Synthesis and evaluation of biodegradable PCL/PEG nanoparticles for neuroendocrine tumor targeted delivery of somatostatin analog

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1 Drug Delivery, 2012; 19(3): ISSN print/issn online DOI: / ORIGINAL articles Synthesis and evaluation of biodegradable PCL/PEG nanoparticles for neuroendocrine tumor targeted delivery of somatostatin analog Nazneen Dubey 1, Raunak Varshney 2, Jaya Shukla 3, Aditya Ganeshpurkar 4, Puja P. Hazari 2, Guru P. Bandopadhaya 3, Anil K. Mishra 2, and Piyush Trivedi 1 1 School of Pharmaceutical Sciences, Drug Discovery Lab, Rajiv Gandhi Technological University, Bhopal, India, 2 Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, New Delhi, India, 3 Department of Nuclear Medicine, All India Institute of Medical Sciences, New Delhi, India, and 4 Department of Pharmacy, Shri Ram Institute of Technology, Near I.T.I., Madhotal, Jabalpur, India Abstract Purpose: Neuroendocrine tumors often present a diagnostic and therapeutic challenge. We have aimed to synthesize and develop biodegradable nanoparticles of somatostatin analogue, octreotide for targeted therapy of human neuroendocrine pancreatic tumor. Methods: Direct solid phase peptide synthesis of octreotide was done. Octreotide loaded PCL/PEG nanoparticles were prepared by solvent evaporation method and characterized for transmission electron microscopy, differential scanning calorimetery (DSC), Zeta potential measurement studies. The nanoparticles were evaluated in vitro for release studies and peptide content. For biological evaluations, receptor binding & cytotoxicity studies were done on BON-1 neuroendocrine tumor cell line. Biodistribution of radiolabeled peptide and nanoparticles, tumor regression studies were performed on tumor-bearing mouse models. Results: We have synthesized and purified octreotide with the purity of 99.96% in our laboratory. PEG/PCL nanoparticles with an average diameter of nm having peptide loading efficiency of 66 84% with a negative surface charge were obtained with the formulation procedure. Octreotide nanoparticles have a negative action on the proliferation of BON-1 cells. In vivo biodistribution studies exhibited major accumulation of octreotide nanoparticles in tumor as compared to plain octreotide. Octreotide nanoparticles inhibited tumor growth more efficiently than free octreotide. Conclusions: Thus, it was concluded that the PCL/PEG nanoformulation of octreotide showed high tumor uptake due to the enhanced permeation and retention (EPR) effect and then peptide ligand imparts targetability to the sst2 receptor and there by showing increase tumor growth inhibition. Selective entry of nanoparticles to the tumor also give the reduce side effects both in vivo and in vitro. Keywords: Octreotide, PCL, nanoparticles, neuroendocrine pancreatic tumors, targeting Introduction Polymeric nanoparticulate drug delivery system made from natural and synthetic polymer in cancer therapies provide better penetration of therapeutic and diagnostic substances within the body at a reduced risk in comparison with conventional cancer therapies (Gou et al., 2011a). Nanoparticles received the majority of attention because it could reduce the multidrug resistance that characterizes many anticancer drugs by a mechanism of internalization of the drug (Davda et al., 2002). Limitation of conventional chemotherapy is typically by the toxicity of drugs to normal tissues, short circulation half-life in plasma, limited aqueous solubility, and nonselectivity restricting therapeutic efficacy. Nanoparticles distribution within the body is based on various parameters such as their relatively small size resulting in longer Address for Correspondence: Prof. Piyush Trivedi, School of Pharmaceutical Sciences, Drug Discovery Laboratory, Rajiv Gandhi Technology University, Gandhi Nagar, Airport Bypass Road, Bhopal , India. Tel: (Office) prof_piyushtrivedi@yahoo.com (Received 27 April 2011; revised 17 November 2011; accepted 11 January 2012) 132

2 Synthesis and evaluation of biodegradable PCL/PEG nanoparticles 133 Abbreviations Fmoc: 9-fluorenylmethoxycarbonyl Boc: tert-butyl carbamates Trt: triphenylmethyl Otbu: O-tertiary butyl Thr-ol: threoninaol Phe: phenylalanine Cys: cysteine Thr: threonine Lys: lysine Trp: tryptophan HOBt: 1-hydroxybenzotriazole circulation times and can extravasate through the EPR effect in endothelium of inflammatory sites, epithelium tumors, or penetrate microcapillaries (Kim, 2007). Some recently discovered carcinogenic and noncarcinogenic agents such as certain dietary substances, natural hormones and synthetic compounds have been proposed to stop or reverse the process of carcinogenesis to alleviate the limited efficacy and considerable toxicity of conventional chemotherapy (Morse et al., 1993), in which increasing attention has been paid to somatostatin and its analogs that have been reported to exert antineoplastic effects in a wide range of tumor types such as carcinoid, osteosarcoma, leukemia, and cancers of thyroid, breast, lung, liver, pancreas, colon, as well as gastric carcinomas (Scarpignato et al., 2001; Ain et al., 1997; Marschke et al., 1999; Setyono et al., 1987; Shan et al., 2003; Gencosmanoglu et al., 2002; Lee et al., 2002; Giannetti et al., 2000; Yuen et al., 2002; Gou et al., 2011b). Poly(ε-caprolactone) (PCL), biodegradable polyesters, is most commonly used for pharmaceutical applications. These biodegradable polyester polymers possess various advantages such as biocompatibility, predictability of degradation kinetics and their ease of formulation. These do not require follow-up surgical removal once the drug is finished. Poly(ethylene glycol) (PEG) is a hydrophilic and non-toxic material with no antigenicity and immunogenicity. Approval from the Food and Drug Administration makes PEG as one of the promising polymers, for its systemic consumption, which is widely applied in biomedical fields. PEG polymer can prevent protein absorption and improve the biocompatibility for blood contact compound (Lewis et al., 1990; Wei et al., 2009). In the malignancy of the pancreas surgery, radiation, and chemotherapy are of limited effectiveness, and there is essentiality, in developing new therapies (Von Hoff et al., 1998; Van Cutsem et al., 1995; Greenlee et al., 2000; Gou et al., 2009; Chanana et al., 2009). In pancreatic carcinogenesis the role of various hormones, growth factors, and their receptors have been extensively investigated. One of the hormones implicated in the inhibition of malignant transformation of the pancreas is somatostatin (SST) (Pollak et al., 1998). SST is a neuropeptide that demonstrates an inhibitory action against several endocrine systems. SST is recognized as a hormone capable of regulating fundamental processes, such as secretion, cell division, proliferation and apoptosis (Reichlin, 1983; Schally, 1988). Synthetic derivatives of naturally occurring somatostatins have been designed due to the short half-lives in blood circulation (1 3 min). In this context, SST and its clinically available octapeptide analogues such as octreotide, vapreotide, and lanreotide have been synthesized which bind to specific somatostatin receptors on cancer cells and can inhibit cell proliferation. Somatostatin octapeptides and their radiolabeled analogues bind to subtypes SSTR2, SSTR3 and SSTR5 (Krenning et al., 1993; Bruns et al., 1997). Octreotide had been used therapeutically to prevent carcinoid crisis and used also in clinical practice for the scintigraphic visualization of tumors containing a high density of somatostatin receptors (Raynor et al., 1993; Simpkins et al., 1996). The radiolabeled octreotide derivatives 123 I-[Tyr 3 ]- octreotide and 111 In diethylenetriamine penta-acetic acid octreotide have found useful applications in detecting small neuroendocrine tumors that cannot be detected by conventional means, and also for identifying tumors that respond to therapeutic doses of octreotide as demonstrated by Lamberts and Krenning (Lambert et al., 1991; Bakker et al., 1991). An antiproliferative effect mediated by octreotide in tumors that express the type 2 receptor suggested by the data with MIA PaCa-2 and other pancreatic cancer cell lines or human tumors (Radulovic et al., 1993; Fisher et al., 1996). Peptide drugs are usually administered systemically, e.g. parenterally. Because of painful discomfort caused by parenteral administration drug substance, the drug should be administered as a controlled release formulation. For peptides encapsulation in biodegradable polymer, nanoparticles have been highly successful to provide sustained release up to several months after injection. The above in vitro and in vivo studies provided the rationale for assessing the efficacy of octreotide loaded nanoparticles for the therapy of human neuroendocrine pancreatic tumor. The aim of the present work is to synthesize octreotide (Oct) and develop octreotide loaded PCL/PEG based biodegradable nanoparticles (Oct-NP) and evaluating its in vitro and in vivo biological efficacy for targeting towards the neuroendocrine pancreatic cancer. Materials and methods Materials Rink amide resin, dichloromethane (DCM), dimethylformamide (DMF), eight protected amino acids, piperidine, trifluoroacetic acid (TFA), Acetonitrile (HPLC grade), hydroxybenzotriazole (HOBt), dry diethyl ether, PCL 85,000 Da, PEG 5000 Da, Pluronic F-68, Stannous chloride dehydrated (SnCl 2.2H 2 O) and sodium dithionite

3 134 N. Dubey et al. were purchased from Sigma-Aldrich. All the other reagents used were of analytical grade and were used as received. 99m Tc was procured from Regional Centre for Radiopharmaceuticals (Northen Region), Board of Radiation and Isotope Technology (BRIT), Department of atomic energy, India. Unless specified, all the chemicals were purchased from CDH, India. Cell culture Monolayer cultures of human pancreatic carcinoma BON-1 cells (obtained from Institute for biophysics, University of Frankfrut, Germany) was maintained at 37 o C in a humidified CO 2 incubator (5% CO 2, 95% air) in a (1:1) nutrient mixture of Dulbecco s modification of Eagle s medium (DMEM) (Sigma) supplemented with 10% fetal calf serum (GIBCO), 50 µg /ml penicillin, 50 µg/ml streptomycin sulfate and 2 µg /ml nystatin. Methods Direct solid phase peptide synthesis of octreotide Direct solid phase peptide synthesis of octreotide was done as described by Hsing-Pang Hsieh, et al. (1999) with slight modifications. The sequence of octapeptide was synthesized on Rink amide resin. Resin (336 mg, 0.25 mmol) was washed with DCM:DMF (1:1) in reaction vessel for 20 min before starting the synthesis programme of the peptide. Couplings were performed by dissolving the protected amino acid in situ with HOBt in DMF. After 5 min, the solution was added to the Rink amide resin and left for 1 h at 25 C with occasional swirling. Then, Kaiser Ninhydrin test was performed to indicate the completion of couplings. The resin-bound sequence of this peptide was found to be Fmoc-Phe-Cys(Trt)-Phe-Trp(Boc)-Lys(Boc)-Thr(Otbu)- Cys(Trt)-Thr(Otbu)-rink amide resin. After the final Fmoc deprotection with 20% piperidine and programmed wash procedure (with DMF, MeOH and DCM), the resin complex was dried in vacuum and weighed. Peptide side chain deprotection and cleavage were carried out in TFA solution (containing TFA 9 ml, water 0.25 ml, thioanisole 0.5 ml, EDA 0.25 ml, and phenol 0.79 g) at 0 o C. Then the mixture was stirred gently at room temperature for 1.5 h. The peptide was precipitated by addition of ice-cold dry ether, after evaporation of TFA under vacuum (Heidolph Laborota 4000); the precipitate was filtered, washed with cold ether on a sintered glass funnel and lyophilized to a white powder. The peptide product was diluted to about 1 mm with 5% ammonium acetate solution and ph was adjusted to 7.0 with ammonium hydroxide (25%) to accomplish disulfide bond formation. After that solution was dropped over 100 ml of dry and cold diethyl ether and the white precipitate obtained is once again centrifuged. The solid is resuspended in diethyl ether and centrifuged again, repeating the operation five times more. The crude peptide is then purified. Purification and characterization The folding of reduced octreotide was done with the elution gradient of 40 60% A and 60 40% B in min at a flow rate of 1.0 ml/min by RP-HPLC (Agilent 1200 series system), where A was 5% acetonitrile aqueous with 0.1% TFA and B was 90% acetonitrile aqueous with 0.1% TFA. During the folding process, 0.1 ml sample was taken at different times and injected to a C-18, 5 mm analytical column for monitoring the disulfide oxidation of reduced octreotide. Peptide purification was conducted on a C-18 preparative column. The peptide solution was first loaded to the column with a flow rate of 4 ml/min then collected by flowing 75% of acetonitrile aqueous through the column at a flow rate of 8 ml/min, monitored at 220 nm to give a white powder of octreotide. Amino acid analysis was done. Mass spectrum was recorded on an electro spray ionization mass spectrometer on an SL1200 system (Agilent). Preparation of octreotide nanoparticles The nanoparticles were prepared by solvent evaporation technique based on the formation of a W 1 /O/ W 2 -multiple emulsion as described by Herrmann et al. (1998) with slight modifications. An aqueous solution of octreotide dissolved in 1.0 ml of 10 mm ph 5.9 acetate buffer was emulsified into a solution of PEG (5000 Da) and PCL (85,000 Da) (1:20) in methylene chloride (3.0 ml) by sonication for 30 s under ice-cooling (LABSONIC U sonicator).the resulting W 1 /O primary emulsion was reemulsified into distilled water (or with 20 ml of varying concentration of Pluronic F-68 solution) with Silverson L4R at 4000 rpm to form the double emulsion [(W 1 /O)/ W 2 ]. After 30 min, the nanoparticles were collected by filtration, rinsed with 30 ml phosphate buffered saline (PBS) and lyophilized (LABCONCO, Triad TM ). Unloaded nanoparticles were prepared using similar method without incorporating the drug. Formulations were optimized using process variables such as the amount of polymer, different concentrations of aqueous Pluronic F-68 and sonication time. Physicochemical characterization of nanoparticles Surface morphology The morphological examination of nanoparticles was performed using a transmission electron microscope (TEM) following negative staining with sodium phosphotungstate solution (0.2%, w/v). One drop of nanoparticle suspension was placed on a copper grid and air-dried before observation. Zeta potential measurements As an indicator of the colloidal stability, surface charge of the nanoparticle was analyzed by using Malvern zetasizer (DTS Version, 4.10, Malvern Instuments). Nanoparticles were dispersed in PBS buffer (ph 7.4) at concentration of 1.5 ± 0.3 mg/ml. The dispersion was slightly sonicated in a water bath ultrasonicator for 1 min before the analysis. Differential scanning calorimetery Differential scanning calorimetery (DSC) of peptide, polymer and peptide loaded nanoparticles were carried Drug Delivery

4 Synthesis and evaluation of biodegradable PCL/PEG nanoparticles 135 out by heating the sample from 30 o C to 450 o C at the heating rate of 10 o C/min. in a nitrogen environment using TA 3100 thermal analyzer having a 951 DSC module (USA). Particle size determination by DLS The particle size measurements were done by dynamic laser light scattering (DLS) technique, using a 90 Plus Particle Size Analyzer from Brookhaven Instrumets Corp. (BIC), USA and an indigeneous goniometer. The excitation source was a 35 mw He-Ne laser emitting at a wavelength of nm in linearly polarized single frequency mode, which was focused onto the sample cell and the scattered light was detected by a photomultiplier tube (Hamamatsu). The signal was converted into intensity auto correlation function by a digital correlator. Peptide entrapment efficiency Octreotide loaded nanoparticles (10 mg) were dissolved in 1.0 ml methylene chloride followed by addition of 1.0 ml of 1 M acetic acid and gentle agitation for 12 h at room temperature (Herrmann et al., 1995). Then octreotide content was analyzed by reversed phase (RP)-HPLC (Agilent 1200 series system). A gradient elution method was utilized with mobile phase A (0.1% v/v TFA in water) and mobile phase B (0.1% v/v TFA in acetonitrile). The gradient was 80:20 (A:B) to 65:35 (A:B) over 20 min with a flow rate of 1.0 ml/min. UV absorbance was measured at 220 nm. Octreotide solutions of known concentrations ( mg/ml) in the same solvent system were used to generate calibration curves. In vitro peptide release study After addition of 3 ml of acetate buffer (ph 4.0) and PBS (ph 7.2) buffer, in each 15 ml round-bottom glass tube having approximately 15 mg of Oct-NP with continuous mixing at 37 C. At different time points, HPLC analysis of the supernatant of each tube was done. Octreotide was analyzed by reversed phase (RP)-HPLC using C-18 column, (250 mm 460 mm) using mobile phase A (0.1% v/v TFA in water) and mobile phase B (0.1% v/v TFA in acetonitrile). The gradient was 80:20 (A: B) to 60:40(A: B) over 25 min with a flow rate of 1 ml/min. UV absorbance was measured at 220 nm. Radiochemical synthesis of octreotide Octreotide (10 µg) was dissolved in 200 µl acetate buffer (0.1 M, ph 7.2) in a shielded vial. Sodium ascorbate solution (ph 6.2) was added. Freshly eluted (<1-h old) 99m Technetium pertechnetate (82 MBq; 200 µl) was added followed by addition of sodium dithionite solution in acetate buffer (0.1 M, ph 7.2) and the mixture was heated for 15 min and cooled for 20 min at room temperature (Gandomkara et al., 2003). Radiochemical synthesis of Oct-NP In a shielded vial 0.1 ml of stannous chloride solution (1 mg/ml) in 10% acetic acid was mixed with freshly eluted 0.1 ml of 99m Technetium pertechnetate (5 mci/ml) and the ph of the reaction mixture was adjusted to 6.5 with 0.1 M Na 2 CO 3 and purged with N 2 shaken to mix. To this solution 1 ml of octreotide nanoparticles solution (1 mg/ml) was added and the vial was allowed to stand for 15 min at room temperature (25 o C). Radiochemical purity Radiolabeling efficiency was determined by ascending thin layer chromatography on ITLC-SG (Paul Gelman) strips using 100% acetone as developing solvent and simultaneously in pyridine:acetic acid:water (PAW) (3:5:15) and saline. Each TLC was cut in 0.5 cm segment and counts of each segment were taken. The radioactivity was counted on Biodex scintillation well counter; USA. The free 99m Tc, which moved with the solvent (R f = 0.9), was calculated. In vitro biological evaluation Receptor binding assay The specificity of somatostatin analog to bind to cell surface receptors on tumor cells were examined by receptor binding assays on BON-1cell lines grown in DMEM. The cell line cultures were then incubated for 40 min with 99m Tc labeled octreotide at 37 o C in HBSS containing various concentration ( µm) in the absence and presence of the 100-fold excess unlabeled octreotide for estimation of total binding and non-specific binding, respectively. Specific binding was obtained by substracting non-specific binding from total binding. At the end of each experiment, the cells were washed with cold PBS and 0.9% saline 7 8 times. The cell associated radioactivity was determined by γ-scintillation counting. Scatchard plot analysis was done using EQUILIBRATE software from graph pad. Cytotoxicity study Cytotoxicity was determined using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Exponentially growing cells were plated in 96-well microtitre plate at a uniform cell density of 10,000 cells/well 24 h before treatment. Cells were treated with plain octreotide and octreotide nanoparticles in varying concentrations (0.008, 0.004, 1, 0.2, 5, 25 µg/ml range) for various time intervals viz., 24 h, 48 h, 72 h and MTT assays were performed. At the end of treatment, negative control and treated cells were incubated with MTT at a final concentration 0.05 mg/ml for 2 h at 37 o C and the medium was removed. The cells were lysed and the formazan crystals were dissolved using 150 µl of DMSO. The absorbance of individual wells was noted on 570 nm via an ELISA plate reader. The inhibiting rate of cell growth was expressed as (A-B)/A 100%, where A is the absorbance value from the controls and B is that from the experimental cells. In vivo biological evaluation Tumor transplantation Athymic Swiss albino Balb/c mice (10 12 weeks) used in these studies was obtained from the institute s central

5 136 N. Dubey et al. animal facility and weighed g at the time of tumor implantation. The institutional animal ethics committee of INMAS (Institute of Nuclear Medicine and Allied Sciences), New Delhi had duly approved the protocol for in vivo studies. BON-1 tumor cells were maintained by serial passage of tumor cell suspension in the peritoneal cavity of athymic mice. Subcutaneous tumors were implanted by intramuscular injection of cells (in ml volume) into right hind leg. Experiments were performed when the tumor had attained a volume of mm 3 (5 6 days after implantation). Tumor regression Mice bearing subcutaneous tumor were treated with the Oct-NP intravenously through the tail vein. The injection volume of the treatment were prepared in normal saline was ml. Tumor-bearing mice (n = 18; tumor size mm 3 ) were allocated to three groups (n = 6). The first group was taken as control and the second group was injected with octreotide nanoparticles; mice in the third group were treated with plain octreotide. Octreotide and Oct-NP were injected in a dose of 100 µg/kg every second day for 15 days. The animals were kept under observation; tumor volume was measured at every alternate day using vernier caliper. The experiment was repeated two times. Biodistribution studies BON-1 cells that over expresses the SSTR2 receptor was injected subcutaneously in the thigh of the right hind leg of the athymic mice. Through the tail vein of each mice of two groups an intravenous injection of 99m Tc octreotide and its nanoparticles formulation (100 µg/kg) in a volume of 100 µl (3.7 MBq activity) was injected. The third group was taken as control. Mice were dissected at 1, 2 and 4 h post injection; different tissues were taken out, weighted and counted in a γ-counter calibrated for 99m Tc energy. The percentage injected dose per gram of the tissues (%ID/g) was calculated. Statistical analysis For evaluating the extent of a relationship between two data sets, Pearson correlation coefficients were used. ANOVA were used to analyze statistical differences among groups. A p value of 0.05 was considered statistically significant. Results Synthesis and characterization of octreotide The resin-bound sequence of Fmoc-Phe-Cys(Trt)-Phe- Trp(Boc)-Lys(Boc)-Thr(Otbu)-Cys(Trt)-Thr(Otbu)-rink amide resin was synthesized. After the deprotection of amino acids, the time course for folding of reduced octreotide was followed by RP-HPLC monitored at 280 nm. The oxidized form had a retention time of 30.3 min (Figure 1). HPLC analysis showed the disulfide oxidation was above 90% complete in 47 h. Finally, octreotide Figure 1. RP-HPLC of oxidized octreotide. with 99.96% purity was obtained. Amino acid analysis of octreotide after hydrolysis with 6 N HCl:TFA (4:1; v/v) for 3 h at 13 o C was found as: amino acid/found/calcd, Lys/1.0/1; 1/2Cys/0.96/1; Phe/1.93/2; Thr/0.66/1 showing m/z (M) + (Figure 2). Preparation of octreotide nanoparticles Table 1 shows particle size and the encapsulation efficiency of the octreotide loaded in PEG/PCL nanoparticles. The optimization of method was done to produce nanoparticles of small size with high entrapment efficiency. Increasing the amount of PCL from 10 to 30 mg and of PEG ( %) caused nanoparticle s size shift toward a higher size range from 130 to 195 nm, and entrapment efficacy was increased from 63.76% to ± The size of the nanoparticles varied from 130 to 195 nm when the concentration of Pluronic F-68 increased from 0.0 to 1.0% but entrapment efficacy was increased from ± 0.67 to ± We have found maximum drug encapsulation efficiencies with sonication time at 15 s. Physicochemical characterization of octreotide loaded nanoparticles Surface morphology The nanoparticles were found to be spherical with average size range of nm (Figure 3). DSC The DSC thermograms of pure octreotide, PCL/PEG nanoparticles and Oct-NP are shown in Figure 4. The DSC thermograms of peptide loaded nanoparticles, prepared in the presence of Pluronic F-68, were shifted toward lower temperature as compared with an endothermic peak at 58 o C of control nanoparticles. Pure octreotide showed endothermic melting peak at o C. Zeta potential measurements Stabilization of nanoparticles can be predicted by zeta potential. Here the zeta potential is ± 1.75 mv for Drug Delivery

6 Synthesis and evaluation of biodegradable PCL/PEG nanoparticles 137 Figure 2. ESI mass spectrum of octreotide. Table 1. Process variables of octreotide nanoparticles. Mean Process variables Fabrication variables diameter (nm) % Drug entrapment Amount of PCL (mg) ± 5* ± 0.45* ± 4* ± 0.66* ± 3* ± 0.34* % Of PEG ± 6* ± 0.23* ± 4* ± 0.21* ± 3* ± 0.46* % Of surfactant ± 8* ± 0.67* ± 3* ± 0.71* ± 4* ± 0.33* Sonication time (s) ± 4* ± 0.42* ± 4* ± 0.56* ± 7* ± 0.21* *Mean ± SD (n = 3); (p 0.05). octreotide loaded nanoparticles. The zeta potential is sufficient to keep the system stable under the steric stabilization offered by PEG chains (data not shown). Particle size determination by DLS Semi quantitative analysis of particles size distribution of the nanoparticles determined by DLS is shown in Figure 5. In vitro peptide release studies In vitro release profiles of octreotide nanoparticles in 0.1 M acetate buffer (ph 4.0) and PBS (ph 7.2) buffer was shown in Figure 5. The in vitro release profile of a small Figure 3. TEM pictograph of octreotide loaded nanoparticles. initial release of 5 15% in PBS (ph 7.2) was obtained as compared to acetate buffer (ph 4.0). Peptide entrapment efficiency The peptide content results are summarized (Table 1). The peptide entrapment efficiency was found to be 65.53% to ± 0.33% (w/w). Radiochemical purity Radiochemical purity of 99m Tc octreotide and its nanoparticles was estimated chromatographically using ITLC-SG

7 138 N. Dubey et al. Figure 6. In vitro release pattern of octreotide from PCL/PEG nanoparticles. Figure 4. DSC thermogram of (a) PCL nanoparticles, (b) Oct-NP (c) Plain octreotide (d) Pluronic F-68. Figure 7. Scatchard plot of the specific binding data to the ratio of bound to free (B/F) for BON-1 cell line. Figure 5. DLS Studies of nanoformulation. (instant thin layer chromatography silica gel) paper as the stationary phase and 100% acetone as the mobile phase. Percentage radiolabeling was calculated for 0, 2, 4, 6, and 24 h. Even up to 24 h labeling efficiency was found to be 97.67%. In vitro biological evaluation Receptor binding assay BON-1 was examined by saturation binding assay using 99m Tc labeled octreotide nanoparticles for the ability of octreotide to bind somatostatin receptors on the surface of tumor cell lines (Figure 6). Scatchard plot analysis revealed affinity of the labeled nanoparticles on tumor cell lines having K d 3.54 ± 0.03 (mean ± SE) to a single class of site. Cytotoxicity study Growth inhibition of human pancreatic cancer cell lines after exposure to octreotide nanoparticles for 72 h showed that the inhibition rate of growth of all cell lines used in this experiment was in a dose-dependent manner. The percentage inhibition rate of cells upon incubation of plain octreotide and its nanoparticle formulations in varying concentrations are shown in Figure 7. The cytotoxicity of free octreotide and octreotide loaded nanoparticles was similar at the concentration of 1 µg/ml. As the concentration increased, the difference in cytotoxicity between the free octreotide and the nanoparticles become obvious. BON-1 cell line (pancreatic neuroendocrine tumor cell line) exhibited a dose-dependent inhibition of growth with the inhibiting rate as high as 54.03% when 25 µg/ ml solution of Oct-NP was added as compared to the free octreotide having the inhibiting rate of 33.06%. In vivo biological evaluation Tumor regression studies In vivo tumor growth inhibition study in BON-1 tumorbearing mice was carried out using the single dose of formulation at every second day (Figure 8). The size of tumor volume in case of control (injected with normal saline) was increased to ± 3.3 mm 3. The volume of tumor was reduced to 92.9 ± 1.5 mm 3 and thus the tumor reduction rate was only 8.42 ± 0.3% when treated with plain octreotide. The tumor volume was found to be reduced to 91.3 ± 1.3 mm 3 and the tumor inhibition rate Drug Delivery

8 Synthesis and evaluation of biodegradable PCL/PEG nanoparticles 139 Figure 8. Colorimetric estimation of the mitochondrial activity for cytotoxicity of octreotide nanoparticle and octreotide (MTT assay) in BON-1 cell line after an incubation time of 72 h at different concentrations. Figure 9. In vivo tumor growth inhibition study of octreotide loaded nanoparticles on BON-1 implanted tumor-bearing mice. was 14.6 ± 0.3% when Oct-NP was injected as compared to control. Biodistribution studies The biodistribution of octreotide with 99m Tc either as free peptide or encapsulated in PCL/PEG nanoparticles were performed in BON-1 cell bearing mouse models. The percentage of ID/g distribution to tissues in different organ at different time intervals for plain 99m Tc octreotide and 99m Tc labeled nanoparticles are shown in Figures 9a and 10. The studies were performed to evaluate the drug delivery at various tissues. There was a comparative higher concentration of octreotide in the tumor when delivered in PCL/PEG nanoparticles than plain octreotide as compared with the aqueous solution. Results were even more pronounced at 2 h after administration. Discussion The ultimate goal of somatostatin analog therapy is either to kill or inhibit the growth of tumor cells. Use of octreotide nanoparticles for pancreatic cancer is an innovative technique. The concept is advantageous as it not only allows targeted drug delivery but also reduces associated side effects. The proposed study is envisaged to synthesize, formulate and characterize octreotide loaded nanoparticle formulation for cancer therapy and explore its potential for delivery of drug to target site. The synthesized octreotide has H 2 N-D-Phe-Cys-Phe- D-Trp-Lys-Thr-Cys-Thr-ol amino acids with one disulfide linkage. The peptide was found to be 99.96% pure and characterized. Octreotide nanoparticles were prepared by double emulsion solvent evaporation method. The addition of peptide and polymer solution to the aqueous phase promotes rapid diffusion of solvent across solventpolymer in to the aqueous phase and results in immediate formation of nanoparticles (Herrmann et al., 1995). It is clear from the Table 1 that the processing variables influenced the size and peptide loading capacity of nanoparticles. Higher concentration of polymer produced a more viscous solution that caused a reduction in the rate of diffusion of polymer solution in the water phase. Faster the diffusion rate of organic solvent to the outer aqueous phase, the smaller the particles would result. Surfactant is added to keep good steric stability of the formed nanoparticles. The size of the nanoparticles decreased gradually with increasing concentration of surfactant (Pluronic F-68). As the sonication time increases the size of the nanoparticle decreases (Herrmann et al., 1998). The nanoparticles were found to be of nanometer size range, these results were also evident from DLS studies. Octreotide melting peak totally disappeared in the calorimetric curve of loaded nanoparticles, evidencing the absence of crystalline drug in the nanoparticle samples (Dubernet et al., 1995). The high zeta potential would be beneficial to the stabilization of a nanoparticle system and preventing aggregation because the charged nanoparticles can repel one another. One of the advantage of using Pluronic (F-68) as surfactants include an effective means to stabilize PCL nanoparticles resulting in preferential targeting of drugs to tumors (Shenoy et al., 2005). As nanoparticles get solidified in aqueous phase, surfactants attach to the hydrophobic nanoparticle surface and the drug is prevented to get diffused back to solid core (Muller et al., 2000). Pleuronic is also known to play an essential role drug encapsulation. When Pleuronic (F-68) is incorporated in nanoparticles, an adequate size of nanoparticles is developed that can be directed for targeting to tumors. (Chawla et al., 2002). With increase in the concentration of surfactant, particle size of nanoparticles decreased with increase in entrapment efficiency. Same results were observed in our studies. Pleuronic (F-68) as surfactants are non-toxic and enhances nanoparticle-cell interaction. (Kerleta et al., 2009) We also observed such results in terms of in vivo studies. However, this rule cannot be applied for stealth nanoparticle, because hydrophilic PEG segment results in a shift of the hydrodynamic phase of shear to greater distances from the nanoparticles surface. Zeta potential decreases with the increasing content of the hydrophilic

9 140 N. Dubey et al. Figure 10. Biodistribution of 99m Tc-Oct-NP and 99m Tc-Oct in BON-1 cell tumor grafted in athymic mice injected with 3.7 MBq activities after (1, 2 and 4 h). PEG segment. Drug content was decreased by increasing the amount of polymer but increased by increasing surfactant concentration. Octreotide is hydrophilic it may possibly have partitioned more in the hydrophilic domains than into the hydrophobic domains. The proper selection of surfactant efficiently reduces the leakage of the peptide molecules from the nanoparticles and hence improves peptide loading (Mu et al., 2002).Then the release of the peptide is in the sustained way. This may be due to the contribution of hydrophilic polymer. The faster release of peptide may possibly be due to increased hydration and opening of the hydrophilic framework with increased penetration of water and consequent dissolution of peptide as well as hydrophilic block. The results correlate well with the similar studies (Jeon et al., 2000). Previously, Bodmer et al. (1992) reported the maximal solubility and stability of octreotide in acetate buffer (ph 4.0) (Liebow et al., 1989). Likewise, when Octreotide microspheres were subjected to in vitro release in acetate buffers, 100% drug release with insignificant degradation products while testing were observed. On the contrary, another research showed that at physiological ph (ph 7.4), no instability was shown by Octreotide (Murty et al., 2003). Therefore in this study two buffers were used to study drug release. Release rate from both buffers was correlated. The regression coefficient was found to be The receptor binding was determined as a function of radiolabeled nanoparticles concentrations. Non-specific binding was linear function of peptide concentration, whereas specific binding was saturable. The cell viability diminished when the concentration of octreotide either in free form or inside the nanoparticles was increased. Using an MTT cytotoxicity assay, we have observed that Drug Delivery

10 Synthesis and evaluation of biodegradable PCL/PEG nanoparticles 141 octreotide nanoparticles is able to specifically deliver octreotide into BON-1 cells without harming co-cultured normal cells (Liebow et al., 1989). Although the free-octreotide dose was initially efficient in suppressing further tumor growth inhibition but activity did not last long. After treatment with nanoparticles at successive alternate days, it was observed that the total tumor volume started declining as compared to the control volume (105.4 ± 3.3 mm 3 to 86.3 ± 1.3 mm 3 ). Octreotide nanoparticles were found to be more effective than plain octreotide, and lead to the significant reduction in tumor volume in tumor-bearing mice. This may be explained on the enhanced permeation and retention (EPR) effects prevented the entry of nanoformulation in the normal cell at the same time favored selective entry in tumor. The perfect mechanism for this antiproliferative effect is not known but the increased glutathione reductase and superoxide dismutase activity is probably the reason for significant reduction of tumor volume (Wenger et al., 2001). Rapid blood clearance, high tumor uptake, predominately renal excretion, and low uptake in tissues which do not express somatostatin receptors were demonstrated by biodistribution studies. Conclusion The diagnosis and management of neuroendocrine tumors remains a clinical challenge. In this context PCL/ PEG nanoparticles loaded with octreotide could have a substantial impact in the area of diagnosis and therapy. The optimal therapeutic response may be achieved with the present nanoformulation. Acknowledgements The authors thank Mrs. Krishna Chuttani for her immense help in radiopharmacy aspect of research work. The authors would like to acknowledge Mr. C. Karthikeyan, Dr. Jasleen Uppal and Dr. Anand Pandey for their valuable guidance throughout the work. 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