Carboxymethyl-Chitosan-Tethered Lipid Vesicles - American

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Carboxymethyl-Chitosan-Tethered Lipid Vesicles: Hybrid Nanoblanket for Oral Delivery of Paclitaxel Nitin Joshi, Rama Saha, Thanigaivel Shanmugam, Biji Balakrishnan, Prachi More, and Rinti Banerjee* WRCBB, Department of Biosciences and Bioengineeering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India S Supporting Information *

ABSTRACT: We describe the development and evaluation of a hybrid lipopolymeric system comprising carboxymethyl chitosan (CMC), covalently tethered to phosphatidylethanolamine units on the surface of lipid nanovesicles, for oral delivery of paclitaxel. The bioploymer is intended to act as a blanket, thereby shielding the drug from harsh gastrointestinal conditions, whereas the lipid nanovesicle ensures high encapsulation efficiency of paclitaxel and its passive targeting to tumor. CMC-tethered nanovesicles (LN-CPTX) in the size range of 200−300 nm improved the gastrointestinal resistance and mucoadhesion properties as compared with unmodified lipid nanovesicles (LN-PTX). Conjugation of CMC did not compromise the cytotoxic potential of paclitaxel yet facilitated the interaction and uptake of the nanovesicles by murine melanoma (B16F10) cells through an ATP-dependent process. CMC-conjugated nanovesicles, upon oral administration in rats, improved the plasma concentration profile of paclitaxel, with 1.5 fold increase in its bioavailability and 5.5 folds increase in elimination half life in comparison with Taxol. We also found that CMC in addition to providing a gastric resistant coating also imparted stealth character to the nanovesicles, thereby reducing their reticuloendothelial system (RES)-mediated uptake by liver and spleen and bypassing the need for PEGylation. In vivo efficacy in subcutaneous model of B16F10 showed significantly improved tumor growth inhibition and survival with CMC-tethered nanovesicles as compared with unmodified nanovesicles, both administered orally. LN-C-PTX exhibited therapeutic efficacy comparable to Taxol and Abraxane and also showed reduced toxicity and improved survival. Overall, these results suggest the therapeutic potential of CMC tethered nanovesicles as a platform for oral administration of paclitaxel and also unravel the ability of CMC to impart stealth character to the nanoparticles, thereby preventing their RES clearance.

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INTRODUCTION Paclitaxel, a potent anticancer drug with poor aqueous solubility is commonly administered intravenously in the form of Taxol, which is formulated in 50:50 (v/v) Cremophor EL (polyoxyethylated castor oil) and dehydrated alcohol.1 Oral administration of paclitaxel results in poor bioavailability (99% were purchased from Lipoid (Germany). Paclitaxel (purity >99%) was purchased from Fresenius Kabi India (India). Abraxane was purchased from Biocon (India). Taxol, the marketed formulation of paclitaxel, was purchased from Cipla (India). Dialysis membrane (molecular wt. cutoff 5000−10 000), EDC, NHS, Dulbecco’s modified Eagle medium (DMEM), RPMI-1640, fetal bovine serum (FBS), antibiotic antimycotic solution, phosphatebuffered saline (PBS), sodium azide, and trypsin-EDTA solution were purchased from Himedia Laboratories, Mumbai (India). Trinitrobenzene sulfonic acid (TNBS), sulphorhodamine-B (SRB), and mucin (type III) from porcine stomach mucosa were purchased from Sigma Aldrich, Mumbai (India). BCA protein assay kit was purchased from Thermo Scientific, Pierce, Rockford, IL (USA). High-performance liquid chromatography (HPLC) grade methanol, chloroform, and acetic acid were purchased from Merck, Mumbai (India). All tissue culture plates and tissue culture flasks were purchased from NUNC (USA). High-purity water purified by a Milli Q Plus water purifier system (Millipore, USA), with a resistivity of 18.2 MΩcm, was used in all experiments. Preparation of CMC Tethered Lipid Nanovesicles. Lipid nanovesicles were prepared by modified thin-film hydration method,26 with DPPC/DSPE in 7:3 molar ratio and paclitaxel/phospholipid in 1:2 molar ratio. Subsequently, the suspension was sonicated at 50% amplitude for 2 min using Branson Sonifier 450 probe sonicator to form small unilamellar vesicles. Nanovesicles were then passed through 0.4 and 0.2 μm polycarbonate membranes (Avanti Mini Extruder, Avanti Polar Lipids) with 11 extrusion cycles to separate free paclitaxel and result in their homogeneous size distribution.21 Further separation of paclitaxel was achieved by centrifuging the nanovesicle suspension at 25 000g, 4 °C for 10 min, after which the pellet was reconstituted in PBS pH 7.4 to maintain final concentration of paclitaxel as 1 mg/mL. This resulted in unmodified lipid nanovesicles (LN-PTX). CMC was prepared from low-molecular-weight chitosan as per the method reported by Chen and Park27 and was characterized using FTIR spectroscopy (Magna 550 FTIR spectrometer, Nicolet Instruments Corporation) and 1H NMR spectroscopy (Plus 300 MHz spectrometer, Varian, USA). Degree of substitution for CMC was determined by CHN analysis using CHNS (O) analyzer (FLASH EA 1112 series, Thermo Finnigan, Italy). CMC was tethered on LN-PTX using EDC/NHS coupling reaction to form an amide bond between free −NH2 groups of DSPE on the surface of nanovesicles and free −COOH groups on CMC,28 thereby forming CMC-tethered lipid nanovesicles (LN-C-PTX), which were separated from unreacted CMC by centrifuging the reaction suspension at 25 000g, 4 °C for 10 min, resulting in the purified pellet of LN-C-PTX, while unreacted CMC was removed with the supernatant. Blank nanovesicles (LN-B and LN-C-B) were prepared by a similar method without the addition of paclitaxel. Conjugation of CMC onto the nanovesicles was characterized by performing FTIR spectroscopy of LN-B and LN-CB. The degree of coupling between CMC and lipid nanovesicles was determined by estimating the free amine groups on CMC-tethered lipid nanovesicles and free amine groups on CMC-coated lipid nanovesicles, which were prepared without the use of EDC/NHS and were a result of electrostatic interaction between CMC and lipid nanovesicles. Free amine groups were determined using TNBS assay, and the degree of coupling was calculated as per the equation previously reported by Balakrishnan and Jayakrishnan.29 LN-PTX and LN-C-PTX were characterized for size distribution by dynamic light scattering (DLS) using laser particle analyzer (BI 200SM, Brookhaven Instruments Corporation, USA). The nanovesicles were also characterized for surface charge by determining their zeta potential using zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corporation, USA). Transmission electron microscopy (TEM) of nanovesicles was done as per the negative staining protocol using

% viability = absorbance of sample/absorbance of control × 100 IC50 values for all the formulations were calculated using GraphPad Prism 4 software. Blank nanovesicles (LN-B and LN-C-B) were also evaluated for biocompatibility in murine fibroblast cells (L929) using SRB assay.36 Cellular Uptake. Cellular uptake of both CMC-tethered (LN-C) and unmodified nanovesicles (LN) by B16F10 cells was studied at three different time points, viz. 0.5, 1, and 3 h, by loading calcein (indicator grade) dye in these nanovesicles. Cells were observed using a confocal laser scanning microscope (CLSM) (Olympus FluoView, FV500, Tokyo, Japan) using an excitation wavelength of 495 nm and C

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Figure 1. (A) FTIR spectra of chitosan and carboxymethyl chitosan (CMC). (B) 1H NMR spectrum of CMC. (C) Possible three derivatives for CMC due to substitution at C6 (a), substitution at C3 (b), and substitution at amino group (c), respectively. (D) FTIR spectra for unmodified nanovesicles (LN-B) and CMC-tethered nanovesicles (LN-C-B). ad libitum. The study was carried out in accordance with the protocols approved by the institutional animal ethical committee. Pharmacokinetics and Biodistribution. Pharmacokinetic and biodistribution studies were performed in Wistar rats (200−250 g) by dividing the animals into two groups with six animals in each. Animals were fasted for 12 h before the experiments but had free access to water. Group I received LN-C-PTX (30 mg PTX/kg) p.o. and group II received Taxol (10 mg PTX/kg) as i.v. bolus, administered through tail veil. Blood samples (90% cell viability were used for the inoculation. Mice were inoculated subcutaneously in the right flank region with 2.5 × 106 cells of B16F10 in 200 μL of PBS using a 27-gauge needle. Following the cell inoculation, mice were examined daily for tumor development and tumor size. When the tumor volume reached 60−100 mm3, mice were randomized into five groups (7 mice/group) (designated as day 0). Group A received Taxol, 10 mg paclitaxel/kg i.v. once in 3 days; group B received LN-PTX 30 mg paclitaxel/kg p.o. every day, group C received LN-C-PTX 30 mg paclitaxel/kg p.o. every day, group D received Abraxane 10 mg paclitaxel/kg i.v. once in 2 days; and group E was left untreated to serve as control. Body weights and tumor volumes were examined once in 3 days. Tumor dimensions were measured using Vernier Callipers, and the final tumor volume was calculated using following equation:40

V = (E2 × L)/2 where V = tumor volume in mm3, L = tumor length (the longest diameter of the tumor), and W = tumor width (diameter perpendicular to the length). Animals were also observed for survival and were finally sacrificed by cervical dislocation on day 20 due to significantly large tumor burden in the control group. Tumors were dissected, and their weights and volumes were recorded. Statistical Methods. All of the studies were done in triplicate, and the results were expressed as mean ± standard deviation. Statistical significance of the data was analyzed by Student’s t test. In all of the cases p < 0.05 was considered to be significant.

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RESULTS AND DISCUSSION Characterization of CMC. CMC was prepared from lowmolecular-weight chitosan as per the method reported by Chen and Park.27 The FTIR spectra (Figure 1A) of both chitosan and CMC show peaks at 3455 cm−1 (OH stretch), 2923−2867 cm−1, (C−H stretch), and 1154 cm−1 (bridge-O stretch). The intense peak at 1733 cm−1 in the spectrum of CMC is due to the COOH group, indicating the carboxymethylation of chitosan. Compared with chitosan, the peaks at 1650 and 1418 cm−1 corresponding to carboxy group (overlapping with NH bend) and CH2COOH group, respectively, are intense in CMC also, suggesting carbyoxymethylation on both the amino and hydroxyl groups of chitosan.41 The 1H NMR spectrum of CMC (Figure 1B) in D2O at 300 MHz shows a chemical shift at δ 2.049, which corresponds to proton of acetamido group of the chitosan. Chemical shift at δ values between 3.1 and 3.4 is assigned to mono and di-carboxymethylation on the amino groups according to Muzzarelli et al.42 However, it is difficult to determine whether all amino groups are carboxymethylated, as the signal of C2 hydrogen overlaps the signals assigned to Ncarboxymethyl sites. Chemical shift at δ value at 4.567 corresponds to O-carboxymethylation at C6 or C3 position.43 Because both chemical shifts are present in the spectrum, the resultant product can be assumed to be a mixture of three different derivatives (Figure 1C) and can be referred to as N,OCMC. The degree of substitution for CMC, as determined from CHN analysis, was found to be 1.4%.

Figure 2. Transmission electron microscopy (TEM) images of LNPTX (A) and LN-C-PTX (B).

showed 200−300 nm sized, uniformly distributed nanovesicles. Paclitaxel was encapsulated in the nanovesicles with highencapsulation efficiencies of 85.2 ± 3.3 and 83.8 ± 7.5%, as observed for LN-PTX and LN-C-PTX, respectively. Wettability and Mucoadhesiveness. Contact angle of water on LN-PTX-coated glass slides was found to be 30.4 ± 0.4°, which reduced to 15.2 ± 1.5° in the case of LN-C-PTX coated glass slides (Figure 3A). Almost 50% reduction in the contact angle was found to be statistically significant (p < 0.05) E

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and therefore confirms that CMC was successfully coupled to the nanovesicles. Both LN-PTX and LN-C-PTX were also evaluated for mucoadhesiveness by incubating the nanovesicles with mucin (from porcine stomach mucosa) and quantifying its adsorption onto their surface. As shown in Figure 3B, mucin adsorption on LN-PTX and LN-C-PTX was found to be 54.4 ± 1.9 and 83.1 ± 8.8%, respectively, which suggests that conjugation of CMC on the nanovesicles significantly (p < 0.05) improved their mucoadhesion, thereby improving their potential as an oral drug delivery system. Increased mucoadhesion of the drug carrier with the intestinal mucosal layer can prolong its residence time, thereby increasing its systemic absorption and bioavailability. Becuase the typical turnover time for intestinal mucus is around 50−270 min,44 mucoadhesive nanoparticles administered orally are expected to remain adhered to mucus for a sufficiently long time (∼4 h) to result in their systemic absorption. Therefore, the strategy of engineering mucoadhesive nanoparticles is well-suited for oral administration of drugs. Recently reported mucus-penetrating nanoparticles may be an alternative strategy for delivery of drugs to the mucosal surfaces.45 However, they may be more relevant to the surfaces like nasal tract, respiratory tract, and so on, which have a relatively thin mucus layer with faster turnover time of 10−20 min,45 which may result in the faster clearance of the adhered nanoparticles, thereby limiting the use of mucoadhesive nanoparticles. Higher mucoadhesion in the case of LN-C-PTX wellcorrelates with their higher zeta potential values as compared with LN-PTX, which reflects an increase in the positive charge

Figure 3. (A) Contact angles for CMC-tethered and unmodified nanovesicles. *p < 0.05 in comparison with LN-PTX. (B) Percentage mucin adsorption on CMC tethered and unmodified nanovesicles. *p < 0.05 in comparison with LN-PTX.

and is an indication of LN-C-PTX-coated surface being more hydrophilic as compared with LN-PTX-coated surface. This enhanced hydrophilicity can be attributed to the presence of CMC, a hydrophilic polymer, on the surface of nanovesicles

Figure 4. (A) In vitro release of paclitaxel at 37 °C in PBS, pH 7.4. *p < 0.05 in comparison with LN-PTX and Taxol. (B) In vitro release of paclitaxel at 37 °C in simulated gastric fluid (SGF), pH 1.2 for 2 h, followed by release in simulated intestinal fluid (SIF), pH 6.8 for the next 46 h. Vertical arrow at 2 h signifies the time point at which release medium was changed from SGF to SIF. *p < 0.05 in comparison with LN-PTX and Taxol. (C) Percentage cell viability of HeLa cells following 48 h of incubation with different concentrations of the formulations. (D) Percentage cell viability of B16F10 cells following 48 h of incubation with different concentrations of the formulations. (E) IC50 values for different formulation in HeLa and B16F10 cells. *p < 0.05 in comparison with Taxol; **p < 0.05 in comparison with the IC50 value of same formulation in B16F10 cells. F

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Figure 5. (A) CLSM images of B16F10 cells after 0.5, 1, and 3 h of incubation with calcein-loaded LN and LN-C. (B) CLSM images of B16F10 cells incubated with calcein-loaded LN-C for 3 h in Z scan mode with the scanning done from −10 to +10 μm (top to bottom). (C) Cellular levels of calcein after incubation of B16F10 cells with calcein-loaded LN under normal and ATP-depleted conditions. *p < 0.05 in comparison with cells treated with ATP-depleted groups. (D) Cellular levels of calcein after incubation of B16F10 cells with calcein-loaded LN-C under normal and ATPdepleted conditions. *p < 0.05 in comparison with cells treated with ATP-depleted groups.

on CMC-tethered nanovesicle surface, as a result of which, they can interact more with the negatively charged sialic acid residues of mucin. This also concurs with the previous finding where researchers demonstrated that increase in positive zeta potential of the polymers increases the detachment force between the polymer and mucin.46 In Vitro Release. In vitro release of paclitaxel in PBS, pH 7.4 was found to be sustained for unmodified nanovesicles (Figure 4A), which may be attributed to the strong association of paclitaxel with the lipid bilayer, and is desirable because unlike pulse kinetics of the current formulations, it can have minimum adverse toxic effects. LN-C-PTX showed further decrease in the drug release rate (Figure 4A), which again confirms the conjugation of CMC on the nanovesicles. Quantitatively, 0.51 ± 0.04 and 0.2 ± 0.05 mg cumulative releases were observed for LN-PTX and LN-C-PTX, respectively, in 48 h in PBS, pH 7.4. In contrast, free paclitaxel (Taxol) showed a burst release profile in PBS, with almost 90% cumulative release (2.7 ± 0.1 mg) in 8 h. In vitro release of paclitaxel from LN-C-PTX in SGF, pH 1.2 for the first 2 h, followed by release in simulated intestinal fluid (SIF), pH 6.8 for the next 46 h was found to be sustained with 0.13 ± 0.05 and 0.66 ± 0.06 mg cumulative releases observed in 2 and 48 h, respectively (Figure 4B). Releases from LN-PTX, that is, unmodified nanovesicles in SGF (2 h), followed by release in SIF (48 h), were found to be 0.99 ± 0.01 and 2.1 ± 0.12 mg, respectively. Release from unmodified nanovesicles increased drastically in SGF and SIF as compared with the release observed in PBS, which indicates spontaneous degradation of the nanovesicles as a result of adverse pH and enzymatic conditions in simulated GI fluids. However, CMC conjugation on the nanovesicles enhanced their stability under gastric and

intestinal conditions, as evident from the sustained release observed with LN-C-PTX in SGF and SIF, which proves the ability of CMC to withstand the pH and enzymatic degradation in GI tract. It also concurs with previously reported findings wherein coating liposome surface with polymers such as PEG, mucin, O-palmitoylpullulan, and so on has been found to impart resistance against the GI conditions.22,47 We speculate that CMC being a hydrophilic polymer can be well-hydrated in the aqueous solutions, resulting in a thick water layer on the surface of the nanovesicles, thereby preventing the direct contact of the nanovesicle surface with the degrading components. Taxol, on the extreme, exhibited almost 100% cumulative release (2.9 ± 0.1 mg) within 6 h in simulated GI fluids. In Vitro Cytotoxicity. Cytotoxicity was evaluated in human cervical cancer cells (HeLa) and murine melanoma cells (B16F10) by SRB assay over an exposure period of 48 h. As shown in Figure 4C,D, which gives the dose response curves for the study, it can be observed that all of the formulations but LN-C-B exhibited a concentration-dependent cytotoxic effect in both cell lines. LN-C-B did not show any cytotoxicity, even at a concentration as high as 10 μM, which confirmed that the nanovesicle itself did not contribute to the cytotoxic effects observed in the case of LN-PTX and LN-C-PTX. Moreover, in vitro biocompatibility of blank nanovesicles (both LN-B and LN-C-B) evaluated in murine fibroblast cells (L929) showed greater than 80% viability of cells at concentrations as high as 1 mg/mL, indicating the biocompatible nature of both CMC tethered and unmodified nanovesicles (Supporting Information, Figure S1). IC50 values for LN-PTX, LN-C-PTX, Taxol, and Abraxane were found to be 90.7 ± 11.7, 84.2 ± 3.4, 157.3 ± 15.3, and 84.8 ± 12.7 nM, respectively, in B16F10 cells G

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Figure 6. (A) Plasma concentration profile of paclitaxel after intravenous administration of Taxol and oral administration of LN-C-PTX in normal Wistar rats. *p < 0.05 in comparison with Taxol. (B) Biodistribution of paclitaxel in Wistar rats after 24 h of administration of Taxol (i.v.) and LN-CPTX (p.o.). *p < 0.05 in comparison with Taxol.

may be attributed to the ability of lipids to fuse with the cell membrane, thereby fluidizing it and hence facilitating the endocytosis mechanism.31 In the case of CMC-tethered nanovesicles, this effect may be due to CMC, as there are recent reports suggesting improved interaction and uptake of CMC nanoparticles by cells.48 CMC conjugation on the nanovesicles also increased their cellular uptake under normal physiological conditions by B16F10 cells at all time points in comparison with unmodified nanovesicles (Figure S3 in the Supporting Information). This might be due to higher positive charge on the surface of CMC-tethered nanovesicles, because of which they can exhibit better interaction with the negatively charged cell membrane. This concurs with a recently reported finding that suggests that increase in positive surface charge of the nanoparticles improves their interaction and uptake by cells and at the same time allows them to escape the lysosomes and exhibit perinuclear localization.49 Improved cellular internalization and lysosomal escape of LN-C-PTX might be a reason for its similar cytotoxic potential as LN-PTX, despite the fact that in vitro release was found to be less in LN-C-PTX as compared with LN-PTX. Pharmacokinetics and Biodistribution. Taxol, administered i.v. at 10 mg PTX/kg dose, showed a rapid distribution phase with t1/2α = 0.05 ± 0.01 h. LN-C-PTX, administered p.o. at 30 mg PTX/kg dose, showed maximum plasma concentration (Cmax = 118.1 ± 1.3 ng/mL) at tmax= 0.9 ± 0.1 h, with 5.5 fold increase in the elimination half life (t1/2β = 17.3 ± 1.4 h) as compared with that of Taxol (t1/2β = 3.2 ± 0.1 h). Higher dose of paclitaxel in the case of orally administered LN-C-PTX was based on the previous reports, suggesting significant bioavailability of oral formulations of paclitaxel administered at three to four fold higher dose of paclitaxel in comparison with the standard dose of intravenously administered Taxol.50 For LN-C-PTX, the plasma concentration of paclitaxel was maintained above 85 ng/mL, the defined therapeutic concentration of paclitaxel,5 for greater than 7 h, while it decreased within 7 h in the case of Taxol. In addition to this, LN-C-PTX also increased the area under curve (AUC) and mean residence time (MRT) significantly (p < 0.05) as compared with that of Taxol. Overall, an increase of 1.5 fold was observed in the bioavailability of paclitaxel with orally administered LN-C-PTX in comparison with the intravenously administered Taxol. Improved bioavailability of orally administered LN-C-PTX may be attributed to the improved GI stability and mucoadhesiveness of nanovesicles due to covalently conjugated CMC on their surface. Also, it has been reported that low-molecular-weight chitosan (LMWC) could reversibly open the tight junctions between intestinal epithelial cells.17 Because the CMC in the present study has been prepared from LMWC, we speculate that it might retain

(Figure 4E). In HeLa, IC50 values for LN-PTX, LN-C-PTX, Taxol, and Abraxane were found to be 203 ± 41, 170.8 ± 48.6, 299.4 ± 42.7, and 219.2 ± 5.8 nM, respectively (Figure 4E). As can be observed, IC50 values for LN-PTX, LN-C-PTX, and Abraxane were found to be significantly less (p < 0.05) as compared with the IC50 observed for Taxol, indicating a clear advantage of nanoparticle-based formulations over free paclitaxel in terms of cytotoxicity. IC50 values for LN-PTX and LN-C-PTX were comparable to each other, suggesting that conjugation of CMC to nanovesicles, while improving their gastric stability and mucoadhesion, does not reduce their anticancer potential. Moreover, the cytotoxic potentials of LNPTX as well as LN-C-PTX were found to be similar to those of Abraxane, commercially available albumin nanoparticle-based formulation of paclitaxel. Furthermore, IC50 values for both LN-PTX and LN-C-PTX suggested better cytotoxic potential of the nanovesicles in B16F10 cells as compared with HeLa. Therefore, further evaluation of the nanovesicles was done on B16F10 cells. Cellular Uptake. B16F10 cells incubated in the presence of calcein-loaded nanovesicles showed bright fluorescence at all time points, colocalized uniformly inside the cells (Figure 5A). In contrast with this, cells incubated with free calcein showed relatively less or no fluorescence (Supporting Information, Figure S2). To further confirm the complete cellular internalization of the nanovesicles and eliminate the probability of any fluorescence as a result of nanovesicles localized on the surface of the cells, Z scan or depth scan of cells was done. Z scan for 3 h internalization of LN-C over a range of −10 to +10 μm showed the highest fluorescence intensity near 0 μm (Figure 5B). This confirmed that the nanovesicles were completely internalized by the cells and were not present at the surface. Furthermore, as a part of the mechanistic study, it was interesting to note that cells pretreated with 0.1% sodium azide and cells incubated at 4 °C showed significantly less (p < 0.05) intracellular calcein content at all time points as compared with those incubated under normal conditions, that is, 37 °C without azide (Figures 5C,D). Sodium azide being a metabolic inhibitor depletes the cells of ATP, and hence no active process is possible thereafter. A similar effect is also caused by the incubation of cells at 4 °C rather than 37 °C. This suggests that the cellular uptake of both CMC tethered and unmodified nanovesicles is an energy -dependent or active process and therefore a statistically significant reduction (p < 0.05) in the intracellular calcein content was observed in the case of cells pretreated with 0.1% sodium azide and cells incubated at 4 °C. This study therefore revealed the roles of both modified and unmodified nanovesicles in facilitating the uptake of encapsulated material through an ATP-dependent process. The facilitated endocytosis in the case of unmodified nanovesicles H

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Figure 7. (A) Tumor volume variation for different groups after randomization. n = 7; *p < 0.05 in comparison with control; **p < 0.05 in comparison with LN-PTX-treated group. (B) Tumor weights for different groups as observed on sacrifice day. n = 7; *p < 0.05 in comparison with control; **p < 0.05 in comparison with other treatment groups. (C) Percentage tumor growth inhibitions for different groups. n = 7; *p < 0.05 in comparison with other treatment groups. (D) Kaplan−Meier survival curves for different groups after tumor implantation. The vertical arrow indicates the point of sacrifice.

arguments suggest that the classic RES uptake of nanoparticles does not have a role in increased accumulation of LN-C-PTX in lungs. Yang et al. has reported lung specific accumulation of chitosan-modified PLGA nanoparticles due to their enhanced trapping in lung capillaries, which is solely mediated by chitosan. A similar mechanism may be involved with LN-CPTX as well because of the presence of CMC, a derivative of chitosan. Also, once trapped in the lung capillaries, LN-C-PTX can undergo enhanced uptake across the endothelial cells due to their positive surface charge as previously reported,53 resulting in their increased accumulation in lungs. Increased lung deposition of LN-C-PTX may have implications toward its application for targeting pulmonary metastasis, often observed in melanoma cases.54 However, this may be of concern in applications other than pulmonary metastases, wherein the increased lung deposition of LN-C-PTX may result in significant pulmonary toxicity. In Vivo Efficacy in Subcutaneous Murine Melanoma (B16F10) Model. After demonstrating the bioavailability of LN-C-PTX upon its oral administration and its cytotoxicity in B16F10 cells in vitro, we evaluated its antitumor efficacy in vivo as an oral formulation in subcutaneous model of murine melanoma (B16F10) and compared against the in vivo efficacy of LN-PTX p.o, Taxol i.v. and Abraxane i.v. Figure 7A gives the mean tumor volumes observed over the entire study after randomization. Tumor volumes as measured on day 20 were 1983.3 ± 384.2, 623.2 ± 67.3, 1332.3 ± 168.3, 667.2 ± 50.3, and 700.3 ± 77.3 mm3, respectively, for control (no treatment), Taxol, LN-PTX, LN-C-PTX, and Abraxane, respectively. Statistically significant reduction (p < 0.05) in tumor volumes in comparison with control was evident after day 9 for LN-CPTX-, Taxol-, and Abraxane-treated groups. However, delayed inhibition in tumor growth was observed for LN-PTX-treated animals, with significant inhibition (p < 0.05) evident after day 15. Figure 7B gives the mean tumor weights for different groups as measured on day 20 after sacrifice. Statistically significant inhibition (p < 0.05) in tumor weights was observed in all treatment groups in comparison with the tumor weight of

chitosan’s ability to open the tight junctions between intestinal epithelial cells, thereby resulting in improved bioavailability, as observed in the case of CMC tethered nanovesicles. Figure 6A shows the plasma concentration profile of paclitaxel for Taxol and LN-C-PTX, and Table S2 in the Supporting Information summarizes the key pharmacokinetic parameters, as obtained from the two-compartment analysis of the data. Tissue distribution of paclitaxel at the end of 24 h showed a significant decrease (p < 0.05) in its accumulation in liver, spleen, and brain with LN-C-PTX as compared with Taxol (Figure 6B). This can substantially contribute toward reducing the adverse toxic effects of paclitaxel in these organs, which are otherwise observed with Taxol. Because liver and spleen are the major organs of the reticuloendothelial system (RES), these findings also imply that CMC conjugation imparts a stealth character to the nanovesicles, allowing them to escape the uptake by RES, as evident from decreased accumulation of paclitaxel with LN-C-PTX in liver and spleen. Monocytic macrophage-mediated uptake of nanoparticles by RES during their systemic circulation has always been a major concern.51 Surface modification of nanoparticles with hydrophilic polymers like PEG, commonly known as PEGylation, has been previously suggested to reduce their classic RES uptake.52 However, in our case, CMC itself acts as a stealth coating and therefore bypasses the need for further PEGylation. Improved plasma concentration profile of paclitaxel with LN-C-PTX, as indicated by its increased blood circulation time and reduced clearance, may also be attributed to the stealth coating of CMC. Moreover, CMC-imparted stealth character may also help the nanovesicles to bypass monocytic macophages upon systemic absorption, thereby allowing them to enter into systemic circulation. CMC conjugation on nanovesicles therefore, in addition to improving their GI stability and mucoadhesiveness, also blankets the nanovesicles and paclitaxel from monocytic macrophages-mediated RES uptake and clearance. In addition to liver and spleen, lungs are also a part of RES, but, to our surprise, paclitaxel accumulation while decreasing in liver and spleen increased significantly in lungs. These I

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control group (Figure 7C). However, inhibition in LN-PTX group, although statistically significant, was also significantly less (p < 0.05) as compared with the inhibitions observed in LN-C-PTX, Taxol and Abraxane treatment groups. Also, growth inhibition for LN-C-PTX was found to be comparable to that of Taxol and Abraxane. Quantitatively, 69.8 ± 3.2, 39.2 ± 6, 67.8 ± 7.1, and 67.1 ± 6% tumor growth inhibitions were observed for Taxol, LN-PTX, LN-C-PTX, and Abraxane groups. No significant changes in body weights were observed during the entire study (data not shown). Kaplan−Meier survival curves (Figure 7D) showed 28.6, 43, 85, 57.1, and 57.1% survival proportions for control, LN-PTX, LN-C-PTX, Taxol, and Abraxane treated groups, as observed on the sacrifice day, that is, day 35 after tumor implantation. This suggests that LN-C-PTX (p.o.) significantly improved the survival proportion of the animals as compared with the survival proportions observed with LN-PTX (p.o.), Taxol (i.v.), and Abraxane (i.v.). These results clearly indicate improved therapeutic efficacy and reduced toxicity of paclitaxel with CMC-conjugated nanovesicles in comparison with unmodified nanovesicles, both administered orally. Increased antitumor efficacy observed with orally administered CMC-tethered nanovesicles may be attributed to their improved GI resistance and improved mucoadhesion, resulting in increased bioavailability, increased blood circulation, reduced plasma clearance, ability to escape RES uptake, and improved cellular interaction as compared with unmodified nanovesicles. It is noteworthy that LN-PTX, even at a higher dose in comparison with Taxol and Abraxane, showed significantly less tumor growth inhibition in comparison with other treatments, which may be attributed to the degradation of LN-PTX by GI conditions. On the contrary, LN-C-PTX, administered orally at higher dose, showed tumor growth inhibition comparable to Taxol (i.v.) and Abraxane (i.v.), and also showed reduced toxicity and improved survival as compared with Taxol and Abraxane.

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Corresponding Author

*Tel: +912225767868. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Dr. Kishori Apte, National Toxicology Centre, Pune, India for providing facilities to carry out animal studies.

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REFERENCES

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For the first time we here developed carboxymethyl chitosan tethered lipid nanovesicles and showed that with the integrated advantages of lipid nanovesicles and carboxymethyl chitosan these nanovesicles offer a promising strategy for oral delivery of paclitaxel. The nanovesicles exhibit significant tumor growth inhibition as well as reduced toxicity as compared with the commercially available formulations of paclitaxel, that is, Taxol and Abraxane. Tethering CMC imparted significant gastric resistance, mucoadhesiveness, and stealth character to the nanovesicles. Further optimization of these properties may be achieved in the future by varying phospholipid/CMC ratio during the preparation of hybrid nanovesicles.

* Supporting Information S

Detailed experimental methods, Figure S1 (Percentage cell viability of L929 murine fibroblast cells incubated with LN-PTX or LN-C-PTX for 24 h), Figure S2 (CLSM image showing B16F10 cells incubated with free calcein), Figure S3 (Cellular levels of calcein after incubation of B16F10 cells with calcein loaded LN or calcein loaded LN-C under normal physiological conditions), Table S1 (Physiochemical properties of LN-PTX and LN-C-PTX) and Table S2 (Pharmacokinetic parameters of paclitaxel for Taxol and LN-C-PTX). This material is available free of charge via the Internet at http://pubs.acs.org. J

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