Ligand-Directed Stearic Acid Grafted Chitosan Micelles to Increase

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Ligand-Directed Stearic acid Grafted Chitosan Micelles to Increase Therapeutic Efficacy in Hepatic Cancer Yuan Yang, Sheng-Xian Yuan, Ling-Hao Zhao, Chao-Wang Wang, JunSheng Ni, Zhen-Guang Wang, Chuan Lin, Meng-Chao Wu, and Wei-Ping Zhou Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500723k • Publication Date (Web): 11 Dec 2014 Downloaded from http://pubs.acs.org on December 16, 2014

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Molecular Pharmaceutics

Ligand-Directed Stearic acid Grafted Chitosan Micelles to Increase Therapeutic Efficacy in Hepatic Cancer Yuan Yang1,2#, Sheng-Xian Yuan1#, Ling-Hao Zhao1#, Chao-Wang1, Jun-Sheng Ni1, ZhenGuang Wang1, Chuan Lin1, Meng-Chao Wu1, Wei-Ping Zhou1,3*

1

Department of Hepatobiliary, Eastern Hepatobiliary Surgery Hospital, Second Military

Medical University, Shanghai 200438, China 2

Department of Health Statistics, Second Military Medical University, Shanghai 200433,

China 3

Department of Hepatobiliary, National Innovation Alliance for Hepatitis & Liver Cancer,

Shanghai, 200438, China

#

These authors contributed equally to this article.

Corresponding author: Wei-Ping Zhou, M.D Department of Hepatobiliary, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, No. 225 Changhai Road, Shanghai 200438, China. Tel & Fax: 0086-21-81875521. Fax: 0086-21-81875529, Email: [email protected]

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ABSTRACT Targeted delivery system would be an interesting platform to enhance the therapeutic effect and to reduce the side effects of anticancer drugs. In this study, we have developed lactobionic acid (LA)-modified chitosan-stearic acid (CS-SA) to deliver doxorubicin (DOX) to hepatic cancer cells. The average particle size of CSS-LA/DOX was ~100 nm with a high entrapment efficiency of >95%. Drug release studies showed that DOX release from pHsensitive micelles is significantly faster at pH 5.0 than at pH 7.4. The LA conjugated micelles showed enhanced cellular uptake in HepG2 and BEL-7402 liver cancer cells than free drug and unconjugated micelles. Consistently, CSS-LA/DOX showed enhanced cell cytotoxicity in these two cell lines. Annexin-V/FITC and PI based apoptosis assay showed that the number of living cells greatly reduced in this group with marked presence of necrotic and apoptotic cells. LA-conjugated carrier induced typical chromatic condensation of cells; membrane blebbing and apoptotic bodies were begin to appear. In vivo, CSS-LA/DOX showed an excellent tumor regression profile with no toxic side effects. The active targeting moiety, long circulation profile, and EPR effect contributed to its superior anticancer effect in HepG2 based tumor. Our results showed that polymeric micelles conjugated with lactobionic acid (LA) increased the therapeutic availability of DOX in the liver cancer cell based solid tumor without any toxic side effects. The active targeting ligand conjugated nanoparticulate system could be a promising therapeutic strategy in the treatment of hepatic cancers.

KEYWORDS Polymeric micelles, doxorubicin, lactobionic acid, apoptosis, antitumor efficacy


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INTRODUCTION Hepatic cancer (HC) is one of the most lethal malignancies recorded in human history with highest rate of incidence and mortality worldwide.1 China alone accounts for more than 60% of global liver cancer burden with nearly 60 million cases diagnosed each year. The liver cancer developed in a rather asymptomatic manner wherein early diagnosis and treatment is difficult.

2,3

Moreover, frequent relapse and reoccurrence of liver cancer makes it difficult to

treat. Surgical resection is the most potential tool for the treatment of HC, however practical difficulty makes the resection rate to less than 30%.4,5 Chemotherapy is regarded as the adjuvant or main alternative treatment to HC however issues such as lack of specific cancer targeting, accumulation of large proportion of drugs in the normal tissues, and severe systemic side effects limits its therapeutic applications.6 Unfortunately, no effective systemic therapy for HC has been developed till date and there is urgent need to develop a new therapeutic strategy.7 Nanotechnology-based drug delivery system has been reported to improve the pharmacological and anticancer property of chemotherapeutic drugs.8 Polymer or lipid based drug delivery system (DDS) have been demonstrated to possess various favourable features including tunable sizes, controlled release kinetics, high drug loading, and uniform pharmacokinetics.9 Specifically, enhanced permeability and retention effect (EPR) process will allow preferential accumulation of nanoparticles to the leaky vasculature of tumor tissues.10 The liver tumor consisted of fenestrated endothelium will allow the nanoparticles to be taken by the liver tissue leading to an increasing antitumor efficacy and decreased side effects. In terms of nanocarriers, polymeric micelles have attracted significant attention as a promising delivery system towards cancer therapy.11 The nanosized polymeric micelles offer many advantages including protection of drug in the core of micelles (in adverse



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environment), reducing the non-specific uptake by reticuloendothelial system (RES) system, prolonging the blood circulation, and possibility of surface modifications.12,13 In the present study, we have employed chitosan (CS)-stearic acid (SA) conjugate as novel grafted polymer. CS was selected due to its excellent biocompatibility and biodegradability that made it an excellent choice for in vivo applications.14 However, CS as such could not self-assemble into micellar architecture; therefore it was chemically conjugated with SA, a hydrophobic chain. Moreover, conjugation of tumor specific targeting ligands to micelles is expected to increase the therapeutic efficacy of delivery system.15 In this regard, lactobiotin (LA) molecule is recognized by asialoglycoprotien receptor (ASGP-R) which is densely spread in the hepatocytes. Nearly ~500,000 ASGP-R are present per liver cancer cells.16 The LA-conjugated CS-SA micelles (CSS-LA) is expected to actively taken up by liver cells wherein high binding capacity and efficient cellular internalization of carrier will increase the accumulation of drug within the cancer cells.17 Anthracycline-based anticancer drug such as doxorubicin (DOX) is indicated as the first line treatment for liver cancer; however severe adverse effects such as cardiotoxicity and myelosuppression limited its clinical application.19 In the present study therefore, DOX was loaded in CSS-LA micelles to improve its chemotherapeutic efficacy against HC. For this purpose, CS-SA conjugate was prepared and surface modified with LA as an active targeting moiety. The physicochemical characteristics of CSS-LA/DOX were studied in terms of size and release kinetics. Anticancer effect of nontargeted and targeted micellar drug was studied in human hepatic carcinoma cell lines, HepG2 and BEL-7402.18 These cells overexpress receptors for LA molecule. Biological characterizations such as cellular uptake, cytotoxicity assay, and apoptosis assays were performed in these cancer cells. Active targeting ability of CS-SA micelles was studied in HepG2 cancer cell bearing xenograft nude mice and H&E staining was performed.


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EXPERIMENTAL SECTIONS Materials Chitosan (LMW, 95% deacetylated), stearic acid, and doxorubicin hydrochloride was purchased from Sigma-Aldrich (China). Lactobionic acid (LA), N, N’-dicyclohexyl carbodimide (DCC), N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), ethylene diamine (EDA) and 1-(3-dimethylaminopropyl)-3 ethylcarbodimide hydrochloride (EDC) was also purchased from Sigma Aldrich, China. All other chemicals were of reagent grade and used without further purification. Preparation of DOX-loaded CSS-LA nanoparticles Chitosan-stearic acid (CS-SA) conjugate was synthesized by coupling –COOH group of SA with –NH2 group of CS via EDC reaction.20 Briefly, 1.52 g of SA and 10.7 g of EDC was dissolved in ethanol-acetone mixture (2:1) and stirred at 60°C for 1h. The organic mixture was mixed with 120 ml of water and stirred for additional 24h. The chemical conjugate formed was dialyzed (MW cut off: 3500, Spectrum laboratories, CA) against distilled water for 24h at 30°C to remove the by-products (3 dialysis cycles were performed). The concentrated product was removed and freeze dried and stored for further use. LA was introduced on the CS-SA conjugate by a chemical reaction. Briefly, specified amount of LA was dissolved in DMSO, to which DCC/DMSO was added and stirred for 60 min to activate the carboxylic acid group. NHS/DMSO was in turn added to above mixture and stirred for 24h. The –COOH activated LA was added to CS-SA conjugate and stirred at 40°C for 4h. The resulting CS-SA-LA was isolated, dialyzed, and freeze dried. The DOX-loaded CSS-LA micelles were prepared by sonication method. DOX.HCL was converted into base form by adding twice the molar weight of triethylamine. Briefly, 28 mg



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of CSS-LA polymer was dissolved in 10 ml of distilled water and subjected to probesonication for 3 min. Followed by which, DOX was added to the polymer solution at a weight ratio of 10% w/w of polymer. The polymer and drug mixture was dialyzed against distilled water for 12h. The drug-loaded micelles were formed while being in dialysis process. The micelles were collected and stored in a cold place (4°C). Particle size and zeta potential analysis The mean diameter and surface charge was analyzed using dynamic light scattering technique by Zetasizer (Nano-ZS 90, Malvern, Worcestershire, UK). The samples were measured at 25°C at a fixed angle of 90°C. Each sample was measure in triplicate. Morphology analysis The morphological examination of nanoparticles was carried out using transmission electron microscope (TEM) (JEM-2010; JEOL, Japan). Nanoparticle dispersion was placed on the carbon-coated copper grid and negatively stained with 2% (w/v) phosphotungstic acids and air dried. The morphology and surface texture was further confirmed by scanning electron microscopy (SEM; FEI Nova NanoSEM 230). The samples were freeze dried and coated with platinum before the SEM analysis. Drug-loading and encapsulation efficiency UV-Vis Spectrophotometer (Shimadzu® UV-1601 double-beam spectrophotometer) was used to calculate loading capacity and entrapment efficiency of DOX in CSS-LA micelles. Centrifugal tubes were used to calculate the loading of drug in micelles. 2 ml of micellar formulation was added to centrifugal tubes and ultra-centrifuged at 12000 rpm for 15 min. The filtrate was collected and analyzed for free DOX via spectrophotometrically at 481 nm.

DOXtotal - DOXsuper

DL% =

Mass of NP

× 100%

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Actual drug loading EE% =

Theoretical drug loading

× 100%

Drug release study Phosphate buffered saline (PBS; pH 7.4) and acetate buffered saline (ABS; pH 5.0) was used to carry out the release study. The study was carried out at 37°C in a rotary shaker. For this 1 ml of CSS/DOX and CSS-LA/DOX (containing 2 mg of DOX equivalent) was placed in a dialysis tube (MW cut off: 3500 kDa) and then placed in a plastic tube containing 30 ml of release media. The whole assembly was kept in a shaker bath (at 100 rpm) at 37°C. 1 ml of release samples was collected at specific time points and replaced with equal amount of the fresh medium. The amount of drug released in the release media was calculated from the spectrophotometer. Amount of drug released was plotted against time point. Cell culture and cytotoxicity assay BEL-7402 (human hepatocellular carcinoma cell line), HepG2 (human liver tumor cell line) were maintained in DMEM medium containing 10% foetal bovine serum and 1% antibiotic mixture (penicillin-streptomycin). The cells were maintained at 37°C and 5% CO2 in an automated incubator. The cytotoxicity assay was carried out in aforementioned cancer cells using 3-(4,5dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, cells were seeded at a density of 1 × 104 in a 96-well plate. After 24h, cells were exposed to blank polymer, free DOX, CSS/DOX and CSS-LA/DOX at different dosing level. The cells were incubated for 24h. At each time point, plate was removed and treated with 100 µl of MTT solution (5 mg/ml) to each 96-well plate and incubated for 4h. The formed formazan crystals 7 ACS Paragon Plus Environment


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were extracted by adding DMSO and incubated for additional 30 min. The absorbance of each plate was read at 570 nm using a microplate reader (Thermo-Fisher, USA).

All

experiments were repeated 6 times. The cytotoxicity assay is based on the reduction of yellow MTT by mitochondrial succinate dehydrogenase. MTT enters the live cells and reduced into insoluble formazan complex. Quantitative cellular uptake analysis The cellular uptake of free drug and drug loaded micelles were evaluated by HPLC method. Briefly, 5×105 cells were seeded in a 12-well plate and incubated for 18h to attach the cells. Formulations (free DOX, CSS/DOX, and CSS-LA/DOX) were exposed to BEL-7402 and HepG2 cancer cells (final concentration of 20 µg/ml of DOX). The cells were treated with formulations for specified time point to observe the time-dependent cell uptake. The cells were washed twice with PBS and treated with lysis buffer. The cells were harvested, sonicated, and centrifuged at high speed. The cell lysate was collected and subject to HPLC analysis. Earlier, standard calibration of DOX was developed and used to determine the concentration of drug in the cell lysate. Qualitative cellular uptake analysis The mechanism of cellular uptake was evaluated using a fluorescence microscope (Eclipse TE300 Microscope). 5×105 cells were seeded in a 12-well plate containing a cover slip. The cells were allowed to attach overnight and in the next day, media was replaced with CSSLA/DOX containing growth medium and incubated for 1h and 6h. Subsequently, the nuclei of cells were stained with Hoechst33342 for 5 min. Cells were then washed with PBS two times and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were washed and viewed under fluorescence microscope.


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Apoptosis measurement Hoechst 33258 was used to observe the cell apoptosis. During cell apoptosis, condensation of chromatin takes place and DNA gets cleaved into small fragments. Generally, it enters the live cells and binds with adenosine-thymidine (AT) part of DNA while in apoptotic cells, it binds to condensed chromosome. Normal cells and apoptotic cells were different in their size and distinct morphology. The drug treated cells were washed with PBS and stained with Hoechst 33258 for 10 min. The cells were washed and fixed with 4% paraformaldehyde and observed under fluorescence microscope. Apoptosis analysis The quantitative cell apoptosis was evaluated by annexin V/PI double stain assay. Briefly, HepG2 cells were seeded (1×106) in 24-well plates and incubated at 37 °C in a 5% CO2 incubator overnight. The cells were treated with free DOX, CSS/DOX, and CSS-LA/DOX and incubated for 18h. The cells were harvested and washed with PBS and fixed in 75% ethanol. The pellets were resuspended with 100 µl of binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2). The cells were then treated with FITC-Annexin V and incubated for 20 min and then PI was added and incubated for additional 10 min. The cells were analysed for apoptotic cells using flow cytometry (BD Biosciences, USA) and analysed using the CELL Quest Version 3.3 software.

In vivo antitumor efficacy study The animal study was approved by ‘Ethics Committee for Animal’; Second Military Medical University, China. In vivo antitumor efficacy study was performed in 7-week old xenograft nude mice. Briefly, 1×106 HepG2 (100 µl PBS) were subcutaneously injected into the right 9 ACS Paragon Plus Environment


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flank of nude mice to establish hepatic carcinoma models. The formulations (100 µl) were administered through tail vein injection. Treatments were initiated when the tumor volume reached around ~50 mm3 and designated as day 0. The mice were divided into 5 groups with 8 mice in each group. The mice were treated with saline, DOX, CSS/DOX, and CSSLA/DOX at day 0, 2, 4, and 6 at a dose of 5 mg/kg. Tumor sizes were measured with digital caliper and the tumor volume was calculated using the formula: volume = 1/2 × Dmax×(Dmin)2. The body weight was measured simultaneously as an indicator of the systemic toxicity. At the end of the study period, tumors were surgically removed and fixed in 10% neutral formalin and embedded in paraffin. The histopathology of tumor sections was evaluated by hematoxylin and eosin (H&E) method. The embedded paraffin tumor sections were cut into 5 µm slices and stained with H&E staining agent and viewed by microscope (Nikon TE2000U). The mice were humanly sacrificed (using CO2) at the end of study period following the ethics protocol. Statistical analysis Data are expressed as the mean ± standard deviation, and statistical analysis was performed by SPSS. *P< 0.05 was considered significant in the present study.

RESULTS Formulation of CSS-LA/DOX micelles The DOX-loaded micelles were formed by dialysis method. The drug was stably entrapped in the core of the micelles and resulted in nanosized micelles of ~100-120 nm in diameter (Figure 2a). The size of drug loaded micelles was slight bigger than the blank micelles as the hydrophobic drug consumed the internal volume. Moreover, the particle size distribution was 10 ACS Paragon Plus Environment

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uniform with polydispersity index 90% at all the concentrations indicating its excellent safety profile. The least or negligible cytotoxicity of blank polymer makes it ideal for in vivo cancer targeting. To investigate the cytotoxicity of free DOX, CSS/DOX, and CSS-LA/DOX, respective cells were treated with formulations between 0.001 to 10 µg/ml and incubated for 24h. The cell proliferation was estimated by MTT assay. As shown in Figure 6c,d, all formulations inhibited the cell proliferation in a similar manner to both the cell lines (HepG2 and Bel7402). It can be seen that the order of cytotoxicity is CSS-LA/DOX> CSS/DOX> free DOX indicating the superior performance of LA-conjugated micellar formulations. Apoptosis assay Annexin V FITC and PI staining was used quantitate the apoptosis rate in HepG2 cancer cells. The scatter plot has four quadrants: lower left quadrant (Q3) indicates viable cells (Annexin −ve, PI −ve), lower right quadrant (Q4) indicates early apoptotic cells (Annexin +ve, PI −ve), upper right quadrant (Q2) indicates late apoptotic cells (Annexin +ve, PI +ve) and upper left quadrant (Q1) indicates necrotic cells (PI +ve). As shown in Figure 7a, almost all cells were confined in viable chamber in case of untreated group. The free DOX and CSS/DOX however increased the cell proportion in early and late apoptosis chamber. Most marked results were observed from CSS-LA/DOX treated cell group which showed significant presence of cells in early, late and necrosis chamber. Specifically, ~10% in early 13 ACS Paragon Plus Environment


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apoptosis phase, ~8% in late apoptosis phase, and ~18% of cells in necrosis chamber were observed. The results clearly showed that the number of living cells greatly reduced in this group with marked necrotic and apoptotic cells. The enhanced apoptosis effect of LAconjugated carrier was consistent with its remarkable cytotoxic effect in HepG2 cells. Qualitative apoptosis analysis Apoptosis of cells were further confirmed by fluorescence microscope after staining with Hoechst 33342. The cells were treated with respective formulations and observed under the microscope after 24h incubation. Changes in cell morphology are regarded as one of the prominent hallmark of apoptosis. As shown in Figure 7b, untreated cells did not show any changes in morphology and maintained good integrity. Slight change in morphology was observed after exposing free DOX, however remarkable changes have been observed in case of CSS/DOX and CSS-LA/DOX. Importantly, LA-conjugated carrier induced typical chromatic condensation of cells; membrane blebbing and apoptotic bodies were begin to appear. The nuclei shrink and fewer numbers of cells were observed on the cover slip indicating the potent effect of CSS-LA/DOX on the cancer cells. Pharmacokinetic and biodistribution analysis The plasma concentration-time profile of formulations following single dose intravenous administration is shown in Figure 8a. As shown, free DOX rapidly cleared from the blood circulation with 3-4h, whereas, drug-loaded nanoparticles significantly enhanced the circulation profile of DOX. The long blood circulation profile of NP will maintain an effective therapeutic level of drug in the systemic circulation. The biodistribution of drug loaded formulations will clearly depict the amount of drug in each tissue (Figure 8b). According to the results, distribution of NP was significantly lower in liver, spleen, and heart comparing to that of free DOX. Most remarkably, CSS-LA/DOX achieved highest 14 ACS Paragon Plus Environment

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concentration in the tumor by comparison to either free drug or CSS-DOX. The significantly higher concentration of CSS-LA/DOX in tumor might be attributed to its ligand-targeting ability to the ASGP-R expressing cancer cells and long circulating property of NP. Antitumor efficacy study The therapeutic efficacy of formulations was investigated on HepG2 cancer cell bearing xenograft nude mice. After subcutaneous injection of liver cancer cells on the right flank, tumor was allowed to grow and the treatment was started after 10 days. The mice were treated with free DOX, CSS/DOX, and CSS-LA/DOX at a dose of 5 mg/kg (3 times). Tumor volume and body weight of mice were periodically measured (Figure 9a). It can be seen that tumor treated with saline and blank nanoparticle did not control the growth of tumor and grew up rapidly. The tumor in free DOX treated group also grew steadily however with lesser rate than that of control groups. CSS/DOX was successful to certain extent in controlling the progression of cancer cells. Importantly, mice treated with LA-conjugated carried significantly reduced the tumor progression and displayed a remarkable tumor inhibition effect on the tumor. The body weight was simultaneously monitored to confirm the safety profile of individual formulations (Figure 9b). It can be seen that free DOX treated group experienced a 10% reduction in body weight indicating its drug-related organ toxicity. CSS/DOX and CSSLA/DOX however maintained the body weight of mice and did not show any loss in body weight. The decrease in body weight in case of free DOX treated group might be due to the unspecific distribution of DOX leading to toxic effect in mice.



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Histopathological analysis H&E staining was performed to stain the tumor sections wherein nucleus was stained with hematoxylin (blue) and extracellular matrix was stained with eosin (pink). The cells were intact with clear morphology and chromatin in case of tumor from untreated mice group (Figure 9c). The cell morphology was displaced with irregular outline and signs of necrosis in case of free DOX and CSS/DOX administered mice group. As expected, LA-conjugated nanocarriers resulted in marked necrosis with fewer cells present in the observed tumor sections.

DISCUSSION Hepatic cancer is one of the most lethal malignancies recorded in human history with highest rate of incidence and mortality worldwide. At present surgical resection is the main treatment option however practical difficulty makes the resection rate to less than 30%. Chemotherapy is employed as alternative or adjuvant therapy.4,5 Specifically, anthracycline-based anticancer drug such as doxorubicin (DOX) is indicated as the first line treatment for liver cancer; however severe adverse effects such as cardiotoxicity and myelosuppression limited its clinical application. Additionally, much of the free drugs accumulate in the normal tissues causing severe damage to normal physiological processes and vital organs. In this regard, DDS has been reported to improve the pharmacological and anticancer property of chemotherapeutic drugs. Although nanocarriers improved the biological performance of anticancer drugs yet it was not satisfactory. In the present study therefore we investigated the potential of ligand-targeted polymeric micelles for the delivery of DOX to the cancer cells. For this purpose, chitosan (CS)-stearic acid (SA) conjugate as novel grafted polymer (Figure 1). CS was selected due to its excellent biocompatibility and biodegradability that made it an 16 ACS Paragon Plus Environment

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excellent choice for in vivo applications, while SA was selected to increase its hydrophobicity.14 Importantly, lactobiotin (LA) molecule was conjugated with CS-SA to increase its specificity towards asialoglycoprotien receptor (ASGP-R) of liver cancers.16 The release study was performed in phosphate buffered saline (pH 7.4) and acetate buffered saline (pH 5.0). None of the formulations showed burst release profile and released the drug in a sustained manner for up to 72. It should be noted that both the micellar formulations released the drug in the same level and presence of LA did not affect the drug release. It could be expected that at physiological pH conditions, core will be intact and DOX would be blocked in the highly hydrophobic core leading to low release rate. However when the pH decreased accelerated release was observed due to the protonation of molecular residue. Specific and fast drug release from acid-triggered micelles was very effective inside the cancer cells. The core-shell architecture was expected to protect the drug in the systemic blood circulation, but would be destabilized in the acidic environment of cancer cells. The DOX released following processes; firstly, the media entered the interior of micelles and the drug dissolved, secondly, dissolved drug spread to the release media gradually. Overall, release study showed that micelles can effectively release the loaded drug in the intracellular environment while protecting its bioactivity. Cellular internalization capacity of free DOX, non-targeted CSS/DOX, and CSS-LA/DOX was studied in HepG2 and BEL-7402 liver cancer cells. For example, ~450 ng/ml of DOX was internalized from CSS-LA micelles in HepG2 cell line comparing to ~300 ng/ml from CSS micelles at the end of 6h. Similar trend was observed in Bel-7402 cells where in LA conjugated micelles showed enhanced cellular uptake. This is because the targeting ability of the LA moieties in CSS micelles is higher than in non-conjugated CSS micelles. The results indicate the strong and specific binding of LA moiety to both cancer cells which overexpress abundant ASGP-R receptor.17 Ligand receptor complex is rapidly internalized and receptor 17 ACS Paragon Plus Environment


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recycles back to surface, which would allow the high binding capacity and efficient cellular uptake of galactosylated carriers. The enhanced cytotoxic effect of CSS-LA/DOX was consistent with its higher cellular uptake in both the cell lines. It is well known that free drug easily diffuse into the cell membrane whereas micellar drug takes a specific cellular internalization pathway and releases the drug in a systemized manner. In the present study, ligand receptor complex is rapidly internalized and receptor recycles back to surface, which would allow the high binding capacity and higher cell cytotoxicity.17 It could be expected that both HepG2 and Bel-7402 expresses abundant ASGP-R receptor which is densely spread in the hepatocytes. IC50 value was calculated to quantitate the amount of drug required to kill 50% of cancer cells. The IC50 value was calculated from GraphPad prism (version5) software. The CSS/DOX reduced the IC50 value by around ~35% and ~32% in HepG2 and Bel-7402 cells, respectively comparing to that of free DOX. More importantly, CSS-LA/DOX reduced the IC50 value of free DOX by around ~85% and ~90% in HepG2 and Bel-7402 cells, respectively, indicating its superior performance via ligand-mediated cellular uptake. The study indicated that the cell inhibition was mainly caused by the cell internalization of the micelles through LA-mediated targeting. The therapeutic efficacy of formulations was investigated on HepG2 cancer cell bearing xenograft nude mice. The final tumor volumes were ~2400 mm3, 1500 mm3, 1100 mm3, 650 mm3 for saline, free DOX, CSS/DOX, CSS-LA/DOX treated mice, respectively. The remarkable tumor reduction effect of CSS-LA/DOX was attributed to the targeting ability of the LA moieties in CSS micelles. The results indicate the strong and specific binding of LA moiety to both cancer cells which overexpress abundant ASGP-R receptor.15 It could be expected that ligand receptor complex is rapidly internalized and receptor recycles back to


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surface, which would allow the high binding capacity and efficient cellular uptake of galactosylated carriers. Other reasons might be the prolonged blood circulation time and efficient accumulation of micelles in the tumor region via EPR effect. H&E staining was performed to stain the tumor sections wherein nucleus was stained with hematoxylin (blue) and extracellular matrix was stained with eosin (pink). As expected, LAconjugated nanocarriers resulted in marked necrosis with fewer cells present in the observed tumor sections. The extent of necrosis followed the order of >CSS-LA/DOX>CSS/DOX>free DOX. The results clearly indicate the greater therapeutic index of ligand-conjugated carried than that of free drug. The results are consistent with the enhanced cellular uptake potential of CSS-LA/DOX and greater ability to induce apoptosis.

CONCLUSION In summary, we have designed and fabricated LA-based polymeric drug delivery system and characterized its physicochemical and in vivo properties. DLS and TEM showed a nanosized spherical particle with excellent physical stability. The CSS-LA/DOX exhibited a sustained release profile with pH-responsive characteristics. Accelerated release pattern was observed under acidic conditions. The LA conjugated micelles showed enhanced cellular uptake in HepG2 and BEL-7402 liver cancer cells than free drug and unconjugated micelles. Consistently, CSS-LA/DOX showed enhanced cell cytotoxicity in these two cell lines. Annexin-V/FITC and PI based apoptosis assay showed that the number of living cells greatly reduced in this group with marked presence of necrotic and apoptotic cells. LA-conjugated carrier induced typical chromatic condensation of cells; membrane blebbing and apoptotic bodies were begin to appear. In vivo, CSS-LA/DOX showed an excellent tumor regression profile with no toxic side effects. The active targeting moiety, long circulation profile, and 19 ACS Paragon Plus Environment


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EPR effect contributed to its superior anticancer effect in HepG2 based tumor. Our results showed that polymeric micelles conjugated with lactobionic acid (LA) increased the therapeutic availability of DOX in the liver cancer cell based solid tumor without any toxic side effects. The active targeting ligand conjugated nanoparticulate system could be a promising therapeutic strategy in the treatment of hepatic cancers. ACKNOWLEDGEMENT This work supported by State key infection disease project of China (NO: 2012ZX10002010) and Science Fund for Creative Research Groups (NSFC, No: 81221061, No: 81201940 and No: 81372207).

DISCLOSURES The authors report no conflicts of interest about this work.

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Figure captions Figure 1: Schematic illustration of conjugation of chitosan (CS), stearic acid (SA), and lactobionic acid (LA). Formation of polymeric micelles via self-assembly process has been depicted. Figure 2: Physicochemical characterization of CSS-LA/DOX. (a) Particle size distribution of CSS-LA/DOX (b) transmission electron microscope (TEM), (c) scanning electron microscope (SEM) image. Figure 3: In vitro drug release study of formulations. Drug release study has been performed in phosphate buffered saline (pH 7.4) and acetate buffered saline (pH 5.0). The sampling has been done at specific time points. *P

Ligand-Directed Stearic Acid Grafted Chitosan Micelles to Increase

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