Oral Delivery of Doxorubicin Using Novel Polyelectrolyte-Stabilized

Article pubs.acs.org/molecularpharmaceutics

Oral Delivery of Doxorubicin Using Novel Polyelectrolyte-Stabilized Liposomes (Layersomes)† Sanyog Jain,* Swapnil R. Patil, Nitin K. Swarnakar, and Ashish K. Agrawal Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali) Punjab-160062, India

ABSTRACT: The present study explores the potential of polyelectrolyte-coated liposomes for improving the oral deliverability of doxorubicin (Dox). As a part of formulation strategy, stearyl amine was selected as a formulation component to provide positive charge to liposomes, which were subsequently coated with anionic poly(acrylic acid) (PAA) followed by coating of cationic polyallyl amine hydrochloride (PAH) in a layer by layer manner and led to the formation of a robust structure “layersomes”. Optimization of various process variables were carried out, and optimized formulation was found to have particle size of 520.4 ± 15.0 nm, PDI of 0.312 ± 0.062, ζ potential of +30.4 ± 5.32 mV, and encapsulation efficiency of 63.4 ± 4.26%. Layersomes were not only stable in simulated gastrointestinal fluids but also presented sustained drug release (∼35%) as compared to both Dox-liposomes and PAA-Dox-liposomes (∼67%), the release pattern being Higuchi kinetics. The in vivo pharmacokinetics studies revealed about 5.94-fold increase in oral bioavailability of Dox as compared to free drug. In vivo antitumor efficacy in a DMBA-induced breast tumor model further exhibited significant reduction in the tumor growth as compared to control and IV-Dox, while results were comparable to IV-LipoDox. Layersomes also exhibited a marked reduction in cardiotoxicity in comparison with IV-doxorubicin and IV-LipoDox (marketed formulation), as evidenced by the reduced levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine phosphokinase (CK-MB) and increased levels of glutathione (GSH) and superoxide dismutase (SOD). The reduced cardiotoxicity of layersomes was further confirmed by comparative histopathological examination of heart tissue after treatment with various formulations. The positive results of the study strengthen our expectation that the developed formulation strategy can be fruitfully exploited to improve the oral deliverability of poorly bioavailable drugs and can open new vistas for oral chemotherapy. KEYWORDS: doxorubicin, cardiotoxicity, liposomes, layersomes, polyelectrolytes, oral delivery

1. INTRODUCTION

The advancement in drug delivery led to emergence of a variety of nanocarriers which have been well recognized for their specialized uptake through intestinal M cells, which serves as an effective means to overcome the drug efflux by P-gp efflux transporters while bypassing the effect of first-pass metabolism.8 Subsequently, nanoformulations have tremendous potential to improve the oral bioavailability of drugs that, otherwise, are poorly bioavailable. They are also reported to reduce side effects of the drugs.3 Over the past few years, a variety of approaches have been tried for improving the oral deliverability of Dox.

Doxorubicin belongs to the class of anthracycline antibiotics with a well-established therapeutic profile against a wide range of cancers.1 However, Dox shows very low oral bioavailability (∼5%) due to inherent low permeability, substrate specificity to P-gp efflux pump, acid catalyzed hydrolysis in the stomach,2 and susceptibility to cytochrome P450 (CYP450), resulting in high first-pass metabolism.3 Therefore, Dox is available only as injectables under the trade name Adriamycin and Rubex (Dox hydrochloride in solution form), Myocet (non-PEGylated liposomal formulation), and Doxil, Caelyx, and LipoDox (PEGlyated liposomal formulations),4,5 but the clinical efficacy of these intravenous formulations is often fraught with the Dox induced cardiotoxicity.6,7 © 2012 American Chemical Society

Received: Revised: Accepted: Published: 2626

April 12, 2012 June 24, 2012 August 7, 2012 August 7, 2012 dx.doi.org/10.1021/mp300202c | Mol. Pharmaceutics 2012, 9, 2626−2635

Molecular Pharmaceutics

Article

These include: (i) encapsulation in PAMAM dendrimers,9 lipidbased systems,10 or other particulate carriers, and (ii) coadministration with quercetin11 and cyclosporin A12 or inhibitors of CYP3A4 and P-gp. Although these systems/approaches reported were successful to some extent, still they were not free from one or more limitations including poor entrapment, insufficient bioavailability, and complexity in scalability and suppression of body’s immune system.13 In our previous report, we have demonstrated efficacy of Dox loaded PLGA nanoparticles following oral administration.3 However, relatively low entrapment and scalability due to high cost of PLGA restricts their further commercial development. Among various other drug delivery systems, liposomes seem to be one of the most prospective candidates not only because of their biocompatibility and biodegradability but because they also provide high drug loading for both hydrophilic and hydrophobic drugs with well established technology for industrial scalability. The only major hurdle in oral delivery of liposomes lies in their instability due to aggregation in the gastric environment and degradation in the presence of bile salts and intestinal lipases.14 A number of attempts have been investigated for the stabilization of liposomes including polymerization,15 coating with chitosan16 and other polymers, and addition of gangliosides GM1 and GM type III.17 In yet another approach, layer by layer coating of oppositely charged polyelectrolytes over the charged liposomal core leads to the formation of a highly robust structure, often termed as “layersomes”.18 Layersomes exhibit the advantages of both particulate systems (escaping P-gp efflux pump, increasing storage stability and robustness) and vesicular system (high drug load). Previously, layersome formulation had been successfully explored by our group for improving the oral deliverability of a combination of antibiotics (amoxicillin and metronidazole) against Helicobacter pylori infection.19 Encouraged by our previous results, we sought to extend the concept for improving the oral deliverability of poorly bioavailable anticancer agents. In line with that approach, a polyelectrolyte-coated liposomal formulation of Dox (Dox-layersomes) was prepared through layer by layer deposition of poly(acrylic acid) (PAA) and polyallyl amine hydrochloride (PAH) on Dox-loaded liposomes. In addition to optimization of process variables, extensive physicochemical characterization, pharmacokinetic, and pharmacodynamic profiles of Dox-layersomes were evaluated in normal and tumor-bearing rats, respectively. Finally, in vivo safety profile of the formulation was assessed in mice by measuring the level of different cardiotoxicity markers and hispathological examination of excised heart tissues.

phase. The resultant mixture was subjected to sonication for 2−5 min in an ice bath which resulted in formation of w/o (reverse) emulsion, which was further processed by vortexing at 35 °C for 30−45 min in a specially designed chamber to evaporate the organic solvent. This resulted in formation of a gel followed by a liposome dispersion containing Dox. This dispersion was further diluted with phosphate buffer (pH 5.0) up to 5 mL. 2.2.1. Optimization of Process Variables. Various process variables of PC:CH mole ratios and % w/w of drug (Table 1) Table 1. Optimization of Process Variables for Preparation of Dox Liposomes optimization parameters

variables

PC:CH mole ratio

2:1 1:1 1:2

% w/w of Dox: 12.5 stearyl amine: 2 mg

remark

% w/w of Dox

12.5 25 37.5 50

PC:CH mole ratio: 1:1 stearyl amine: 2 mg

were optimized to get the liposomes of quality attributes in terms of size, polydispersity index (PDI), ζ, and entrapment efficiency. 2.3. Coating of Liposomes. Coating of Dox-liposomes was done by alternate coating of PAA and PAH as polyanion and polycation, respectively. Briefly, cationic Dox-liposomes were taken as the template, and coating of anionic polyelectrolyte PAA was implemented over the template. Coating optimization was done at different concentrations (0.01−0.5%) and different volumes (50−1800 μL) of PAA by mixing with liposomal dispersion (5 mL) under constant stirring at 2000 rpm for 1 h. Extra polymer was removed at 20000g, followed by single washing with water. These PAA-Dox-liposomes were taken as a template and subjected to the next coating of cationic polyelectrolyte PAH. Coating optimization was done at different concentrations (0.01−0.5%) and different volumes (50−500 μL) of PAH by mixing with PAA-Dox-liposomal dispersion (5 mL) under constant stirring at 2000 rpm for 1 h. The dispersion was subjected to centrifugation at 20000g, followed by single washing with water in order to get the final formulation (Dox-layersomes). 2.4. Characterization. 2.4.1. Size and Zeta Potential Measurement. The Dox-liposomes, PAA-Dox-liposomes, and Dox-layersomes were characterized for size, PDI, and ζ potential. Size and PDI were measured by dynamic light scattering (DLS) (Nano ZS, Malvern, UK), while ζ potential was estimated on the basis of electrophoretic mobility under an electric field. 2.4.2. Transmission Electron Microscopy (TEM). Surface morphology was checked using TEM (Morgagni 268D. Fei Electron Optics) operated at 80 kV. Briefly, a drop of each sample was placed over the Formvar-coated grid, followed by drying and negative staining with phosphotungstic acid (1% w/v) solution. 2.4.3. Encapsulation Efficiency. The percentage of drug incorporated was determined by direct method. Liposome dispersion was subjected to centrifugation at 40000 rpm for 1 h at 4 °C to pelletize the liposomes. Pellet was subjected to washing to remove surface adsorbed drug. It was dissolved in a minimum quantity of triton-X 100 (0.5% v/v) and centrifuged, and liberated drug was analyzed by UV spectrophotometer at

2. MATERIALS AND METHODS 2.1. Materials. Dox was a kind gift from Sun Pharma Advanced Research Centre (SPARC), Vadodara, India. Egg phosphotidylcholine (>90% PC) was supplied as a generous gift from Cargill corporation, Germany. Cholesterol (CH), stearylamine (SA), poly(allylamine hydrochloride) (PAH, MW ∼ 56 kD), poly(acrylic acid) (PAA, MW ∼ 5100 D), and 7,12-dimethylbenz[α]-anthracene (DMBA) were procured from SigmaAldrich, St. Louis, MO. All other reagents used were of analytical grade. 2.2. Preparation of Positively Charged Liposomes. Liposomes were prepared by the well documented reverse phase evaporation technique.20 Briefly, egg PC and CH (1:1 molar ratio) (total lipid, 36.0 mg) and SA (2 mg) were dissolved in 5 mL of diethyl ether. Dox (12.5% w/w) was dissolved in phosphate buffer (pH 5.0, 2 mL) and added to the organic 2627

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of blood). Plasma was separated by centrifuging the blood samples at 15000 rcf for 5 min at 15 °C. Methanol (250 μL) was added to plasma (125 μL) to precipitate the plasma proteins, followed by addition of internal standard (methyl paraben) (50 μL) in 10 μg/mL concentration. The samples were further vortexed and centrifuged at 15000 rcf for 15 min. The supernatants were collected and analyzed for drug content by validated RP-HPLC method.3 The pharmacokinetic parameters of plasma concentration−time data were analyzed by one-compartmental model using Kinetica software (Thermo Scientific). Pharmacokinetics parameters like total area under the curve (AUC)0−∞ and peak plasma concentration (Cmax) were determined. The relative bioavailability of formulations after oral administration was calculated as follows:

480 nm. The percentage of drug entrapped was calculated as follows: entrapment efficiency(%) mass of drug in pellet = mass of drug initially taken for liposomes reparation × 100

Entrapment efficiencies for PAA-Dox-liposomes and Doxlayersomes were determined using same procedure except for breakdown of the pellet, which was done by sonicating the pellet for 1 h in a bath sonicator. 2.5. Stability in SGF and SIF. To determine the protective effect and stability provided by the polyelectrolytes coatings, all the liposomal formulations were incubated in simulate gastric fluid (SGF), pH 1.2, and simulated intestinal fluid (SIF), pH 6.8. SGF was prepared by dissolving 100 mg of pepsin in 5 mL of water containing 0.35 mL concentrated HCl followed by addition of sodium chloride (100 mg) and volume adjustment (up to 50 mL) with water. Finally, pH of the solution was adjusted to 1.2 by adding concentrated HCl. SIF was prepared by dissolving 340 mg of monobasic potassium phosphate in 10 mL of water followed by addition of 3.85 mL of 0.2 M NaOH and 500 mg of pancreatin was added. Final volume was made up to 50 mL, and pH was adjusted to 6.8 by NaOH. To simulate the effect of bile salts, 3 mM sodium taurocholate was added additionally to the SIF.21 Each mL of formulation was added to 9 mL of simulated media followed by its incubation for 2 and 6 h in SGF and SIF, respectively. The overall effect was evaluated by measuring the effect on size, PDI, ζ potential, and entrapment efficiency. 2.6. In Vitro Drug Release. In vitro drug release from Doxliposomes, PAA-Dox-liposomes, and Dox-layersomes was studied using the dialysis membrane method. Dialysis membrane (Sigma, MWCO ∼ 12000) was activated by washing under the running water tap for 6 h to remove glycerol, followed by its treatment with sodium sulfide (0.3% w/v) for 1 min and acidification using sulfuric acid (0.2% v/v). The excess of the acid was removed by washing with water. Phosphate buffer saline (PBS) (pH 7.4) was taken as the release medium.22 Doxliposomes, PAA-Dox-liposomes, and Dox-layersomes equivalent to 2 mg of Dox were poured into dialysis bags. The dialysis bags were suspended in 15 mL of PBS at 37.0 ± 0.5 °C in a shaking water bath maintained at 50 rpm. The samples (500 μL) were withdrawn at predetermined intervals up to 24 h and replaced with an equal quantity of fresh medium to maintain the sink conditions. Sample analysis was done using a UV spectrophotometer at 480 nm. 2.7. In Vivo Studies. 2.7.1. Pharmacokinetics. Female Sprague−Dawley rats of 200−250 g were used for the study. Animals were supplied by the central animal facility of NIPER, India. All the animal study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC), NIPER, India. Throughout the experiments, the animals were housed in laminar flow at a temperature of 25 ± 2 °C and relative humidity of 50−60% under 12 h light/dark cycles. Animals were randomly distributed into four groups each containing six animals. Free Dox, Dox-liposomes, PAA-Dox-liposomes, and Dox-layersomes were administered to the overnight fasted animals by oral gavage at an equivalent drug dose of 10 mg/kg body weight. The blood samples (∼0.5 mL) were collected from the retro-orbital plexus under mild anesthesia into heparinized microcentrifuge tubes (containing 30 μL of 1000 U heparin/mL

relative bioavailability =

AUC(formulation) × 100 AUC(drug solution)

2.7.2. Tumor Growth Inhibition Study. Female Sprague− Dawley rats of 200−250 g were used for the induction of chemical-induced breast cancer. DMBA in soy bean oil was administered orally to rats at 45 mg/kg dose at weekly interval for three consecutive weeks.3,22,23 Drug treatment was given after 10 weeks of the last dose of DMBA and tumor-bearing animals were separated and divided randomly into different treatment groups (n = 6) viz control (oral administration of saline), Oral-Dox, IV-Dox, Oral-Dox-liposomes, Oral-PAA-Dox-liposomes, OralDox-layersomes, and IV-LipoDox (PEGylated liposomes). All the formulations were given at a dose equivalent to 5 mg/kg body weight of Dox.3 The tumor width (w) and length (l) were recorded with an electronic digital caliper, and tumor size was calculated using the formula (l × w2/2). Additionally, to evaluate the effect of multiple dosing through oral route, Free-Dox, Dox-liposomes, PAADox-liposomes, and Dox-layersomes were administered in three doses (at a dose of 5 mg/kg) after every 48 h to separate groups of animals. Tumor volume was measured up to 15 days. 2.7.3. Toxicity Study. To address the toxicity issues pertaining to the use of Dox-layersomes, levels of different biochemical markers were determined in normal mice. Healthy female mice (Swiss strain) of uniform body weight (25−30 g) with no prior drug treatment were procured from central animal facility of NIPER, India. The mice were kept on a commercial diet and given water ad libitum. The cages were placed in a conventional room, air conditioned at 23 °C and 55−70% humidity with a 12 h light/dark cycle. The animals were divided into six groups (six mice per group) and treated with control (saline), Oral-Dox, IV-Dox, Oral-Dox-layersomes, IV-Dox-layersomes, and IVLipoDox. Different formulations were administered at a dose equivalent to 25 mg/kg (in case of oral route) or 5 mg/kg (in case of IV route). The animals were sacrificed on 15th day, and toxicity was determined by measuring the levels of different biochemical markers viz LDH, CK-MB in plasma, and MDA, GSH, and SOD in heart homogenate. The levels of these biochemical parameters were estimated spectrophotometrically according to the manufacturer guidelines provided with the diagnostic kits (Accurex biomedical Pvt. Ltd., India). Heart tissues from each group were excised and fixed in 10% (v/v) formalin and processed for histopathological examination. Paraffin embedded specimen were cut into 5 μm sections, followed by staining with hematoxylin and eosin (H&E) for histopathological evaluations.3 2.8. Statistical Analysis. All the results were presented as the mean ± standard deviation (SD). Statistical analysis was performed with Sigma Stat (Version 2.03) using one-way analysis of 2628



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Table 2. Optimization of PC:CH Ratioa

a

PC:CH

vesicle size

ζ potential

PDI

entrapment efficiency (%)

2:1 1:1 1:2

206.4 ± 3.01 213.5 ± 4.03 272.2 ± 16.85

+22.9 ± 2.43 +23.9 ± 2.46 +25.8 ± 2.51

0.256 ± 0.001 0.231 ± 0.009 0.327 ± 0.024

65.5 ± 1.32 78.4 ± 2.89 72.6 ± 1.71

All values are expressed as mean ± SD (n = 6).

Table 3. Optimization of Drug Loadinga

a

loading (% w/w)

vesicle size (nm)

ζ potential (mV)

PDI

entrapment efficiency (%)

12.5 25.0 37.5 50.0

213.5 ± 4.03 226.2 ± 16.85 250.0 ± 4.76 290.7 ± 8.90

+23.9 ± 2.46 +24.2 ± 2.92 +24.8 ± 2.51 +24.4 ± 2.60

0.231 ± 0.009 0.245 ± 0.016 0.256 ± 0.009 0.307 ± 0.010

78.4 ± 2.89 76.6 ± 2.49 77.4 ± 3.13 64.1 ± 1.67


Figure 1. Effect of PAA coating on (A) size, (B) PDI, (C) ζ potential.

Figure 2. Effect of PAH coating on (A) size, (B) PDI, (C) ζ potential.

(from positive to negative), PDI, and size after coating. As evident from Figure 1, the PAA solution (0.01 to 0.5% w/v) in the volume range (50−1000 μL) resulted in partial charge reversal and aggregation. However, PAA (>0.1%) in the volume range (1200−1800 μL) resulted in satisfactory charge reversal (∼−25 mV). Furthermore, particle size and PDI were the limiting factors and acceptable (particle size 0.05) changes were recorded in formulation attributes. 3.5. In Vitro Drug Release. In vitro drug release profiles of Dox-liposomes, PAA-Dox-liposomes, and Dox-layersomes are depicted in Figure 4. Significant retardation in drug release was observed in case of Dox-layersomes. Up to 67% Dox release was

Figure 4. In vitro drug release profile of Dox-liposomes, PAA-Doxliposomes, and Dox-layersomes.

observed within 12 h in the cases of Dox-liposomes and PAADox-liposomes, while it was approx 35% in the case of Doxlayersomes. The values of correlation coefficient calculated based on different drug release kinetics are given in Table 6. Doxliposomes and PAA-Dox-liposomes were found to follow Hixson−Crowell model, while Dox-layersomes followed Higuchi kinetics of drug release. 3.6. In Vivo Studies. 3.6.1. Pharmacokinetics. Mean plasma concentration time profiles of different Dox loaded formulations are shown in Figure 5. It is worthy to note that oral administration of free-Dox, Dox-liposomes, and PAA-Dox-liposomes led to very low 2630



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Table 6. Correlation Coefficient of Various Drug Release Models for Dox Loaded Formulationsa release model

Doxliposomes

PAA-Doxliposomes

Doxlayersomes

zero-order first-order Higuchi model Hixon−Crowell Korsmeyer−Peppas

0.616 0.711 0.899 0.919 0.965*

0.614 0.693 0.900 0.914 0.941**

0.602 0.632 0.900 0.891 0.875

a

Slope: *0.408, **0.436.

Figure 6. Tumor growth inhibition study of different formulations following single oral dose (a = in comparison with control, ***p < 0.001).

in the case of Dox-layersomes in comparison with Oral-Dox, Doxliposomes, and PAA-Dox-liposomes (Figure 7). Interestingly, the reduction in tumor volume was significantly higher (p < 0.01) upon multiple oral dosing of Dox-layersomes in comparison with IV-Dox and IV-LipoDox up to 7 days. After 7 days, the difference between Dox-layersomes and IV-LipoDox was comparable (p > 0.05). 3.6.3. Toxicity Study. The toxicity profiling of the developed formulation was carried out using different toxicity markers. Orally administered Dox-layersomes exhibited marked reduction in cardiotoxicity as evident from levels of different cardiotoxicity markers (Figure 8A−E). A significant increase (p < 0.001) in LDH, CK-MB, and MDA levels while a significant decrease (p < 0.001) in GSH and SOD levels was observed in case of IV-Dox and IV-LipoDox in comparison with control and oral Dox. An insignificant (p > 0.05) difference in levels of all these biochemical markers was observed in Oral-Dox and Oral-Dox-layersomes in comparison with control. It is also worthy to note that significant difference (p < 0.001) in levels of all biochemical markers was observed in the case of Dox-layersomes administered through oral/ IV routes, in comparison with IV-Dox, while the difference was insignificant (p > 0.05) in comparison with IV-LipoDox. These results confirmed reduced and comparable toxicity of Doxlayersomes in comparison with IV-Dox and LipoDox, respectively. Histopathological examination of the representative heart tissue from each treatment group was also carried out for the presence of any possible toxicity. Normal striated cells were observed with one or two nuclei centrally in the cells in the control group (Figure 9A). A marked degeneration of cardiac muscles characterized by the disorganization of the cardiac muscles, edema, and vacuolization of cells was observed in IV-Dox as evident from Figure 9D. Moreover, focal fragmentation and scanty number of macrophages were observed in IV-Dox. No significant changes were noticed in the heart histopathology in IV-LipoDox (Figure 9C). On the contrary, minimum changes in terms of disorganization of the cardiac muscles, edema, and vacuolization were observed in Dox-layersomes administered through oral/IV routes (Figure 9E,F).

Figure 5. Plasma concentration vs time profile of Dox solution, Doxliposomes, PAA-Dox-liposomes, and Dox-layersomes following single oral administration in dose equivalent to 10 mg/kg of free Dox.

peak plasma concentration (Cmax) ,while significantly higher (p < 0.05) Cmax was achieved in the case of Dox-layersomes which was 5.58-, 5.28-, and 3.0- fold higher in comparison with free-Dox, Dox-liposomes, and PAA-Dox-liposomes, respectively. Similarly, AUC for the Dox-layersomes was 2829.61 ± 62.16 ng/mL·h, which was 5.94-, 5.72-, and 3.0-fold higher than that for free-Dox, Doxliposomes, and PAA-Dox-liposomes, respectively (Table 7). Table 7. Pharmacokinetic Parameters of Various Dox Loaded Formulations upon Single Oral Administration in Dose Equivalent to 10 mg/kg of Free Doxa

a

parameters

Cmax (ng/mL)

(AUC)0−∞ (ng mL−1 h−1 )

Free-DOX Dox-liposomes PAA-Dox-liposomes Dox-layersomes

19.40 ± 3.44 20.51 ± 7.09 25.83 ± 8.34 108.34 ± 11.12

475.84 ± 69.11 493.88 ± 32.27 941.51 ± 36.81 2829.61 ± 62.16


3.6.2. Tumor Growth Inhibition Study. Percentage change in tumor volume with respect to time in different treatment groups is shown in Figure 6. It is evident that tumor volume was not controlled but rather increased in all the treatment groups except IV-Dox. An insignificant difference (p > 0.05) was observed in Oral-Dox, Dox-liposomes, and PAA-Dox liposomes, while the difference was significant (p < 0.05) in Dox-layersomes in comparison with control. Very significant (p < 0.001) reduction in tumor volume was observed in IV-Dox in comparison with Oral-Dox-layersomes. Because the single dose of various formulations through the oral route was not effective, multiple dosing (3 doses at the interval of every 48 h) was tried for better understanding the in vivo behavior of the formulations. Insignificant change (p > 0.05) in tumor volume was observed in Oral-Dox, Dox-liposomes, and PAA-Dox-liposomes even after multiple dosing in comparison with control, while a very significant reduction (p < 0.001) in tumor volume was observed

4. DISCUSSION Extensive optimization of the critical process variables involved in the preparation of Dox-liposomes and further 2631



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Figure 7. Tumor growth inhibition study of different formulations following multiple oral dose (a = in comparison with control, b = in comparison with IV-Dox, ***p < 0.001, **p < 0.01).

Figure 8. Plasma levels of biochemical markers in mice after treatment with various Dox loaded formulations (A) LDH, (B) CK-MB, (C) MDA, (D) GSH, (E) SOD (a = in comparison with control, b = in comparison with Oral-Dox, c = in comparison with IV-Dox, d = in comparison with IV-LipoDox, ***p < 0.001, **p < 0.01, *p < 0.05).

polyelectrolyte-coated systems was performed. The PC:CH ratio was found to have a significant impact on formulation quality attributes. As evident from Table 2, the particle size, PDI, and encapsulation efficiency improved to an extent, with the increase

in the cholesterol concentration in the liposomes (1:1 mol ratio). This could be attributed to the rigidity provided due to assembly of cholesterol molecules in between the phospholipid molecules and was in coordination with previous reports.24 However, 2632



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Figure 9. Histopathology of heart tissue (A) control, (B) Oral-Dox, (C) IV-LipoDox, (D) IV-Dox, (E) IV-Dox-layersomes, (F) Oral-Dox-layersomes (arrows indicates the vacuolization and structural changes in heart tissue).

cholesterol in higher concentration (beyond 1:1 mol ratio) led to detrimental effects on formulation quality attributes which could be due to the disruption of the membrane vesicles. Therefore, PC:CH (1:1 mol ratio) was selected for further optimization. Subsequently, maximum drug carrying capacity of the liposomes was assessed and 37.5% w/w of Dox resulted in formation of liposomes of desired quality attributes (Table 3). The developed liposomes were able to carry such a high amount of drug, which might be attributed to the aqueous core that provided a separate compartment capable of carrying a large amount of drug. Incorporation of stearyl amine imparted high positive charge on the order of +24.8 mV and provided a template for electrostatic deposition of oppositely charged polyelectrolytes. The adequate amount of polyelectrolytes is essential for uniform coating over liposomes and has a significant effect on formulation quality attributes. PAA in the volume range of 1−1.2 mL at a concentration of 0.1% w/v was found to be optimal, as particles with desired quality attributes were obtained in this region (Figure 1A−C). Aggregation was observed at lower concentration, 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. Subsequent coating of PAH was found optimum in the volume range of 200− 400 μL at a concentration of 0.05% w/v. As discussed previously, the reason for aggregation above/below a particular level could be attributed to the charge imbalances. A slight decrease in entrapment was also observed during each coating step, which might be attributed to high hydrophilicity of Dox which resulted in leaching during the coating steps. Polyelectrolytes layers were clearly visualized in TEM image of PAA-Dox-liposomes and Dox-layersomes (Figure 3B,C). The spherical shapes of the liposomes were maintained upon polyelectrolytes coatings, and the results were in coordination with the results of DLS analysis. To further evaluate the protective effect of polyelectrolytes coatings, the developed formulations were subjected to incubation in different simulated gastrointestinal fluids. Doxlayersomes were found to be stable in both the simulated biological media, i.e., SGF and SIF, and could be attributed to protective effect of alternative coatings, which prevents the exposure of phospholipids to a harsh gastrointestinal environment, suggestive of robustness of formulation. Dox-lipsomes and 2633



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repeated multiple dosing. Even after multiple dosing, free-Dox, Dox-liposomes, and PAA-Dox-liposomes were found ineffective in reducing the tumor burden, which revalidates the instability of liposomes and inability of PAA coating to provide adequate stability to the liposomes. A significant higher reduction in tumor volume observed in Dox-layersomes upon multiple doses could be attributed to the maintenance of the drug plasma level within the therapeutic window for prolonged duration. Additionally, enhanced permeation and retention at tumor site due to particulate nature of the carrier may also be the possible reason for better results in terms of tumor suppression rather than single dosing. The developed formulation was found safe, as no detectable toxicity was perceived with Oral-Dox-layersomes. However, as argued in the case of oral Dox, low toxicity of Dox layersomes could be attributed to its insufficient bioavailability in comparison with IV standards (100% bioavailability). To resolve this issue, the toxicity of Dox-layersomes was evaluated after IV injection and compared with IV-Dox and IV-LipoDox. Impressively, toxicity of IV-Dox-layersomes was significantly lower in comparison to IV-Dox (p < 0.05). The toxicity was also comparable with the marketed formulation of IV-LipoDox. This improved safety profile of IV-Dox-layersomes over IV-Dox could be attributed to better control over drug release provided by polyelectrolytes coatings. Histopathological examination further confirmed that Dox-induced myocardial morphological changes were markedly suppressed or almost eliminated in Doxlayersomes. Extensive sarcoplasmic vacuolization and severe disruption of fine structures were observed in IV-Dox, while sarcoplasmic vacuolization and mitochondrial structural changes were almost completely prevented in Dox-layersomes. The overall data in hand suggests that Dox-layersomes developed in the course of the study are less toxic than IV-Dox and IVLipoDox and can be a viable option for oral chemotherapy.

PAA-Dox-liposomes were quite unstable. The excess of hydrogen ions present in the external environment can diffuse in the inner aqueous phase of the liposomes, which might have led to disruption and destabilization of the liposomes. Other constituents of the GI fluids like bile salts act as surfactants, thereby leading to solubilization of lipids. Pancreatic lipases have the digestive action on phosphotidylcholine, which also contributes in destabilization of the liposomal system.25 Thus the reduction in size and drug loss observed in Dox-liposomes and PAA-Dox-liposomes might be the result of cumulative disruptive effect of the constituents. In the case of PAA-Dox-liposomes, the negative charges on the surface might have led to adsorption of oppositely charged ions from the surrounding media, which in turn resulted in aggregation and destabilization of negatively charged PAA-Dox-liposomes. In vitro drug release studies were performed in PBS (pH 7.4). The selection of the medium was based on its simulating characteristic with other biological fluids and the reports regarding higher stability of Dox in PBS in comparison to tris buffer of same pH.26 Significant retardation in drug release (Figure 4) was obtained in case of Dox-layersomes. An approximately similar release profile in the case of Dox-liposomes and PAA-Doxliposomes could be attributed to the inability of the single coat of PAA to retain the Dox within the system as it is highly water soluble. Higher correlation coefficient was evident for the Hixson−Crowell and Korsemeyer−Peppas models in the case of Dox-liposomes and PAA-Dox-liposomes (Table 6). The results were in coordination with previous findings in which liposomes have been reported to follow diffusion and erosion based drug release pattern.27 However, release kinetics suggested the Higuchi model for Dox-layersomes, indicative of drug diffusion from the matrix system. These results further supported the fact that coating of polyelectrolytes provides particulate character to the vesicular system. Results of pharmacokinetic studies further exhibited improved performance of Dox-layersomes in comparison with free-Dox, Dox-liposomes, and PAA-Dox-liposomes. Low bioavailability of the free-Dox could be attributed to its low permeability, acid catalyzed hydrolysis, high first-pass metabolism, and susceptibility to P-gp efflux pump.3,26 Similarly, poor pharmacokinetic profiles of Dox-liposomes and PAA-Dox-liposomes could be attributed to their poor stability in the gastrointestinal milieu, revalidating that the single coating of PAA was not sufficient to stabilize the liposomes. Significantly better pharmacokinetic profiles obtained in the case of Dox-layersomes in comparison with other formulation could be attributed to number of factors. First, multiple polyelectrolytes coatings provided strong protection against the acid catalyzed hydrolysis of Dox in stomach. Second, selective uptake of the system through intestinal epithelial M cells makes possible the direct entry into the systemic circulation. Additionally, particulate character of the Dox-layersomes protect the system from the effect of the P-gp efflux pump. The encouraging results of pharmacokinetic study were further supported by in vivo antitumor efficacy of the developed formulation. Rats treated with Dox-layersomes showed a significant reduction (p < 0.001) in tumor volume in comparison with control. However, it was significantly lower (p < 0.001) when compared to that of IV-Dox. This could be attributed to the inherent low bioavailability associated with oral route, which at the same dose can never be comparable to that of IV administration. Thus, the oral route can be paralleled with IV either by increasing the dose or dosing frequency. Increasing the dose may aggravate other problems related to dose-dependent toxicity, hence increasing dosing frequency was chosen in terms of

5. CONCLUSION Layer by layer assembly of oppositely charged polyelectrolytes over the liposomes core not only provide a robust structure which protect the entrapped drug from the harsh gastric environment but also increase the bioavailability due to specialized uptake mechanisms. Sustained release obtained from the formulation increase the chances of continuous exposure of the tumor to a modest concentration of Dox within the therapeutic range. Moreover, effective tumor growth inhibition upon multiple dosing with low toxicity presents this system as a lucrative approach for oral Dox delivery. Already well established technology for liposomes at large scale is another hidden advantage of the system which can make this approach a clinical reality. Further studies may be directed to investigate the deposition pattern at the molecular level number of layers and their effect on release from the system. Multiple dose kinetics, targeting potential, and intracellular trafficking of this system is currently underway and will be reported in due course.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0172-2292055. Fax: 0172-2214692. E-mail: sanyogjain@ niper.ac.in; sanyogjain@rediffmail.com. Notes

The authors declare no competing financial interest. † This work is the part of the study for which an Indian patent application no. 3246/DEL/2011 has been filed on November 15, 2011. 2634



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Article

(18) Ciobanu, M.; Heurtault, B.; Schultz, P.; Ruhlmann, C.; Muller, C. D.; Frisch, B. Layersome: development and optimization of stable liposomes as drug delivery system. Int. J. Pharm. 2007, 344 (1−2), 154− 157. (19) Jain, P.; Jain, S.; Prasad, K. N.; Jain, S. K.; Vyas, S. P. Polyelectrolyte coated multilayered liposomes (nanocapsules) for the treatment of Helicobacter pylori infection. Mol. Pharmaceutics 2009, 6 (2), 593−603. (20) Szoka, F., Jr; Papahadjopoulos, D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu. Rev. Biophys. Biol. 1980, 9 (1), 467−508. (21) Vertzoni, M.; Fotaki, N.; Nicolaides, E.; Reppas, C.; Kostewicz, E.; Stippler, E.; Leuner, C.; Dressman, J. Dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects. J. Pharm. Pharmacol. 2004, 56 (4), 453−462. (22) Upadhyay, K. K.; Bhatt, A. N.; Mishra, A. K.; Dwarakanath, B. S.; Jain, S.; Schatz, C.; Le Meins, J. F.; Farooque, A.; Chandraiah, G.; Jain, A. K. The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly ([gamma]-benzyl L-glutamate)-b-hyaluronan polymersomes. Biomaterials 2010, 31 (10), 2882−2892. (23) Jain, A. K.; Swarnakar, N. K.; Godugu, C.; Singh, R. P.; Jain, S. The effect of the oral administration of polymeric nanoparticles on the efficacy and toxicity of tamoxifen. Biomaterials 2011, 32 (2), 503−515. (24) Lee, S. C.; Lee, K. E.; Kim, J. J.; Lim, S. H. The effect of cholesterol in the liposome bilayer on the stabilization of incorporated retinol. J. Liposome Res. 2005, 15 (3−4), 157−166. (25) Rowland, R. N.; Woodley, J. F. The stability of liposomes in vitro to pH, bile salts and pancreatic lipase. Biochim. Biophys. Acta Lipids Lipid Metab. 1980, 620 (3), 400−409. (26) Janssen, M. J. H.; Crommelin, D. J. A.; Storm, G.; Hulshoff, A. Doxorubicin decomposition on storage. Effect of pH, type of buffer and liposome encapsulation. Int. J. Pharm. 1985, 23 (1), 1−11. (27) Rudra, A.; Deepa, R. M.; Ghosh, M. K.; Ghosh, S.; Mukherjee, B. Doxorubicin-loaded phosphatidylethanolamine-conjugated nanoliposomes: in vitro characterization and their accumulation in liver, kidneys, and lungs in rats. Int. J. Nanomed. 2010, 5, 811−823.

ACKNOWLEDGMENTS We are thankful to the Director, NIPER, for providing necessary infrastructure facilities, and the Department of Science & Technology (DST), New Delhi, India, for financial support. Technical assistance rendered by Dinesh Singh and Rahul Mahajan is also duly acknowledged.

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REFERENCES

(1) Travis, L. B.; Curtis, R. E.; Boice, J. D., Jr; Hankey, B. F.; Fraumeni, J. F., Jr Second cancers following non-Hodgkin’s lymphoma. Cancer 1991, 67 (7), 2002−2009. (2) Beijnen, J. H.; Van der Houwen, O.; Underberg, W. J. M. Aspects of the degradation kinetics of doxorubicin in aqueous solution. Int. J. Pharm. 1986, 32 (2−3), 123−131. (3) Jain, A. K.; Swarnakar, N. K.; Das, M.; Godugu, C.; Singh, R. P.; Rao, P. R.; Jain, S. Augmented anticancer efficacy of doxorubicin loaded polymeric nanoparticles after oral administration in breast cancer induced animal model. Mol. Pharmaceutics 2011, 8 (4), 1140−1151. (4) Perez, A. T.; Domenech, G. H.; Frankel, C.; Vogel, C. L. Pegylated Liposomal Doxorubicin (Doxil) for Metastatic Breast Cancer. Cancer Invest. 2002, 20 (S2), 22−29. (5) Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nature Rev. Drug Discovery 2005, 4 (2), 145−160. (6) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56 (2), 185−229. (7) O’Brien, M. E. R.; Wigler, N.; Inbar, M.; Rosso, R.; Grischke, E.; Santoro, A.; Catane, R.; Kieback, D. G.; Tomczak, P.; Ackland, S. P. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann. Oncol. 2004, 15 (3), 440−449. (8) Jain, A. K.; Das, M.; Swarnakar, N. K.; Jain, S. Engineered PLGA Nanoparticles: An Emerging Delivery Tool in Cancer Therapeutics. Crit. Rev. Ther. Drug. Carrier Syst. 2011, 28 (1), 1−45. (9) Ke, W.; Zhao, Y.; Huang, R.; Jiang, C.; Pei, Y. Enhanced oral bioavailability of doxorubicin in a dendrimer drug delivery system. J. Pharm. Sci. 2008, 97 (6), 2208−2216. (10) Benival, D. M.; Devarajan, P. V. Lipomer of doxorubicin hydrochloride for enhanced oral bioavailability. Int. J. Pharm. 2011, 423 (2), 554−561. (11) Choi, J. S.; Piao, Y. J.; Kang, K. W. Effects of quercetin on the bioavailability of doxorubicin in rats: role of CYP3A4 and P-gp inhibition by quercetin. Arch. Pharm. Res. 2011, 34 (4), 607−613. (12) Sonneveld, P.; Suciu, S.; Weijermans, P.; Beksac, M.; Neuwirtova, R.; Solbu, G.; Lokhorst, H.; Van Der Lelie, J.; Dohner, H.; Gerhartz, H. Cyclosporin A combined with vincristine, doxorubicin and dexamethasone (VAD) compared with VAD alone in patients with advanced refractory multiple myeloma: An EORTC-HOVON randomized phase III study (06914). Br. J. Hamaetol. 2001, 115 (4), 895−902. (13) Kalaria, D. R.; Sharma, G.; Beniwal, V.; Ravi Kumar, M. N. V. Design of biodegradable nanoparticles for oral delivery of doxorubicin: in vivo pharmacokinetics and toxicity studies in rats. Pharm. Res. 2009, 26 (3), 492−501. (14) Rowland, R. N.; Woodley, J. F. The stability of liposomes in vitro to pH, bile salts and pancreatic lipase. Biochim. Biophys. Acta 1980, 620 (3), 400−409. (15) Chen, H.; Torchilin, V.; Langer, R. Lectin-bearing polymerized liposomes as potential oral vaccine carriers. Pharm. Res. 1996, 13 (9), 1378−1383. (16) Wu, Z. H.; Ping, Q. N.; Wei, Y.; Lai, J. M. Hypoglycemic efficacy of chitosan-coated insulin liposomes after oral administration in mice. Acta Pharm. Sin. 2004, 25 (7), 966−972. (17) Taira, M. C.; Chiaramoni, N. S.; Pecuch, K. M.; AlonsoRomanowski, S. Stability of liposomal formulations in physiological conditions for oral drug delivery. Drug Delivery 2004, 11 (2), 123−128. 2635


Oral Delivery of Doxorubicin Using Novel Polyelectrolyte-Stabilized

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