time was also noticed which led to the selection of 30 s as optimized time interval for desired quality attributes in the prepared liposomes. 3.2. Coating of PTX-liposomes 3.2.1. PAA coating PAA was implemented as the anionic polyelectrolyte polymer for coating of cationically charged PTX-liposomes. Fig. 1 reflects the effect of strength of PAA solution on the zeta potential, particle size and PDI of the PTX-liposomes. The selection of the appropriate strength of PAA solution was based on the charge reversal, particle size and PDI after coating. As evident from Fig. 1, the lesser volume of the lower concentration of the PAA was able to achieve only partial charge reversal in the order of 0e20 mV and in fact precipitation was observed. This phenomenon was found from 0.01% to 0.05% (w/v)

3.3. Shape and surface morphology Fig. 3 demonstrates the photomicrographs of PTX-liposomes and PAH-PAA-PTX-layersomes, which revealed layered structure of layersomes. The obtained size was in well correlation with that from zeta sizer. Additionally the spherical structure of these formulations was also demonstrated.

Table 2 Effect of phospholipid to cholesterol ratio on the PTX-liposomes. Phospholipid:Cholesterol

Size (nm)

3:1 2:1 1:1 1:2

329 209 121 472

Values are expressed as Mean
SD (n ¼ 6). The optimized values are represented in bold.







15.52 23.89 11.55 24.72

PDI 0.453 0.397 0.274 0.475







0.057 0.092 0.029 0.080

Zeta potential (mV)

% Encapsulation efficiency

þ48.0 þ45.0 D44.9 þ48.0

59.19 75.33 85.03 43.22







3.1 2.3 4.1 6.2







07.76 02.02 03.75 11.34

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in case of PTX-liposomes. PAH-PAA-PTX-layersomes on the other end were found to be stable in both simulated gastrointestinal fluids. Insignificant changes (p > 0.05) in the particle size, PDI, zeta potential and encapsulation efficiency were observed upon exposure to SGF, pH 1.2 and SIF, pH 6.8.

Table 4 Effect of sonication time on PTX-liposomes. Sonication time

Size (nm)

15 30 45 60

219 127 110 107

s s s s


7.69 ± 9.32
7.42
5.45

PDI

Zeta potential (mV)

% Encapsulation efficiency

0.509
0.024 0.263 ± 0.030 0.340
0.070 0.312
0.048

þ43.2
3.5 D48.0 ± 4.3 þ49.8
6.5 þ53.0
3.2

90.85
01.31 83.04 ± 02.74 41.38
11.12 38.20
03.19

3.5.2. Storage stability studies Table 8 represents the storage stability studies conducted for paclitaxel loaded formulations. After 6 months of storage at 4  C and at room temperature, freeze dried formulations were found to be stable without any collapse or shrinkage of dried cake. Insignificant changes were observed in the physical appearance, encapsulation efficiency, particle size and PDI before and after storage.

Values are expressed as Mean
SD (n ¼ 6). The optimized values are represented in bold.

3.4. Lyophilization Lyophilization of the prepared formulations was performed for stabilization purpose. Among the various cryoprotectants which were implemented during preliminary screening experiments, mannitol yielded the most optimum results in retaining the original quality attributes. Hence mannitol was used as cryoprotectant in all cases. Table 6 reveals the effect of freeze drying on the various formulations. The Sf/Si ratio (ratio of the particle size after and before freeze drying) in all cases was found approximately about 1, indicative of insignificant changes upon reconstitution. Furthermore, the cake which was formed was intact fluffy in nature with reconstitution time less than 30 s in all tested formulations. 3.5. Stability studies 3.5.1. Stability in simulated gastrointestinal fluids Table 7 depicts the stability studies of different PTX loaded formulations in simulated gastrointestinal fluids. PTX-liposomes and PAA-PTX-liposomes were found to be unstable in the simulated gastrointestinal fluids. All the parameters were significantly affected upon exposure of PTX-liposomes with SGF, pH 1.2 while insignificant changes in the particle size and zeta potential were observed when exposed to SIF, pH 6.8. Similarly, in case of PAAPTX-liposomes, all the parameters were significantly affected in both media, when exposed to SGF, pH 1.2 and SIF, pH 6.8. However, aggregation was observed in this case in contrast to that observed

3.6. In vitro drug release studies Fig. 4 reveals the in vitro drug release profiles of PTX-liposomes, PAA-PTX-liposomes and PAH-PAA-PTX-layersomes. PTX-liposomes demonstrated relatively rapid drug release with 46% in 1 h and 65% in 4 h. In contrast, PAA-PTX-liposomes sustained the drug release with lag time of 2 h and 45% of drug in 4 h. Furthermore, PAH-PAAPTX-layersomes showed greatest retardation by exhibiting lag time of 2 h followed by release of only 38% of drug in 4 h. Table 9 reflects the values of correlation coefficient obtained for various dissolution models calculated based on drug release kinetics of different PTX loaded formulations. On the basis of this, it was found that PTXliposomes preferentially followed Hixson-Crowell and Korsemeyer peppas (slope n ¼ 0.1499, suggesting fickian diffusion as mechanism of drug release) model whereas PAA-PTX-liposomes and PAHPAA-PTX-layersomes followed Higuchi kinetics of drug release. 3.7. Cell culture experiments 3.7.1. Cell uptake studies The cellular uptake studies revealed that efficient internalization of the tested formulations within A549 cells in 3 h. Green fluorescence of coumarin-6 was clearly visualized in cells treated with all the formulations (Fig. 5). The intensity of fluorescence was slightly lower in case of PAA-PTX-liposomes as compared to PTX-liposomes

Fig. 1. Effect of PAA on the (A) zeta potential, (B) size and (C) PDI on PTX-liposomes.

Table 5 Optimized parameters of various PTX loaded formulations. Formulations

Size (nm)

PDI

Zeta potential (mV)

% Encapsulation efficiency

% Practical drug loading

PTX-liposomes PAA-PTX-liposomes PAH-PAA-PTX-layersomes

114
07.57 144
12.66 226
17.61

0.262
0.027 0.295
0.025 0.343
0.070

þ48.0
2.79 38.9
2.90 þ39.9
3.79

87.55
3.68 79.16
2.62 71.91
3.16

4.37
0.18 3.95
0.13 3.59
0.15

Values are expressed as Mean
SD (n ¼ 6).

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Fig. 2. Effect of PAH on (A) zeta potential, (B) particle size and (C) PDI on PAA-PTX-liposomes.

and PAH-PAA-PTX-layersomes. The 3D images of these cells are presented in the Supplementary Information as Figs. S1eS3. 3.7.2. MTT assay Fig. 6 reflects the % cell viability of the A549 cells treated with free PTX, PTX-liposomes and PAH-PAA-PTX-layersomes at various concentrations. The % cell viability decreased proportionately with increase in concentration of formulations in all cases. The IC50 value of free PTX was found to be 35.42 mg/ml whereas those of PTXliposomes and PAH-PAA-PTX-layersomes were 28.25 mg/ml and 29.37 mg/ml, respectively. The values are suggestive of retention of higher in vitro cytotoxicity of PTX upon incorporation in to liposomes and layersomes formulations. 3.8. In vivo pharmacokinetic studies Fig. 7 reveals the plasma concentration time profile of various PTX loaded formulations whereas Table 10 reflects the various pharmacokinetic parameters of PTX suspension, PTX-liposomes, PAA-PTX-liposomes and PAH-PAA-PTX-layersomes. As evident, about 6 fold increase in the peak plasma concentration (Cmax) was observed in case of PAH-PAA-PTX-layersomes (142.16
12.92 ng/ ml) as compared to the free drug (23.65
11.57 ng/ml). Furthermore, significant differences in AUC0eN and T1/2 were also observed as compared to that of free drug. Upon comparing the AUC0eN of PAH-PAA-PTX-layersomes, about 4.07 fold increase in the oral bioavailability was observed as compared to that of free drug. 3.9. In vivo antitumor efficacy Fig. 8 reveals the percentage change in tumor volume of animal groups treated with control, free PTX (oral), PAA-PTX-liposomes (oral), PAH-PAA-PTX-layersomes (oral), TaxolÒ (i.v.) and NanoxelÔ (i.v.). It is evident that tumor volume was not controlled, rather

increased, in case of control, free PTX and PAA-PTX-liposomes treated animals. However, significant reduction in tumor volume was observed in case of PAH-PAA-PTX-layersomes (oral), TaxolÒ (i.v.) and NanoxelÔ (i.v.). Fig. 9 reflects the residual tumor burden in animals after 15 days and it was found that tumor size was reduced up to 66.24% in PAH-PAA-PTX-layersomes treated animals whereas untreated groups showed an increase in tumor size up to 165.6% as compared to the initial tumor volume. Tumor volume was reduced upto 58.90% with i.v. TaxolÒ and 30.13% with i.v. NanoxelÔ. However, insignificant difference (p > 0.05) in the tumor volume reduction was observed in case of PAH-PAA-PTX-layersomes (oral) as compared to that of i.v. TaxolÒ (p > 0.05). Furthermore, the Kaplan Meier survival plot was constructed by observing animal survival for 60 days post treatment which revealed that highest survival rate (100%) in case of i.v. NanoxelÔ, followed by PAH-PAA-PTX-layersomes (83.3%) in comparison to 50% survival rate observed with i.v. TaxolÒ (Fig. 10); animals observed for 60 days post treatment. 3.10. Toxicity studies The toxicity profiling of the developed formulation was carried out using different toxicity markers. BUN, plasma urea and plasma creatinine levels were determined for evaluating nephrotoxicity while plasma ALT and AST levels were determined for evaluating hepatotoxicity [31]. Fig. 11 reveals the levels of ALT, AST, creatinine, urea and BUN in different plasma samples collected from treatment animal groups. Significantly higher (p < 0.05) ALT and AST levels were found in case of TaxolÒ and PAA-PTX-liposomes as compared to control whereas insignificant changes (p > 0.05) were observed in case of PAH-PAA-PTX-layersomes and NanoxelÔ. Similarly, PAHPAA-PTX-layersomes and NanoxelÔ also showed insignificant changes (p > 0.05) in the values of plasma creatinine, plasma urea and BUN levels as compared to that of control whereas the level of all these nephrotoxicity parameters were significantly increased in case of TaxolÒ. The results are suggestive of comparable toxicity profile of PAH-PAA-PTX-layersomes with that of NanoxelÔ and relatively safer toxicity profile as compared to that of TaxolÒ. 4. Discussion

Fig. 3. TEM photomicrographs of (A) PTX-liposomes and (B) PAH-PAA-PTX-layersomes.

Extensive optimization of the critical process variables involved in the preparation of PAH-PAA-PTX-layersomes has been carried out. The phospholipid to cholesterol ratio was found to have significant impact on the formation of liposomes. As evident from Table 2, the particle size, PDI and encapsulation efficiency improved to a certain extent, with the increase in the cholesterol concentration in the liposomes (1:1 M ratio). This could be attributed to the assembling of cholesterol within the phospholipid molecules to

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Table 6 Physicochemical properties of paclitaxel loaded formulations before and after freeze drying. Parameters

PTX-liposomes

Size (nm) PDI Encapsulation efficiency (%) Ratio (Sf/Si) Physical appearance Reconstitution time

PAA-PTX-liposomes

PAH-PAA-PTX-layersomes

Before

After

Before

After

Before

After

114
7.57 0.262
0.027 87.55
3.68 1.03 Intact fluffy cake <30 s

119
6.37 0.242
0.002 86.64
2.90

144
12.66 0.295
0.025 79.16
2.62 1.05 Intact fluffy cake <30 s

152
8.69 0.268
0.002 76.95
1.78

226
17.61 0.343
0.07 71.91
3.16 1.03 Intact fluffy cake <30 s

238
17.60 0.283
0.05 70.53
4.43

Values are expressed as Mean
SD (n ¼ 6); Reconstitution was performed in 1 ml of the vehicle followed by gentle agitation.

Table 7 Stability studies of formulations in simulated gastrointestinal fluids. Parameters

Size (nm) Initial

PTX-liposomes SGF pH 1.2 114
07.57 SIF pH 6.8 114
07.57 PAA-PTX-liposomes SGF pH 1.2 144
12.66 SIF pH 6.8 144
12.66 PAH-PAA-PTX-layersomes SGF pH 1.2 226
17.61 SIF pH 6.8 226
17.61

PDI

Zeta potential (mV)

% Encapsulation efficiency

Final

Initial

Final

Initial

Final

Initial

Final

62
06.52 100
15.84

0.262
0.027 0.262
0.027

0.39
0.06 0.38
0.04

þ48.0
2.79 þ48.0
2.79

þ41.1
2.47 þ42.5
3.51

87.55
3.68 87.55
3.68

25.58
3.35 29.22
2.90

495
52.36 196
06.69

0.295
0.025 0.295
0.025

0.51
0.05 0.42
0.06

38.9
2.90 38.9
2.90

þ36.1
4.35 þ34.3
4.72

79.16
2.62 79.16
2.62

32.53
6.33 52.57
8.47

235
16.57 213
15.72

0.343
0.07 0.343
0.07

0.22
0.02 0.26
0.04

þ39.9
3.79 þ39.9
3.79

þ44.7
1.52 þ43.3
4.04

71.91
3.16 71.91
3.16

64.75
3.63 66.39
4.25

Values are expressed as Mean
SD (n ¼ 6).

provide rigidity to the resultant vesicular structures. However with increase in the concentration of cholesterol beyond 1:1, leads to detrimental effects on quality attributes of the liposomes, which could be due to disruption of the membrane vesicles at higher concentration. Furthermore, the steep decrease in encapsulation efficiency might be the result of increase competence of cholesterol with PTX to incorporate within the vesicles and leaching of drug molecules owing to disruption of membrane vesicles [32]. Therefore, ratio of phospholipid:cholesterol was selected as 1:1 (mole ratio) for further optimization. Subsequently, maximum drug loading within the liposome composition was assessed and it was found that 5% w/w resulted in desired quality attributes of the liposomes, as shown in Table 3. The higher drug loading leads to the membrane destabilizing effects on the prepared liposomes. The results are in line with the studies reported previously in literature, revealing maximum drug loading in case of PTX as 5% w/w [33]. Therefore, further studies were performed with 5% w/w drug loading. The decreased particle size and encapsulation efficiency with increase in sonication time could be attributed to the higher energy input to the formulations leading to the destabilization via mechanical collision and partially by the heat generated in microenvironment. This results in to breakdown of vesicles and release of entrapped drug. Therefore, on the basis of results obtained

(Table 4), the sonication for 30 s at amplitude 80 with three cycles at 1 min interval was selected as optimum. The charge on plain liposomes was about 2.5 mV which offered very weak ionic interaction with the oppositely charged polyelectrolytes; hence stearyl amine was incorporated in liposomes that imparted very high positive charge in the order of wþ48 mV, thus favoring very strong interactions with anionic polyelectrolyte (PAA) leading to significant changes in the quality attributes of liposomes. The adequate amount of PAA is essential to have uniform coating over liposomes and preliminary studies revealed that optimization in terms of concentration and volume was necessary to have reproducible and reliable results. Upon comparing the contour regions (Fig. 1AeC), it was found that concentration of 0.08e0.12% w/v at 400e500 ml yielded desired results. At lower concentration the precipitation was observed which could be attributed to insufficient coating leading to undesired interaction among partially coated liposomes. However, at higher concentrations, the interaction was so strong that particle size and PDI were significantly affected. The zeta potential of the optimized PAA-PTX-liposomes was found to be w38 mV. Subsequently, Fig. 2AeC demonstrates the contour plots constructed for zeta potential, particle size and PDI upon coating with PAH over PAA-PTX-liposomes. The optimized region in this case was 0.1%e

Table 8 Storage stability studies of paclitaxel loaded formulations. Temperature

Size (nm) Initial

PTX-liposomes 114
07.57 4 C 114
07.57 25  C PAA-PTX-liposomes 4 C 144
12.66 144
12.66 25  C PAH-PAA-PTX-layersomes 226
17.61 4 C 226
17.61 25  C

PDI

Zeta potential (mV)

% Encapsulation efficiency

After 6 months

Initial

After 6 months

Initial

After 6 months

Initial

After 6 months

109
04.32 115
04.64

0.262
0.027 0.262
0.027

0.278
0.01 0.269
0.01

þ48.0
2.79 þ48.0
2.79

þ47.4
3.65 þ46.6
2.12

87.55
3.68 87.55
3.68

84.66
4.30 84.37
3.76

146
07.54 151
17.08

0.295
0.025 0.295
0.025

0.262
0.04 0.272
0.02

38.9
2.90 38.9
2.90

35.8
3.29 37.3
3.64

79.16
2.62 79.16
2.62

78.74
1.22 75.91
1.96

222
08.84 235
18.30

0.343
0.070 0.343
0.070

0.320
0.01 0.297
0.02

þ39.9
3.79 þ39.9
3.79

þ38.5
1.61 þ39.7
2.33

71.91
3.16 71.91
3.16

70.62
2.12 67.76
4.37

Values are expressed as Mean
SD (n ¼ 6).

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Fig. 4. In vitro drug release profile of various PTX loaded formulations.

0.2% (w/v) at volume of 500e800 ml. As discussed previously, the reason for precipitation could be attributed to the charge imbalances leading to aggregation. The zeta potential of the final PAHPAA-PTX-layersomes was found to be wþ39 mV. Loosely held polyelectrolyte layers were visualized in TEM image of PAH-PAA-PTX-layersomes (Fig. 3B). The spherical shape was not altered upon polyelectrolyte coating and observed particle size was in correlation with that obtained from dynamic light scattering analysis. The formulation was further subjected to lyophilization to improve its storage stability utilizing a universal stepwise freeze drying cycle developed and patented by our group [20]. Mannitol at 5% w/v, based on preliminary studies, was selected as suitable cryoprotectant for achieving optimum quality attributes was as shown in Table 6. The selection was further in line with previously reported studies [34]. The lyophilized formulation was spontaneously reconstitutable (<30 s) upon gentle shaking without use of any additional energy input such as sonication or vortexing. In addition, all the original quality attributes of formulation such as particle size, PDI and encapsulation efficiency were unaltered upon reconstitution of lyophilized formulation, clearly revealing the stabilization of nanoparticles. In vitro stability studies in simulated gastrointestinal fluids revealed that PTX-liposomes were unstable in both SGF and SIF (Table 7). The excess hydrogen ions, present in the external environment can diffuse in the inner aqueous phase of liposomes, made up of phospholipids and destabilize them. Whereas the bile salt monomers can also permeate in to lipid bilayers and may lead to disruption of the vesicular structures. Furthermore, the cholic and taurocholic acid in the gastrointestinal tract can also increase the permeability of liposomes, whereas the pancreatin lipases tend to lyse the liposomes [35]. These rupturing and lysing of the liposomes can be accounted for the decreased particle size and encapsulation efficiency. However, PAA-PTX-liposomes also shows instability in these mediums, which could be attributed to the aggregation owing to presence of the opposite charge on the surface of liposomes compared to that of exterior environment. Hence the particle size was increased drastically along with a steep decrease in the encapsulation efficiency, being indicative of the

Fig. 5. Cellular uptake of Coumarin-6 loaded PTX-liposomes, PAA-PTX-liposomes and PAH-PAA-PTX-layersomes in A549 cancer cell lines.

destabilization of the system. On the other hand, PAH-PAA-PTXlayersomes were found stable in all mediums in terms of particle size, PDI, zeta potential and encapsulation efficiency. The reason behind their stability could be the protective role of multiple coatings, which prevents the exposure of the phospholipids from the harsh conditions of the gastrointestinal tract, suggestive of the robustness of formulation [14]. In vitro drug release profiles (Fig. 4) revealed significant retardation in drug release upon coating the liposomes with polyelectrolytes. The drug release kinetics suggested Hixson Crowell and Korsemeyer peppas model in case of PTX-liposomes (Table 9). The results were in line with the previously reported studies showing Korsemeyer peppas model leading to diffusion and erosion based drug release from liposomes [36]. However, the drug release kinetics suggested Higuchi type of drug release profile from PAA-PTX-liposomes and PAH-PAA-PTX-layersomes, indicative of diffusion from matrix systems as mechanism of drug release. These results further correlates with the fact that the vesicular nature of the liposomes is modified to that of particulate type upon coating of polyelectrolytes. These results are in line with our previous findings on polymeric nanoparticles [30]. Similar results with PTX based particulate nanocarriers have been reported previously by other research groups [37].

Table 9 Correlation coefficient of various dissolution models for PTX loaded formulations. Release profile

PTXliposomes

PAA-PTXliposomes

PAH-PAA-PTXlayersomes

Zero order First order Hixson-Crowell Higuchi Korsmeyer-Peppas

0.7515 0.886 0.933 0.8701 0.9089a

0.5687 0.7939 0.8102 0.833 0.569

0.5713 0.5943 0.6811 0.8897 0.5523

a

Slope ¼ 0.1499.

Fig. 6. Cell cytotoxicity of various formulations at different concentrations (** ¼ significant difference at p < 0.01% and * ¼ significant difference at p < 0.05%, a ¼ compared to free PTX at given concentration, b ¼ compared to PTX-liposomes at given concentration).

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Fig. 7. Plasma concentration time profile of various PTX loaded formulations.

Fig. 9. Comparative tumor burden after 15 days (*** ¼ significant difference at p < 0.001%, ** ¼ significant difference at p < 0.01% and * ¼ significant difference at p < 0.05%), a ¼ comparison with control DMBA, b ¼ comparison with PTX suspension, c ¼ comparison with PAA-PTX-liposomes and d ¼ comparison with PAH-PAA-PTXlayersomes.

Table 10 Pharmacokinetic parameters after oral administration of various formulations. Formulations

AUC (ng ml1 h1) T1/2 (h)

PTX suspension PTX-liposomes PAA-PTX-liposomes PAH-PAA-PTX-layersomes

767.15 759.52 999.14 3127.7







207.63 120.92 177.30 508.20

05.66 10.70 10.21 13.23







Cmax (ng/ml) 2.07 25.33 2.12 23.65 3.11 49.16 0.74 142.16







02.41 11.57 04.73 12.92

Values are expressed as Mean
SD (n ¼ 6).

Fig. 5 revealed efficient internalization of prepared formulations within the A549 cells which was slightly higher in case of PTXliposomes and PAH-PAA-PTX-layersomes as compared to PAA coated liposomes. The probable reason might be the cationic surface of PTX-liposomes (due to stearyl amine) and PAH-PAA-PTXlayersomes (due to PAH) in contrast to anionic charge on PAA coated liposomes, which imparts superior binding with the negatively charged cell membrane. The results of MTT assay revealed that cell cytotoxicity of PTX for A549 cells was increased significantly (p < 0.05) upon incorporation in the layersomes (Fig. 6), which could also be corroborated by the lower IC50 value of the layersome formulation as compared to free PTX. In vivo pharmacokinetic studies further revealed that layersome formulation was able to increase the Cmax of PTX by about 6 fold and overall increase in the oral bioavailability by 4.07 fold as compared to that of free drug. The increase in the oral bioavailability could be attributed to enhanced permeation of these lipid based nanocarriers across the gastrointestinal tract [38]. Furthermore, these systems are also eligible for uptake by chylomicron uptake pathway leading to bypass of the p-gp efflux of the drug, which is one of major reason for the poor bioavailability of PTX [39]. Additionally,

Fig. 8. Tumor inhibition study of different formulations (n ¼ 6).

these alternate route of absorption are also able to bypass first pass hepatic metabolism of drugs, which also contributes to increment in the oral bioavailability upon utilizing these nanoformulations [40]. The encouraging results of in vivo pharmacokinetic studies were further supported by in vivo antitumor efficacy of the developed formulation. As evident from Figs. 8 and 9, PAH-PAA-PTXlayersomes via oral route was able to significantly restrict the tumor growth as compared to that of control. The relatively higher tumor restriction capability could be partly attributed to the probable passive targeting of these nanocarriers using enhanced permeation and retention effect [30]. Furthermore, the results of oral PAH-PAA-PTX-layersomes were comparable to that of i.v. TaxolÒ (p > 0.05). However, upon comparison to NanoxelÔ (administered via intravenous route), the restriction in tumor growth was significantly lower (p < 0.001). This could be attributed to the preferential absorption of NanoxelÔ at tumor site via EPR effect and contribution of albumin in tumor targeting [41]. Nevertheless, comparable therapeutic efficacy equivalent to NanoxelÔ can be possibly achieved by dose adjustment or multiple dosage regimens. Furthermore, the inability of the PAA-PTX-liposomes to restrict the tumor growth clearly reveals the significance of multiple polyelectrolyte coating to the surface of liposomes to module its deliverability. Kaplan Meier survival plot showed greater survival rate of the present formulation (w83%) in comparison with TaxolÒ (w50%), however 100% survival rate was achieved in the case of NanoxelÔ. But it could be noticed that the lesser survival rate with oral layersomes is not due to toxicity rather can be attributed to the fact that single dose is insufficient for

Fig. 10. Kaplan Meier survival plot for evaluation of animal death during antitumor efficacy study.

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Fig. 11. Toxicity profiling of various PTX loaded formulations.

complete treatment and multiple dosing can be sought-after. PTX is also reported to cause acute nephrotoxicity and hepatotoxicity which can be a major limitation of its therapy [42e44]. Therefore, toxicity studies were performed and the present formulation showed significantly lesser hepatotoxicity and nephrotoxicity in comparison with TaxolÒ and comparable to that of NanoxelÔ, even when administered intravenously (Fig. 11). The relatively lower toxicity of these formulations could be attributed to their nano size which alters its biodistribution in the body to a significant level, which can be exploited carefully to achieve tailor-made formulations with desired quality attributes [45]. 5. Conclusion The layer by layer approach has been successfully implemented to achieve multiple polyelectrolyte coatings on the surface of liposomes and subsequently its stabilization. The polyelectrolyte coatings, mediated principally by ionic interactions have been

exploited to stabilize these systems and make them eligible to withstand in extremities of the gastrointestinal fluids. Furthermore, the oral administration of developed formulation was found to exhibit comparable antitumor efficacy with safer toxicity profile as that of i.v. TaxolÒ. Therefore, this formulation strategy can be rationally utilized to improve the deliverability of difficult-todeliver drugs such as PTX. Furthermore, a careful consideration of dosage regimen (in terms of multiple dosing) can be future line of action to achieve the comparable therapeutic efficacy as that of nanotechnology based marketed products (i.v. NanoxelÔ). In addition, the targeting potential of these nanocarriers can also be exploited to further improve the therapeutic efficacy of system and decrease their toxic side effects. Acknowledgments Authors are thankful to Director, NIPER for providing necessary infrastructure facilities and Department of Science & Technology

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(DST), Government of India, New Delhi, India, for financial support. Authors are also thankful for the technical support rendered by Mr. Dinesh Singh and Mr. Rahul Mahajan.

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