Design and development of ligand-appended polysaccharidic nanoparticles for the delivery of oxaliplatin in colorectal cancer

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Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 179 – 190

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Original Article

Design and development of ligand-appended polysaccharidic nanoparticles for the delivery of oxaliplatin in colorectal cancer Anekant Jain, MPharma , Sanjay K. Jain, PhDa,⁎, N. Ganesh, PhDb , Jaya Barve, MScb , Aadil M. Beg, BUMSb a

Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar, India b Department of Research, Jawaharlal Nehru Cancer Hospital & Research Center, Iidgah Hills, Bhopal, India Received 9 July 2008; accepted 16 March 2009

Abstract Hyaluronic acid–coupled chitosan nanoparticles bearing oxaliplatin (L-OHP) encapsulated in Eudragit S100–coated pellets were developed for effective delivery to colon tumors. The in vitro drug release was investigated using a USP dissolution rate test paddle-type apparatus in different simulated gastrointestinal tract fluids. In therapeutic experiments the pellets of free drug, and hyaluronic acid–coupled and uncoupled chitosan nanoparticles bearing L-OHP were administered orally at the dose of 10 mg L-OHP/kg body weight to tumorbearing Balb/c mice. In vivo data showed that hyaluronic acid–coupled chitosan nanoparticles delivered 1.99 ± 0.82 and 9.36 ± 1.10 μg of L-OHP/g of tissue in the colon and tumor, respectively after 12 hours, reflecting its targeting potential to the colon and tumor. These drug delivery systems show relatively high local drug concentration in the colonic milieu and colonic tumors with prolonged exposure time, which provides a potential to enhance antitumor efficacy with low systemic toxicity for the treatment of colon cancer. From the Clinical Editor: In this study, a nanoparticle system was developed to deliver oxaliplatin to colorectal tumors. In murine models, the drug delivery system showed relatively high local drug concentration in colonic tumors with prolonged exposure time, which provides a potential for enhanced antitumor efficacy with low systematic toxicity. © 2010 Elsevier Inc. All rights reserved. Key words: Colon-targeted drug delivery; Chitosan nanoparticles; Colorectal cancer; Eudragit S100; Oxaliplatin

Cancer is the second leading cause of death in the United States, exceeded only by heart disease, and accounts for one in four deaths. Cancer of the colon and rectum is one of the most common internal malignancies. An estimated 1,419,000 cancer cases were diagnosed in 2007, and 148,210 Americans died of colorectal cancer in 2007.1 Targeting of drugs specifically to the colon is advantageous for the treatment of diseases associated with the colon such as amebiasis, Crohn disease, ulcerative colitis, and colorectal cancer.2 A drug delivery system is most often associated with particulate carriers such as emulsion, liposomes, and nanoparticles, which are No conflict of interest was reported by the authors of this article. One of the authors, Anekant Jain, is thankful to ICMR New Delhi, India, for funding a Senior Research Fellowship to carry out the project on colonspecific drug delivery. ⁎Corresponding author. E-mail addresses: [email protected], [email protected] (S.K. Jain).

designed to localize drugs at the target site.3-5 The efficacy of present cancer chemotherapy is mainly limited by the toxicity associated with the anticancer drugs to normal tissues. This limitation results from the lack of efficient selectivity toward tumor cells of anticancer drugs presently used in chemotherapy. However, conventional chemotherapy is not as effective in colorectal cancer as it is in other cancers, because the drug does not reach the target site in effective concentrations.6,7 Thus, effective treatment demands increased dose size, which may lead to undue consequences. To improve this situation, pharmaceutical technologists have been working on ways to deliver the drug more efficiently to the colon, where it can target the tumor tissues. Ciftci and Groves showed that it is possible for a colon-targeted delivery system to selectively deliver drug to tissues, not through tissues.8 It is possible that delivery of small quantities of antineoplastic agent to the inner surface of the colon could destroy small tumors that arise spontaneously in this region, reducing the need for

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.03.002

Please cite this article as: A. Jain, S.K. Jain, N. Ganesh, J. Barve, A.M. Beg, Design and development of ligand-appended polysaccharidic nanoparticles for the delivery of oxaliplatin in colorectal cancer. Nanomedicine: NBM 2010;6:179-190, doi:10.1016/j.nano.2009.03.002

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surgery. Drugs are commonly delivered to the large bowel by coating them with polymeric substances such as cellulose derivatives9 or acrylic polymers.10 These polymers protect the drug against the acidic environment of the stomach, because they swell while traveling down the intestine. It is postulated that the drug is released from the coated dosage form when it arrives at the distal ileum.9,10 The performance of such colonic delivery systems may be limited by gastrointestinal (GI) motility and pH variations. To overcome these limitations, new delivery systems with alternative drug release mechanism have been suggested.2,11-13 Oxaliplatin [Oxalato(trans-l,1,2-diaminocyclohexane] platinum(II) (L-OHP) is a third generation of platinum (Pt) antitumor compound, and it is now approved as first-line chemotherapy in combination with 5-fluorouracil for the treatment of advanced colorectal cancer. Despite its better tolerability in comparison to other Pt compounds, like cisplatin and carboplatin, oxaliplatin is associated with a few side effects (acute dysesthesias, cumulative peripheral distal neurotoxicity) that limit the range of usable doses. Systemic chemotherapy with L-OHP, alone or in combination with other chemotherapeutic agents, is effective, but side effects due to drug interactions at sites other than those associated with the tumors often result in low patient compliance and higher failure rates.14 From a drug delivery perspective it would be preferable to deliver smaller quantities of the antineoplastic directly to the tumor site, thereby improving the prospects for a successful treatment outcome. Anticancer drug targeting via active targeting approach exploits specific interactions between receptors on the cell surface and targeting moieties conjugated to the polymer backbone. The active approach therefore takes advantage of the enhanced permeability and retention effect but further increases the therapeutic index through receptor-mediated uptake by targeting cancer cells. In other studies on the transport of macromolecules into tumor tissues, it was demonstrated that the vascular pore size of the LS174T tumor, a human colon adenocarcinoma, could be as large as 400 nm.15 Therefore, nanoparticles well below this particle size range can be effectively targeted via the enhanced permeability and retention effect of passive targeting. In addition to elevated hyaluronic acid (HA) in the environment surrounding tumors, most malignant cell types overexpress the HA receptors (viz. CD44 and RHAMM).16,17 Isoforms of HA receptors, CD44 and RHAMM are also overexpressed in transformed human breast epithelial cells,18 colorectal carcinoma,19-22 and other cancers.23 As a result, malignant cells with the highest metastatic potential often show enhanced binding and internalization of HA.24 Working on this rationale, we selected oxaliplatin as a drug along with HA-coupled chitosan nanoparticles for the study. The present work describes our recent contribution to the design of a drug delivery system bearing L-OHP for colon tumor targeting. Methods Materials Chitosan (purified viscosity grade 50 cps; molecular weight [MW] 150 kDa; deacetylation degree 85%) was procured as a gift sample from M/s Panacea Biotech (Chandigarh, India). Fermentation-derived HA (sodium salt, Mr 1.5 MDa) was purchased from Sigma-Aldrich India Ltd. (Mumbai, India).

L-OHP was received as a gift sample from Khandelwal Laboratories Ltd. (Mumbai, India). Eudragit S100 (Rohm GmbH & Co KG., Darmstadt, Germany) was procured as a gift sample from Degussa, Germany. Triethylcitrate was purchased from Himedia (Mumbai, India). HT-29 cells were purchased from National Center of Cell Science (Pune, India) and cultured in McCoy's Medium (Gibco, Eggenstein, Germany) and supplemented 1:1 (vol/vol) with 10% fetal bovine serum (Gibco), 2 mmol/L glutamine (Sigma-Aldrich, Mumbai, India), and 1000 IU/mL penicillin-streptomycin serum (Gibco). Sodium tripolyphosphate (TPP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, and dialysis membrane (MW cutoff 3500) were purchased from Himedia. All other chemicals used were of analytical grade. Doubledistilled water was used throughout the study. Preparation of chitosan TPP nanoparticles Chitosan nanoparticles were prepared according to the procedure first reported by Calvo et al25with suitable modifications based on the ionotropic gelation of chitosan with TPP anions. Chitosan (2.0 mg/mL) was dissolved in aqueous acetic acid (pH 4.0) solution, and TPP was dissolved in purified water (1.0 mg/mL). Drug (10.0%) was added to the TPP solution (drug dissolved previously in 100 mL distilled water) and stirred for 5 minutes using a magnetic stirrer. Finally, 1.5 mL of drugcontaining TPP solution was added to 4 mL of the chitosan solution through a syringe needle under magnetic stirring at room temperature, thereby leading to formation of drug-loaded chitosan nanoparticles at room temperature (25°C). The milky dispersion formed was centrifuged (Hitachi, Himac CP100 MX; Tokyo, Japan) at 10,000 rpm at 4°C for 30 minutes. The supernatant was discarded, and sediment was poured into a dialysis bag and dialyzed three times with phosphate-buffered saline (PBS; pH 7.4) under strict sink conditions for 10 minutes to remove free drug from the formulation, which was then estimated spectrophotometrically at 254 nm (UV-1601; Shimadzu, Kyoto, Japan) to determine indirectly the amount of drug loaded within the system. The dialyzed formulation was lyophilized and used for further characterization. Conjugation of nanoparticles with HA HA was covalently coupled with its carboxyl group to the free amino group of chitosan present on the surface of nanoparticles using EDC as coupling agent according to the procedure reported previously by our laboratory with few modifications.5 Briefly, drug-loaded lyophilized nanoparticles were suspended into PBS (pH 7.4) containing HA (HA/ nanoparticles ratio,10:100 wt/wt). EDC (20 mg/mL of HA/ nanoparticles mixture) was added and vortexed and incubated for 1.5 hour at room temperature. The ligand-coupled nanoparticles were separated from unconjugated ligand using minicolumn centrifugation (Sephadex G-75 packed column Sigma-Aldrich, Mumbai, India) technique and washed three times with deaerated distilled water. The nanoparticles were collected after ultracentrifugation (Himac CP100 MX, Hitachi) at 60,000 rpm for 45 minutes, and the resultant pellet was redispersed in distilled water and subsequently preserved after

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lyophilization at 4°C. The amount of HA coupled to the surface of the nanoparticles was assessed by change in the zeta potential. The two process variables (incubation time and total nanoparticulate formulation-to-HA weight ratio) were optimized by measuring the change in zeta potential (the surface charge density) of the dispersion (Zetasizer 3000 HS; Malvern Instruments, Worcestershire, United Kingdom). Preparation of enteric-coated nanoparticulate pellets Preparation of nanoparticulate pellets The pellets of HA-coupled (HACTNPOPs) and uncoupled (CTNPOPs) chitosan nanoparticles bearing L-OHP were prepared using extrusion-spheronization technique (UFEE 60; Umang Pharmatech, Ahmedabad, India). Briefly, Avicel (Rankem RFCL Ltd., Okhla, New Delhi, India) (25%) and L-OHP– loaded nanoparticles (75%) were first mixed using a laboratory blender (LB100 Laboratory Blender; REMI, Mumbai, India) for 5 minutes, and deionized water was added with further mixing (10 minutes) for the production of wet mass. The wet mass was extruded with an extruder (sieve opening 1 mm; screen thickness 3.25 mm; 15 rpm; extrudate cut off at a length of approximately 2–3 mm) and spheronization of the extrudate in a spheronizer (spheronization speed 2200 rpm; spheronization time about 5 minutes). Pellets in the size fraction 1.1–1.4 mm (N75% yield in this size range) were used in the subsequent studies. The pellets were dried for 3 hours at 40°C. Radiolabeled enteric pellets of chitosan nanoparticles (CTNPTEPs) bearing technetium 99m–labeled diethylene triamine pentacetate (99mTc-DTPA) were prepared similar to the method as discussed above, and all the ingredients were used in the same quantity, except that the drug was replaced with sodium chloride having radioactive (99m Tc-DTPA) tracer adsorbed on its surface. Coating of nanoparticulate pellets The pellets obtained as above were subjected to enteric coating by conventional coating pan and were termed as L-OHP–loaded HA-coupled (HACTNPOEPs) and uncoupled (CTNPOEPs) enteric pellets and tracer-loaded enteric-coated radioactive pellets (CTNPTEPs). The enteric-coating solution was prepared as reported by Huyghebaert et al.26 In brief, it was prepared by first making a milky latex of Eudragit S100 using 1 M ammonia (1.5% vol/vol) and kept under stirring for 60 minutes; then, after 1 hour triethylcitrate (20%) was added and stirring was continued for another 60 minutes. The enteric-coating dispersion was passed through a 0.3-mm sieve before use. Throughout the coating process the coating dispersion was stirred using a magnetic stirrer. The parameters of the film-coating process were as follows: pan rotating speed 20 rpm; atomizing air pressure 2 bar; inlet air temperature 60°–70°C; outlet air temperature 35°–40°C; pellets bed temperature 38°C; the coating solution was applied through a 1.1-mm spray nozzle. The film-coated pellets were not removed from the pan until complete weight gain was achieved. All the pellets were stored in a vacuum dessicator at room temperature until used. A series of coated products with different film thicknesses were produced, quantified by the percentage total weight gain (% TWG), by varying the amount of coating solution

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Table 1 Characterization of L-OHP–loaded uncoupled and coupled nanoparticles⁎ Formulation

Particle size (nm)

EE (%)

Zeta potential (mV)

CTNPOPs HACTNPOPs

136 ± 6.0 152 ± 5.2

44 ± 4.2 40 ± 3.9

+40.3 ± 1.4 +10.0 ± 0.5

EE, entrapment efficiency. ⁎ EDC (coupling agent), 20 mg/mL nanoparticles; incubation time, 90 minutes; total HA/nanoparticle ratio 10:100. Each value is shown as mean ± SD (n = 4).

sprayed. The coated pellets were removed from the coating chamber and cured in an oven for 1 hour at 40°C to ensure coalescence of the film coat. The film thickness is expressed as the theoretical percentage of the weight gained (% TWG). It was calculated using the following formula:   CP  1  100 TWG ðkÞ = UP where UP is the weight of the uncoated pellets and CP is the weight of the coated pellets. Characterization of nanoparticles Particle morphology, size, and zeta potential analysis The nanoparticles were characterized for their shape and morphology by scanning electron microscopy (SEM; Philips XL 30 scanning microscope; Philips, Eindhoven, The Netherlands). The resolution of 3.5 nm was used with secondary electron image display. The nanoparticles were coated with goldpalladium alloy (150–250 Å) using a sputter coater. The coater was operated at 2.2 kV, 20 mV, 0.1 torr (argon) for 90 seconds at an accelerating voltage of 15 kV. The samples were viewed under a scanning electron microscope. The particle size, size distribution, and polydispersity index of the drug-loaded nanoparticles were assessed by photon correlation spectroscopy using Zeta Sizer (Zetasizer 3000 HS; Malvern Instruments). The morphological examination of the nanoparticles was also performed by transmission electron microscopy (JEM-200 CX; JEOL, Tokyo, Japan). The samples were stained with 2% (wt/vol) phosphotungstic acid for 10 seconds, and excess of the solution was drained off with a filter paper. The grid was allowed to thoroughly dry in air, and samples were viewed under a transmission electron microscope. Particle size and zeta potential of the uncoupled and HA-coupled chitosan nanoparticles was determined using Zeta Sizer (Zetasizer 3000; Malvern Instruments) (Table 1). Percent encapsulation efficiency The entrapment of L-OHP in nanoparticulate formulations was determined using the method reported by Fry et al after suitable modifications.27 The unentrapped drug from nanoparticulate suspension was removed by passing the formulation through a Sephadex G-50 (Sigma-Aldrich, Mumbai, India) minicolumn and centrifuged at 10,000 rpm for 5 minutes. The nanoparticles were then lysed using methanol, and drug content was determined spectrophotometrically at 254 nm (UV 1601; Shimadzu). All

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measurements were performed in triplicate. The percentage drug encapsulation efficiency was calculated by the formula: Encapsulation efficiency ð%Þ ¼

Total drug ðmgÞ−Free drug ðmgÞ  100 Total drug ðmgÞ

Fourier transform–infrared spectroscopy (FT-IR) FT-IR spectral measurement was performed to assess the coupling of HA to the amino group of chitosan of chitosan nanoparticles. The polymer samples were mixed with potassium bromide and compressed on a hydraulic press to make pellets. Spectra were taken by scanning the samples between 400 and 3000 cm–1 on a FT-IR spectrometer (Pyrogon 1000; PerkinElmer, Shelton, Connecticut). Differential scanning calorimetry (DSC) studies DSC study was performed to characterize the physical state of L-OHP in nanoparticles. Thermograms were obtained using a DSC (Shimadzu). About 5 mg of a sample was weighed, crimped into an aluminum pan, and analyzed at a scanning temperature range from 50° to 300°C at the heating rate of 10°C/min. Baseline optimization was performed before each run. X-ray diffraction (XRD) studies Molecular arrangements of drug (L-OHP) in nanoparticulate formulations were compared by powder XRD patterns acquired at room temperature on an x-ray diffractometer (PANalytical X'pert PRO; Lelyweg, Almelo, The Netherlands) using CuKα radiation. The data were collected over an angular range from 3 degrees to 50 degrees 2θ in continuous mode using a step size of 0.02 degree 2θ and step time of 5 seconds. In vitro drug release In vitro drug release in GI fluids of different pH values In vitro drug release studies were carried out according to Souder and Ellenbogen extraction technique28 using modified USP dissolution test apparatus (apparatus 2). The scheme of using the simulated fluids at different pH values was as follows:

Hour 1: Simulated gastric fluid of pH 1.2 Hours 2–3: Mixture of simulated gastric and intestinal fluid of pH 4.5 Hours 4–5: Simulated intestinal fluid of pH 7.5 Hours 6–8: Simulated colonic fluid of pH 7.0 In vitro drug release from the formulations, CTNPOEPs and HACTNPOEPs, was studied using a dialysis bag. Drug-loaded nanoparticles were taken into a dialysis bag (MW cutoff 3500), which was placed in a beaker containing 100 mL of simulated fluids at 37 ± 1°C with slow magnetic stirring under perfect sink conditions, and fluids were changed according to the Souder and Ellenbogen scheme at each time point. Samples (1-mL aliquots) were withdrawn periodically and replaced with the same volume of fresh dissolution medium, and the amount of drug was

quantified spectrophotometrically (UV 1601; Shimadzu) at 254 nm for L-OHP. Induction of colonic tumor In the present study new methodology was chosen for induction of colon cancer in C57BL mice by making these mice immunodeficient via irradiation so that they accept human cell lines easily. The cobalt (60Co) teletherapy unit (Pteraphon 780C; M/s Theraponics Ltd., Ottawa, Ontario, Canada, attached with New 150 RMM radiation source) in the Jawaharlal Nehru Cancer Hospital and Research Center (Bhopal, India) was used for irradiation. HT-29 cancer lines (2 × 106 cells/mouse) were injected into the mucosa of the ascending colon of anesthetized mice. All procedures were performed in a laminar-flow hood using aseptic techniques, and these mice were removed (after a week) from the laminar hood only after confirmation of their well-being. Plasma and tissue distribution study The drug distribution profiles of L-OHP–loaded formulations (pellets of free drug, CTNPOEPs, and HACTNPOEPs) in various organs/tissues after oral administration were investigated. Healthy C57BL tumorigenic mice (male, 30–35 g) were assigned randomly into four groups, with 18 mice in each group for in vivo studies. The first group served as control. Pellets of free drug were given to all mice in the second group, whereas animals of the third and fourth groups received CTNPOEPs and HACTNPOEPs formulations, respectively, by oral gavage. The dose of L-OHP given to animals was equivalent to 10 mg/kg body weight of animals. The animals were kept in well-spaced ventilated cages and maintained on healthy and normal fixed commercial pellet diet (Hindustan Lever, Bangalore, India), and water ad libitum. These studies were carried out according to the guidelines of the Council for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India, and after approval from the University Animal Ethical Committee (Registration No. 379/01/ab/CPCSEA). Two animals from each group were killed at each time point (i.e., 0, 2, 4, 6, 8, 10, 12, 16, and 24 hours. The GI tract (GIT) was removed, and the mesenteric and fatty acid tissues were separated. The GIT was segmented into the stomach, small intestine, cecum, and colon. Tumor was also isolated, and simultaneously the blood was also collected from the heart puncture. The luminal contents were removed by applying gentle pressure with wet scissors to the tissues. Organs and luminal contents were weighed. One gram of each part of the GIT was homogenized with 4 mL PBS (pH 7.4) using tissue homogenizer (MAC Micro Tissue Homogenizer; New Delhi, India) at 4°C. In the case of GIT parts weighing less than 1 g, the whole GIT part/tissue was used. To 150 μL of tissue homogenate an equal volume of 10% (vol/vol) trichloroacetic acid in water was added and mixed by vortexing for 30 seconds. After centrifugation of tissue homogenates (1000g for 10 minutes at 4°C), the fatty layer was discarded and supernatants were collected. Luminal contents were diluted with PBS followed by centrifugation, and supernatants were

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Figure 1. Mechanistic scheme of colon-specific targeting of Eudragit-coated pellets encapsulating nanoparticles (arbitrary scale).

collected. Supernatants from both the above samples were filtered through a 0.45-μm membrane filter, and a 50-μL aliquot was injected into a high-performance liquid chromatography (HPLC) column for the analysis of L-OHP. Similarly, the blood samples were prepared and analyzed for the drug content using the HPLC method. HPLC equipment (Shimadzu) consisted of a Shimadzu LC-10ATvp with a Rheodyne manual sample injector (FCV14AH) fitted with a 50-μL sample loop and a Shimadzu SPDM10Avp diode array spectrophotometric detector. The column was a cation exchange resin in the form of sulfonated styrenedivinylbenzene copolymer (Aminex HPX-87H, 9-μm mean particle diameter, 300 × 7.8 mm internal diameter; Bio-Rad, Ivery-sur-Seine, France) preceded by a similar guard column with UV detection system (254 nm), whereas the mobile phase used was acetonitrile and water (80:20, vol/vol) and filtered through a 0.2-mm membrane filter. The flow rate of the mobile phase was 0.8 mL/min.29 The method was linear in the range of 0.1 to 40 μg/mL; the limit of detection was 0.001 μg/mL. The intraday and interday variations of the HPLC method were found to be less than 3.6% (CV) and less than 10.1% (CV), respectively.

scanning. The camera was linked to an online computer system for acquisition and storage of data. The useful field of view was 256 × 256 mm, and the mean energy window of 99m Tc was 140 ± 14 keV. The camera was set for 100 Kcounts for each acquisition, and anterior images were recorded with the rodents in supine position. Images were recorded just after administration of the formulation and at 10-minute intervals for 1 hour and then at 20-minute intervals up to 4 hours and later on at 60-minute intervals up to the end of the experiment. In between image acquisitions, the rodents were free to move away from the camera. Images were digitally recorded using an integrated computer system and archived onto optical disk for subsequent analysis. Statistical analysis The results were expressed as mean ± SD, and statistical evaluation of data was performed using an analysis of variance. A probability level of P b .05 was considered significant. Results

γ-Scintigraphic study

Preparation and characterization of HA-coupled chitosan nanoparticles (HACTNPs)

The present study was performed to investigate the behavior of drug dosage form and to assess the release pattern of the drug qualitatively within the GIT of mice. Mice (male) weighing between 30 and 35 g were selected and were fasted overnight before administration of the radiolabeled nanoparticles encapsulated in enteric-coated pellets (CTNPTEPs). To obtain the outline of the whole-body image a very low radioactive 99mTc-DTPA quantity (1 μCi) was injected after every 6 hours through the tail vein. Now, to assess the presence of the pellets in different parts of GIT, a high dose (1 mCi) of 99mTc-DTPA was encapsulated in pellets and radioactivity was measured using radioisotope dose calibrator (CRC-127R; Capintech Inc., Pittsburgh, Pennsylvania). These enteric-coated pellets were orally administered to the mice with 2 mL of water. The dosed mice were kept in a restraining cage that was placed under the scintillation camera. Transit of the pellets through the GIT was monitored at different time intervals using a gamma camera (E. Cam; Siemens, Munchen, Germany) fitted with low-energy all-purpose collimeter for

The main goal of this work was to investigate the potential of HACTNPs and to compare them to uncoupled chitosan nanoparticles (CTNPs) for colon delivery of anticancer drug on oral administration. Therefore, we expected to collect information not only about the potential of HACTNPs for increasing concentration of anticancer drug to the site of colon tumors but also for their selective and preferential localization at tumor cells for effective treatment of cancer. Figure 1 describes the scheme of targeting of enteric-coated pellets bearing drugloaded ligand-coupled nanoparticles (HACTNPOEPs) to the site of colon tumor. L-OHP–loaded nanoparticles of chitosan were prepared using ionotropic gelation technique. SEM and TEM revealed the spherical shape of HACTNPs (Figure 2). The mean diameters of HACTNPOP and CTNPOP formulations were 152 ± 5.2 and 136 ± 6.0 nm, respectively. Low polydispersity indexes (0.155 and 0.110) obtained for both of the formulations indicated a narrow size distribution of the nanoparticles suspension and consequently a homogeneous distribution. The

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Figure 3. Formulations with different ratios of nanoparticles (NP) and HA were prepared. (A) Incubation time optimization for coupling of HA at 25°C. (B) Optimization of total NP/HA weight ratio (zeta potential values after time period of 24 hours at 25°C). All values are expressed as mean ± SD (n = 4).

zeta potentials of CTNPOP and HACTNPOP formulations were observed to be 40.3 ± 1.4 and 10.0 ± 0.5 mV, respectively (Figure 3; Table 1). Entrapment efficiency was found to be less in HACTNPs (40% ± 3.9%) as compared with uncoupled nanoparticles (44% ± 4.2%). Coupling efficiency: optimization ratio of nanoparticles and HA HACTNPOPs were prepared by incubating the predrugloaded optimized nanoparticle formulations with HA in the presence of EDC. The HA conjugation to the amine groups present on the surface of nanoparticles was assessed as a function of the carbodiimide concentration (amount of carbodiimide reagent added to the nanoparticle dispersion) and the reaction time (incubation time between the carbodiimide reagent addition and nanoparticle dispersion). Therefore, the concentration of cross-linking agents EDC, incubation time, and HA/nanoparticle ratios were optimized so as to ensure effective coupling of a ligand to the nanoparticle surface. These coupled nanoparticle systems were characterized for the particle size, shape, percentage encapsulation efficiency, and in vitro drug release and compared to the uncoupled nanoparticles. Figure 2. Photomicrographs of chitosan nanoparticles. (A) SEM analysis and (B) TEM analysis of uncoupled nanoparticles. (C) TEM analysis of HA-coupled nanoparticles.

Optimization of coating thickness The coating thickness over the drug-bearing pellets was optimized in terms of TWG of the pellets after coating with enteric-coating dispersion, and their effect on in vitro drug release in simulated GIT fluids was studied. It was observed

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Figure 5. DSC curve of (A) L-OHP, (B) HA, (C) HA with drug-polymer mixture, and (D) L-OHP–loaded HA-conjugated HACTNPOP formulations, (E) L-OHP–loaded HACTNPOP formulations after storage at 40°C and 75% relative humidity for 6 months.

Figure 4. FT-IR spectra of (A) L-OHP, (B) chitosan, (C) drug-bearing uncoupled nanoparticles, and (D) HA-coupled drug-bearing nanoparticles.

that in simulated intestinal fluid (pH 7.4) at hour 5, drug release was observed to be 52.0% ± 1.20%, 37.7% ± 1.19%, 9.1% ± 1.40%, and 4.2% ± 1.29% at 2, 4, 10, and 20% TWG, respectively. On changing the dissolution medium to simulated colonic fluid (pH 7.0), at the end of 8 hours drug release was observed to be 97.9% ± 1.22%, 87.2% ± 2.22%, 60.1% ± 1.66%, and 32.5% ± 2.52% from coating thickness of 2, 4, 10, and 20% TWG, respectively. FT-IR analysis For the IR spectrum of the drug, L-OHP, the peaks at 3264.1 and 3508.6 cm–1 confirm the presence of an NH stretch, and the peak at 811.8 cm–1 shows N-H bending; C=O stretch was observed at 1706.6 cm–1 (Figure 4, A). In the IR spectrum of chitosan, peaks assigned to the saccharide structure at 1085.0 and

770.1 cm–1, and strong amino characteristic peaks at around 3429.1, 1596.1, and 1325.5 cm–1 were observed (Figure 4, B). An FT-IR spectrum of chitosan and chitosan nanoparticles as shown in Figure 4, B and C is compared. A band at 3429.1 cm–1 has been previously attributed to -NH2 and -OH group stretching vibrations in the chitosan matrix.30 In nanoparticles the shoulder peak of 1596.1 cm–1 disappears and a new sharp peak at 1590.2 cm–1 appears, and the 1600 cm–1 peak of -NH2 bending vibration shifts to 1530.9 cm–1. Knaul observed a similar result in the study of chitosan film treated with phosphate (NaH2PO4) and attributed it to the linkage between phosphoric and ammonium ions.31 Therefore, we assume that the tripolyphosphoric groups of TPP are linked with the ammonium group of chitosan, and the inter- and intramolecular activities are enhanced in chitosan nanoparticles. From the IR spectrum of TPP, the vibration of the P=O or P-O at 1217.3 cm–1 was observed. From the IR spectra of the nanoparticles, on comparing with spectra of TPP, some peaks disappeared or became stronger due to interaction or superposition of peaks among groups of chitosan and TPP. Amide I 1596 cm–1 in chitosan and P=O or P-O 1217.3 cm–1 of TPP overlaps with 3508.6, 3264.1, 811.8 cm–1 of the L-OHP–loaded CTNP spectrum, indicating that L-OHP may have been loaded successfully to the CTNPs. On comparing the IR spectrum of chitosan and HACTNPs following conjugation, additional peaks in the spectra of the HACTNPs have been observed (Figure 4, B and D). A new peak appearing at 3430.4 cm–1 corresponded to bonded -NH stretching vibrations. Amide I band at 1632.8 cm–1 is clearly observed, further confirming the grafting reaction. From the IR data it is clear that the HACTNPs had characteristic peaks of amide bond that could constitute evidence of successful coupling reaction between the HA group and the chitosan amine group. DSC analysis DSC is very useful in the investigation of the thermal properties of drug delivery carriers, providing both qualitative

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Figure 6. XRDs of (A) L-OHP, (B) blank nanoparticles (HACTNPs), (C) L-OHP–loaded HACTNPOPs, and (D) L-OHP–loaded HACTNPOPs after storage at 40°C and 75% relative humidity for 6 months.

and quantitative information about the physicochemical state of drug inside the drug delivery systems.32 There is no detectable endotherm if the drug is present in a molecular dispersion or solid-solution state in the polymeric systems loaded with drug.33 In the present investigation, DSC thermograms of pure drug, ligand, drug-ligand-polymer physical mixtures, and drug-loaded ligand-coupled chitosan nanoparticles were taken (Figure 5). DSC tracings of pure L-OHP, HA, HA with drug-polymer physical mixture, and drug-loaded CTNPs were taken. As shown in Figure 5, A-C, melting endotherms of pure L-OHP, HA, and HA with drug-polymer mixture showed the presence of an endothermic peak at 260.2°C, 89.5°C, and 74.1°C, respectively. In addition, significant sharp exothermic peaks were observed at 243.6°C and 258.7°C for HA, indicating the crystalline structure of the polymer, whereas the HA-polymer mixture shows another endothermic peak at 238.2°C. The HA nanoparticulate carrier showed a sharp endothermic peak at 54.1°C, which overlapped with the endothermic peak of HA-polymer mixture and the melting peak of L-OHP (Figure 5, C and D). However, no characteristic peak of L-OHP was observed in DSC of drugloaded chitosan nanoparticles, which showed a broad small peak at 259.3°C, suggesting that the drug was molecularly dispersed in the polymer matrix (Figure 5, D). The reduction of height and sharpness of the endotherm peak is due to the presence of polymers in the nanoparticles. These results correlate well with the similar study.34 XRD analysis XRD is a proven tool to study crystal lattice arrangements and yields very useful information on degree of sample crystallinity. XRD patterns of L-OHP, blank nanoparticles, and L-OHP– loaded nanoparticles were obtained and compared, which revealed marked differences in the molecular state of L-OHP (Figure 6). In the case of L-OHP the diffractogram showed oxaliplatin intense peaks at the 2θ values of 16.32, 19.23, 20.66, 29.79, 36.44, and 54.46 degrees due to its crystalline nature. The diffractogram of blank HA-coupled chitosan nanoparticles

Figure 7. In vitro drug release profile of nanoparticulate pellets (HACTNPOEPs and CTNPOEPs) in different simulated fluids. SCF, simulated colon fluid; SGF, simulated gastric fluid; SIF, simulated intestinal fluid. Results are presented as mean ± SD, n = 4.

shows one intense peak at 2.85 degrees 2θ. When the diffraction pattern of L-OHP in HACTNPOPs was compared with that of LOHP, the pattern differed to a large extent. Several high-angle diffraction peaks that were observed in L-OHP and blank nanoparticles were not observed in L-OHP–chitosan nanoparticles. Only the small hump in the diffraction pattern is observed from 18.12 degrees to 28 degrees 2θ, as observed for HACTNPOPs. These results are well correlated with a similar study done by Babu et al.35 XRD patterns showed typical sharp peaks in drug crystal. However, peaks for plain drug were masked in the drug-loaded nanoparticles, whereas empty nanoparticles showed relatively fewer peaks. Therefore, it can be assumed that the XRD diffractogram of L-OHP–loaded nanoparticles did not show any characteristic peaks for the drug, indicating the amorphous state of the encapsulated drug in the nanoparticles. In view of the potential utility of the HACTNP formulation for targeting 5-fluorouracil to the colon, the stability studies were performed at 40°C and 75% relative humidity for 6 months (climatic zone IV conditions for accelerating testing—i.e., tropical conditions) to assess their long-term stability (2 years). It was observed that there was no change in the DSC thermograms and XRD diffractograms before and after storage of the formulation (Figures 5, E and 6, D). Drug release studies The percentage drug release profile from the HACTNPOEPs and CTNPOEPs in various simulated GIT fluids is presented in Figure 7. It was observed that there was no drug release up to 4 hours in case of both the products, whereas drug begins to be released only after 4 hours at simulated intestinal fluid pH 7.5. This can be explained by the fact that the Eudragit polymer contains carboxyl groups that ionize from neutral to alkaline media. As the ionization takes place, integrity of the film is disturbed and releases the nanoparticles. In the case of the CTNPOEP formulation, drug release was found to be 56.0% ± 2.2% and 76.20% ± 4.40% at the end of 8 and 24 hours, respectively; in the case of the HACTNPOEP formulation, drug release was found to be 48.6% ± 1.1% and 65.56% ± 3.01% at the end of 8 and 24 hours, respectively.

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Plasma and tissue distribution study A tissue distribution study was undertaken to assess the in vivo L-OHP release in different parts of the GIT from enterically protected pellets encapsulating nanoparticles. No drug was recovered from stomachs with Eudragit S100–coated pellets. In the case of Eudragit S100–coated pellets, no drug release was observed in small intestines even after 4 hours. Very slight drug release (0.003 ± 0.001 μg L-OHP/g of tissue) was observed in small intestines after 6 hour (i.e., in the distal part of the small intestine). Eudragit S100 is a pH-dependent polymer, which dissolves as the pH changes toward the alkaline range. The formulation might have entered into the ileum after 4 hours and thus would have a suitable pH to dissolve the Eudragit S100 coating, and nanoparticles exposed to intestinal fluid released a mere 0.003 ± 0.001 μg L-OHP/g of tissue, which could be the surfacial drug present on the nanoparticles. After 12 hours of fasting, minimal contents were observed in the stomach and small intestine of mice. Whole stomachs and small intestines (with fluids) were homogenized, and the content of L-OHP was quantitatively determined using an HPLC assay. The results of drug distribution from coated and uncoated pellets in the upper GIT after oral administration are displayed in Figure 8, A-C. The results represent the total amount of LOHP in the upper GIT. Significant differences have been observed in the distribution of drug from coated and uncoated pellets. The mean peak L-OHP concentration detected in small intestine was 0.003 ± 0.001 and 0.002 ± 0.001 μg of L-OHP/g of tissue from the uncoupled and HA-coupled nanoparticles encapsulated in coated pellets, respectively. After the administration of uncoated pellets, 65.44 ± 6.00 and 1.05 ± 0.50 μg of L-OHP/g of tissue were detected from the stomach and small intestine, respectively, after 2 hours. A small amount of L-OHP was released from the coated pellets in the upper GIT, whereas the majority of L-OHP was released from the uncoated pellets in the stomach and small intestine for about 5 and 6 hours following oral administration. The relatively sharp decrease in concentration in the stomach after 2–4 hours may be attributed to drug absorption through the stomach and intestinal drug transit (Figure 8, A). The biodistribution of the drug after oral administration of uncoated and coated pellets for the colon tissue and tumor are demonstrated in Figure 8, A-C, respectively. In all cases the LOHP concentrations were considerably higher for the coated pellets than for the uncoated pellets. The mean peak L-OHP concentration after 10 hours was observed to be only 0.040 ± 0.02 μg L-OHP/g tissue in the tumor, wheres no drug was detected in colon after the administration of uncoated pellets. In the case of CTNPOEPs and HACTNPOEPs, L-OHP concentration in colon and tumor tissue after 12 hours was observed to be 2.67 ± 1.25 and 3.70 ± 0.98 (CTNPOEPs) and 1.99 ± 0.82 and 9.36 ± 1.10 μg L-OHP/g tissue (HACTNPOEPs), respectively. γ-Scintigraphic studies The gastric transit and colon arrival time of designed pellets in Balb/c mice were recorded using γ-scintigraphy. The mean gastric emptying time of the formulation was found to be 1.46 ± 0.17 hours; mean intestinal transit time was observed to be 2.03 ±

Figure 8. Distribution profiles of free drug and of uncoupled nanoparticles and HA-coupled nanoparticles bearing L-OHP in various tissues as a function of time after oral dose (10 mg/kg) to mice. (A) Pellets of free L-OHP; (B) CTNPOEPs; (C) HACTNPOEPs. represent mean ± SD obtained from two animals per time point. Colon (+C)⁎, includes colon contents and tissue, cecal contents, and tissue; OXA, oxaliplatin; SI, small intestine.

0.17 hours, and mean colon arrival time was 4.57 ± 0.33 hours. In all the rodents the tracer-bearing pellets (CTNPTEPs) entered into the colon between 4.10 and 4.85 hours after the administration of the formulation. Tumor regression studies Tumor was successfully induced after choosing new methodology for induction of colon tumors. Nude mice were difficult to maintain in aseptic conditions for longer periods of time. Therefore, C57 Balb/c mice were irradiated so that they could accept human colon cancer cell lines HT-29 easily. Results of the tumor regression studies showed that HACTNPOEPs were found to have significantly greater tumor-inhibitory effect than free L-OHP and CTNPOEPs. It was observed that there was a delay in tumor growth when treated with HACTNPOEPs as compared with free L-OHP and CTNPOEPs. Treatment with HACTNPOEPs delayed the tumor growth significantly (8 ± 1.7 days)

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(P b.001) in comparison to free L-OHP and CTNPOEPs (1 ± 0.21 and 4 ± 0.11 days), respectively. Life span was increased significantly (P b .001) in the case of treatment with HACTNPOEPs (105.12%) in comparison to free L-OHP (6.18%) and CTNPOEPs (47.57%). From the results it was clearly observed that HACTNPOEPs caused a marked improvement in therapeutic efficacy and growth-inhibitory effect on tumor.

Discussion L-OHP–loaded nanoparticles of chitosan were prepared using ionotropic gelation technique, and their surface was coupled with HA using carbodiimide chemistry. The carboxylic group of the HA was covalently coupled in the presence of EDC with amine group present at the surface of chitosan, forming an amide linkage. The EDC concentration was optimized to ensure that coupling of HA does not result in particle-particle aggregation or extensive cross-linking. The zeta potential of CTNPOPs and HACTNPOPs was measured. The zeta potential was found to be decreased on coupling of HA with chitosan. This may be due to the masking of cationic surface charge by HA coating. Entrapment efficiency was found to be less in HA-anchored chitosan nanoparticles as compared to uncoupled nanoparticles; this may be due to the leaching of drug during HA coating. The two process variables (incubation time and total nanoparticulate formulation-to-HA weight ratio) were optimized by measuring the change in zeta potential (the surface charge density) of the dispersion (Zetasizer 3000 HS; Malvern Instruments) (Figure 3, A and B). During the conjugation process, negatively charged HA becomes conjugated onto the positively charged chitosan nanoparticles, thus reducing the inherent charge of the nanoparticles. This change in zeta potential value can be used to optimize the process variables. For optimization of total nanoparticulate formulation-to-HA ratio, formulations with different ratios were prepared and incubated for a fixed time period of 24 hours at 25°C. The optimum ratio was determined at which no significant change in zeta potential was recorded on further increasing the nanoparticulate formulation-to-HA ratio. Similarly, for optimization of incubation time the formulations with optimum nanoparticulate formulation-to-HA ratio were prepared and incubated for different time periods and zeta potential was recorded. After completion of conjugation no significant change in zeta potential was recorded (Figure 3). The pellets were coated in terms of different TWGs. The formulation with the coating thickness of TWG 10% was deemed the most suitable coating parameter due to its optimum drug release by virtue of ionization, disruption, and dissolution of the coating, in that pellets with 20% TWG show hindered drug release. The rate of release was inversely proportional to the thickness of the coat, thereby implying that the film coat was controlling the release process. Ideally, the coating should be thick enough to resist drug release for a sufficient period of time equivalent to transit through the upper GIT, yet not so thick that drug release under colonic conditions is hindered. Coating thicknesses of TWG = 10% coating appear to fulfill these criteria.

Figure 9. γ-scintigraphs captured at different time intervals. (A) Just after administration of pellet showing integrity of the system in stomach. (B) At 1 hour showing the presence of intact enteric-coated pellet in stomach. (C) At 3 hours showing the enteric-coated pellet in small intestine. (D) At 6 hours showing the release of tracer in ascending colon from enteric-coated pellet. (E) At 10 hours showing the release of tracer in ascending colon and entry of pellet mass into transverse colon. (F) At 18 hours showing the distribution of liberated radioactivity in whole colon from enteric-coated pellet. (G) At 24 hours showing the radioactivity in distal part of colon from enteric-coated pellet.

The in vitro drug release profile of HACTNPOEP and CTNPOEP formulations was studied using a dialysis membrane. The relative drug release was suppressed in case of HA-coupled nanoparticles (Figure 7). The decreased drug release from HACTNPOPs could be due to the structural integrity of HA coupling, which further may lead to a doublebarrier effect for drug diffusion. Similar results were obtained previously in our laboratory while working with ferritincoupled solid lipid nanoparticles.3 The biodistribution studies indicated that HA-coupled nanoparticles reached the tumor and were more effective in treating colon tumors as compared to uncoupled nanoparticles and the free drug. In the studies of γ-scintigraphy, the interesting fact in the transit of the material is that during the initial hours of the study the formulation remains firm, which may cause fast transit; however, as the time passes the rigidity of the formulation becomes weaker due to penetration of GIT fluid, and that may

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lead to slow transit through rest of the GIT. Hardy et al observed gastric emptying values from 0.4 to 4.4 hours with multiparticulate radiolabeled pellets administered to human volunteers, whereas Jain et al13 observed that in case of Eudragit-coated calcium pectinate beads colonic arrival time was around 5 hours in the case of albino rats after oral administration. It is clearly seen from the captured scintigraphs that no amount of the tracer was released in the stomach (Figure 9, A). This corresponds to effective enteric coating to the pellets. It is evident from the scintigraph taken after 3 hours (Figure 9, B, C) that a very minute quantity of tracer has been released in the small intestine during the period from 1.85 to 2.20 hours. The pellets remain intact as they descend through the small intestine. The tracer, which was released in the small intestinal environment, is visible around the pellet. After 4.10– 4.85 hours as the formulation entered into the ascending colon, release of the tracer was increased (Figure 9, D). This was due to change in the pH of the lower GIT, leading to dissolution of enteric coating and thus release of the tracer. After 5 and 6 hours the formulation remained in the ascending colon, but the release of radioactivity from the pellet was considerably improved as the pellets degraded with time in the colon. After 8 hours the pellet was still in the ascending colon, but its shape was distorted with the liberation of considerably higher amount of tracer. After 11 hours the formulation entered the transverse colon, leaving the ascending colon filled with the tracer released from the pellet. The pellet was completely disintegrated after 10 hours, and after 15 hours the liberated radioactivity was distributed across the ascending colon, transverse colon, descending colon, and sigmoid colon (Figure 9, E). The movement of the pellet in intact form from stomach and small intestine to the colon without the tracer being released and complete degradation of the network in the colon reveals the efficiency of this pHdependent system. Although on entering the colon the pellet released tracer, the amount of tracer released was limited. However, the intensity of the tracer was markedly improved upon complete disintegration of the pellet in the colonic milieu. Krishnaiah et al37 reported that the disintegration time of the matrix tablets of guar gum in human volunteers was from 12 to 16 hours. When the tracer was incorporated in the form of sodium chloride in nanoparticles, by virtue of its hydrophilic nature it was quickly distributed in the aqueous environment after liberation from the formulation. After 15–18 hours, radioactivity was observed in the whole colon (Figure 9, F). After 24 hours the intensity of the activity was diminished, and tracer was visible in the distal part of the colon (Figure 9, G). Thus, our results collectively demonstrate that this study favored the Eudragit S100–coated pellets bearing HA-coupled and uncoupled, L-OHP containing chitosan nanoparticles as a potential system for colon tumor targeting. The distribution of coated pellets was markedly different from that of uncoated pellets after oral administration. In contrast to uncoated pellets, which released the drug along the whole upper GIT, L-OHP was predominantly released from the coated pellets in colon and in the vicinity of tumors. In conclusion, colon-specific delivery of L-OHP was achieved after oral administration of nanoparticles bearing enteric-coated pellets to the mice. HA coupling on the surface of nanoparticles was found to make

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them more specific for delivery of drug to tumors of the colon. Therefore, the developed system (i.e., HACTNPs) appears more promising in cancer treatment by targeting to tumor vasculature in a mouse model as compared to noncoupled nanoparticles. The results warrant further evaluation of this delivery system. Detailed in vivo studies will be communicated in the next publication. Acknowledgments The authors are thankful to M/s Khandelwal Laboratories Ltd., Mumbai, India, for generously supplying L-OHP, and to the Indian Institute of Technology, Kanpur, for carrying out SEM studies.

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