Peptide conjugated polymeric nanoparticles as a carrier for targeted delivery of docetaxel

Colloids and Surfaces B: Biointerfaces 117 (2014) 166–173

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Peptide conjugated polymeric nanoparticles as a carrier for targeted delivery of docetaxel Hitesh Kulhari a,b , Deep Pooja b , Shweta Shrivastava c , Naidu V.G.M c , Ramakrishna Sistla b,∗ a

IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Medicinal Chemistry & Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India c National Institute of Pharmaceutical Education and Research, Hyderabad, India b

a r t i c l e

i n f o

Article history: Received 29 August 2013 Received in revised form 14 February 2014 Accepted 16 February 2014 Available online 23 February 2014 Keywords: Bombesin Nanoparticles Conjugation Docetaxel Targeting Breast cancer

a b s t r a c t The aim of this research work was to develop Bombesin peptide (BBN) conjugated, docetaxel loaded nanocarrier for the treatment of breast cancer. Docetaxel loaded nanoparticles (DNP) were prepared by solvent evaporation method using sodium cholate as surfactant. BBN was conjugated to DNP surface through covalent bonding. Both DNP and BBN conjugated DNP (BDNP) were characterized by various techniques such as dynamic light scattering, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimetric analysis. The particle diameter and zeta potential of BDNP were 136 ± 3.95 nm and −10.8 ± 2.7 mV, respectively. The change in surface charge and FTIR studies confirmed the formation of amide linkage between BBN and DNP. AFM analysis showed that nanoparticles were spherical in shapes. In nanoparticles, docetaxel was present in its amorphous form as confirmed by DSC and PXRD analysis and was stable during the thermal studies. The formulations showed the sustained release of DTX over the period of 120 h. During cellular toxicity assay in gastrin releasing peptide receptor positive breast cancer cells (MDA-MB-231), BDNP were found to be 12 times more toxic than pure DTX and Taxotere. The IC50 value for DTX, Taxotere, DNP and BDNP was >375, >375, 142.23 and 35.53 ng/ml, respectively. The above studies showed that Bombesin conjugated nanocarrier system could be a promising carrier for active targeting of anticancer drugs in GRP receptor over expressing cancer cells. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nanoparticle mediated targeted drug delivery has attracted lot of attention in last one decade especially for anticancer drug delivery. Conventional use of anticancer drugs is often hampered by nonspecific toxicity and side effects in non-targeted tissues. Nanoparticle mediated intracellular delivery of anticancer drugs provides the advantages of increased local drug delivery due to enhanced permeability and retention (EPR) effect, specificity, controlled drug release and avoidance of systemic drug toxicity. Apart from passive targeting, nanoparticles also offer an opportunity for active targeting through surface modification [1,2].

∗ Corresponding author at: Medicinal Chemistry & Pharmacology Division, CSIRIndian Institute of Chemical Technology, Hyderabad 500607, India. Tel.: +91 40 27193753. E-mail address: [email protected] (R. Sistla). http://dx.doi.org/10.1016/j.colsurfb.2014.02.026 0927-7765/© 2014 Elsevier B.V. All rights reserved.

Currently various small chemicals such as folic acid [3], beta hydroxybutyric acid [4] and biomolecules like mannose6-phosphate [5], galactose [6,7], peptides [8], glycoproteins [9], aptamers [10] and monoclonal antibodies [11] are being used for active targeting of anticancer drugs [1,3,12]. However, physiologically occurring or regulatory peptides have several unique advantages that make them attractive as targeting ligands. Peptides are small molecules with high permeability and biocompatibility. The small size of peptide minimizes the overall size of nanoconjugate while still maintaining high surface density (number of peptides per nanoparticle). Further, peptides are usually hydrophilic in nature and cannot cross (less than 0.1% of total injected peptide) the blood brain barrier. This property of peptide gives an extra benefit when peripheral tumors are the desired targets [13–15]. Various peptides such as K237, RGD, LyP-1, and I4R have been conjugated to nanocarriers to target tumor neovasculature, endothelium, lymphatic metastatic tumors and other tumor tissues [8,16–18].

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The primary objective of this study was to develop Bombesin (BBN) conjugated biodegradable polymeric drug delivery system for anticancer drug. BBN, a tetradecapeptide, was first isolated from the skin of the frog bombina bombina by Anastasi et al. [19]. BBN contains a mammalian counterpart called gastrin releasing peptide (GRP). BBN and GRP show structural and functional similarities especially on C-terminus residue. Because of this, BBN and its analogs may be used to target BBN receptor subtype 2 i.e. GRP receptors. GRP receptors have been found over expressed on lung, breast, ovarian and prostate cancer cells [19,20]. Docetaxel (DTX) is a semi-synthetic, taxane derived, highly potent anticancer drug. It has shown broad spectrum anti-tumor activity against prostate, breast, pancreatic, lung, gastric and hepatic carcinomas [21–23]. DTX binds irreversibly with ␤-actin and stabilizes the microtubule assembly which is responsible for inhibition of cell division and finally cell death [24]. In this study, BBN was conjugated to poly(lactic-co-glycolic acid) (PLGA) nanoparticles for targeted delivery of docetaxel in GRP receptor over expressing breast cancer cells. PLGA is approved by US FDA and European Medicine Agency (EMA) for use in various therapeutic applications in human. Chemically, it is cyclic dimer of two monomers namely lactic acid and glycolic acid. Both monomers are endogenous and by-products of normal metabolic pathways in the body. It provides excellent biocompatibility and biodegradability to PLGA and makes the polymer of choice for drug delivery system development [25]. PLGA nanoparticles provide the advantages of high structural stability, availability of free surface groups for bioconjugation and require less number of excipients than lipid based nanoparticulate systems. On the other hand, they also avoid the problem of rapid agglomeration and surface drug loading of inorganic nanocarriers [26,27]. BBN was covalently coupled to the PLGA nanoparticle surface using EDC/NHS chemistry. The nanoconjugate was physicochemically characterized and studied for in vitro cytotoxicity studies.

2. Materials and methods 2.1. Reagents and chemicals Docetaxel was obtained as gift sample from TherDose Pharma Pvt Ltd. (Hyderabad, India). Poly(d,l-lactic-co-glycolic acid) (PLGA), 50:50 molecular weight ∼ 30,000–60,000, and Bombesin were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade solvents were purchased from Merck specialties (Mumbai, India). MDAMB-231 (breast cancer) cell line was obtained from American Type Culture Collection (ATCC, Manassas, USA). Dulbecco’s modified eagle’s medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), trypsin, EDTA were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco, USA. 96-Well flat bottom tissue culture plates were purchased from Tarson Ltd. (Mumbai, India).

2.2. Preparation of nanoparticles PLGA nanoparticles were prepared by a modified solvent evaporation (nanoprecipitation) method [28]. Five milligrams of DTX was dissolved in 5 ml of PLGA solution (10 mg/ml in acetone) and then quickly poured in 50 ml distilled water with 0.25% (w/v) sodium cholate. The dispersion was briefly sonicated for 2 min in an ice bath and kept on stirring. After 3 h, the dispersion was centrifuged (15,000 rpm) for 30 min to separate DTX loaded nanoparticles (DNP) from free DTX. Nanoparticles were washed thrice with distilled water, lyophilized and stored at 2–8 ◦ C.

167

Fig. 1. Particle size and surface morphology of BBN conjugated nanoparticles (BDNP) determined by (a) dynamic light scattering and (b) atomic force microscopy analysis.

2.3. Bioconjugation of BBN to DTX loaded nanoparticles Twenty milligrams of DNP was dispersed in 5 ml 0.1 M MES buffer (pH 6.2) and incubated with 14.62 mg N-hydroxysuccinimide (NHS) and 191.22 mg N-(3-dimethylaminopropyl)-N ethylcarbodiimide hydrochloride (EDC). The dispersion was kept on gentle stirring for 1 h at room temperature. To this, 0.5 mg of BBN was added, mixed well and kept for further stirring overnight. BBN conjugated DTX loaded nanoparticles (BDNP) were collected after centrifugation at 10,000 rpm for 10 min and washed thrice with distilled water. Conjugation efficiency was determined by estimating free or unconjugated BBN content in the supernatant. BBN was quantified using standard Bradford protein assay. A calibration curve of standard range of 0.5–10 ␮g/ml was prepared using standard BSA stock solution (2 mg/ml). The supernatant was diluted and absorbance was measured at 595 nm using microplate reader (Synergy 4, Biotek, USA). 2.4. Nanoparticle characterization 2.4.1. Particle size and zeta potential Mean particle size and zeta potential of blank and docetaxel loaded PLGA nanoparticles were measured by proton correlation spectroscopy using Malvern Zetasizer Nano (Malvern Instrument Ltd., Malvern, UK). Samples were diluted 1:9 with distilled water and analyzed at 25 ◦ C with a backscattering angle of 173◦ . 2.4.2. Surface morphology Surface morphology of nanoparticles was examined by atomic force microscopy (AFM) using Digital Nanoscope IV (Veeco Instruments, Santa Barbara, CA). A drop of sample was placed on metal slab, air dried for 24 h and scanned for analysis.

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b)

c)

%T

d)

4000.0

3600

3200

2800

2400

2000

1800

cm-1

1600

1400

1200

1000

800

600

450.0

Fig. 2. FTIR spectra of (a) DTX, (b) Bombesin, (c) docetaxel loaded PLGA nanoparticle (DNP) and (d) Bombesin conjugated-docetaxel loaded PLGA nanoparticles (BDNP).

2.4.3. Differential scanning calorimetry (DSC) DSC scans of blank and DTX-loaded PLGA nanoparticles were carried out on DSC-Q100 (TA Instruments, USA). The samples were scanned from 30 ◦ C to 200 ◦ C at a speed of 10 ◦ C/min, under nitrogen environment. 2.4.4. Thermogravimetric analysis (TGA) The thermal properties of DTX, BNP, DNP and BDNP were investigated by TGA analyzer (TGA/SDTA 851e Mettler Toledo, Switzerland). A weighed amount of the compound was placed in a crucible and heated from 25 ◦ C to 650 ◦ C with a heating rate 10 ◦ C/min in nitrogen environment. The change in weight was plotted as a function of temperature.

analyzed for DTX content using HPLC. Drug loading and encapsulation efficiency were determined as follows: % Drug loading =

W

D

in NP WNP

% Encapsulation efficiency =



W

× 100

D

in NP WI



× 100

whereas WNP is the weight of nanoparticles; WD is the weight of DTX; WI is the initial weight of DTX. 2.7. In vitro drug release

2.4.5. Powder X-ray diffraction (PXRD) analysis X-ray diffractograms of DTX, DNP and BDNP were obtained using X-ray diffractometer (D8 Advance, Bruker, Germany) equipped with Cu-K␣ X-ray radiation source, at 40 kV and 30 mA. The diffraction angle (2) was measured at 2–50◦ at a scanning speed of 2◦ /min and step time 13.6 s. 2.5. Analytical method Docetaxel content was measured using high performance liquid chromatography with photodiode array detector (Waters, USA). An octadecylsilane column (Shodex, 250 mm × 4.6 mm, 5 ␮m) was used for analysis and column temperature was maintained at 25 ± 5 ◦ C. The mobile phase, acetonitrile (60%) and water (40%), was pumped at a flow rate of 1.0 ml/min and monitored at a wavelength of 229 nm. The calibration graph was rectilinear in the concentration range of 0.5–10 ␮g/ml with a correlation coefficient of 0.999. The inter-intraday accuracy and precision were within a relative standard deviation (RSD) of ≤5%. 2.6. Drug loading and encapsulation efficiency Lyophilized DTX-loaded PLGA NPs (10 mg) were completely dissolved in 2 ml acetonitrile, centrifuged and supernatant was

To study the docetaxel release profile, pure DTX, Taxotere, DNP and BDNP (containing 1 mg of DTX) were suspended in 1 ml distilled water and placed in a dialysis tubing (12,000–14,000 molecular weight cutoff, Sigma Aldrich, USA). The tubing was placed individually into 75 ml release media at 37 ◦ C and kept for stirring at 100 rpm. The phosphate buffer saline (PBS, pH 7.4) and sodium acetate buffer (SAB, pH 5.0) containing 0.5% Tween 80 were used as release media. An aliquot of 1 ml was withdrawn from the release medium at different time intervals and was replaced with same volume of fresh medium. The total release medium was replaced every 3rd hour with fresh medium. The samples were appropriately diluted, filtered through 0.22 ␮m nylon filter and analyzed for DTX content by HPLC as described above.

2.8. Cell culture MDA-MB-231 (breast cancer) cell line was grown as adherent in DMEM medium supplemented with 10% fetal bovine serum, 100 ␮g/ml penicillin, 200 ␮g/ml streptomycin and 2 mM l-glutamine. The culture was maintained in a humidified atmosphere with 5% CO2 . Dilutions were made with sterile PBS to get required concentration. Formulations were filtered with 0.22 ␮m sterile filter before adding to the wells containing cells.

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169

Fig. 3. (a) Differential scanning calorimetry (DSC) and (b) thermogravimetric analysis (TGA) of pure DTX, PLGA, and various formulations.

2.9. In vitro cytotoxicity studies

2.10. Statistical analysis

Cytotoxicity of formulations was determined by MTT assay based on mitochondrial reduction of yellow MTT tetrazolium dye to a highly colored blue formazan product. 1 × 104 cells (counted by Trypan blue exclusion dye method) in 96 well plates were incubated with formulations and standard Erlotinib with series of concentrations for 48 h at 37 ◦ C in DMEM with 10% FBS medium. Then the above media was replaced with 90 ␮l of fresh serum free media and 10 ␮l of MTT reagent (5 mg/ml) and plates were incubated at 37 ◦ C for 4 h. After removing the media, 200 ␮l of DMSO was added and incubated at 37 ◦ C for further 10 min. The absorbance at 570 nm was measured using spectrophotometer (Spectramax Plus, Molecular Devices, USA). The mean % of cell viability relative to that of untreated cells was estimated from data of three individual experiments. The IC50 value of each compound was calculated by curve fitting method.

All the studies were performed in triplicate and results are expressed as mean ± SD (standard deviation). Statistical significance was analyzed using Student’s t-test for two groups and one-way ANOVA for multiple groups. The p value <0.05 was considered as significant. 3. Results and discussion 3.1. Preparation of blank and DTX loaded PLGA nanoparticles Blank (BNP) and DTX loaded PLGA nanoparticles (DNP) were prepared using sodium cholate and an anionic surfactant. Nanoprecipitation or solvent displacement method was used for nanoparticle preparation because of its simplicity and high drug loading efficiency. The prepared nanoparticles showed very narrow size distribution and low polydispersity. The size of BNP was

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Fig. 4. Powder X-ray diffraction (PXRD) analysis of pure DTX, DNP and BDNP.

92.71 ± 1.46 nm with 0.083 PDI while the DNP showed particle diameter 111.35 ± 2.19 nm with 0.152 PDI (Table 1). 3.2. Drug loading and encapsulation efficiency DTX loading and encapsulation efficiency of PLGA nanoparticle were determined at two drug/polymer (D/P) ratios – 5% (w/w) and

Cumulative % DTX release

a)

3.3. Bioconjugation of BBN to DNP DTX

100 90 80 70

Tax otere DNP BDNP

60 50 40

Bombesin was decorated on DNP surface by well-known EDC/NHS reaction. About 18.2 ␮g of BBN was conjugated to per mg of nanoparticles. The conjugation efficiency was 72.8%. 3.4. Physicochemical characterization of BBN conjugated and unconjugated drug loaded nanoparticles

30 20 10 0 0

24

48

72

96

120

Time (h)

3.4.1. Particle diameter, surface charge and morphology Bombesin surface conjugation was characterized in terms of mean particle diameter, polydispersity index and zeta potential. After BBN conjugation, the size of nanoparticles was increased to

DTX

b) 100

90

Pure DTX

DNP

80

Taxotere

BDNP

70

DNP BDNP

Taxotere

90 80 70

% Cell viability

Cumulative % DTX release

10% (w/w) (Table 1). Initially, 5% (w/w) DTX was added to polymer solution and observed DTX loading and encapsulation efficiency were 4.58% and 96.12%. Because of high drug encapsulation efficiency, drug to polymer ratio was increased to 10% (w/w). At this level, drug loading was found to be 8.03% whereas encapsulation efficiency was 81.38%. Thus 10% (w/w) D/P ratio was considered as optimum level for DTX loading in PLGA nanoparticles.

60 50 40 30

60 50 40 30

20

20

10

10 0

0 0

24

48

72

96

120

Time (h) Fig. 5. In vitro drug release studies of different DTX formulations in (a) PBS and (b) SAB (mean ± SD; n = 3).

3.125

6.25

12.5

25

100

200

Concentration (ng/ml) Fig. 6. Cell viability studies of DTX, Taxotere, DTX loaded nanoparticles (DNP) and BBN conjugated-DTX loaded nanoparticles (BDNP) (mean ± SD; n = 3).

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Fig. 7. Phase contrast images of MDA-MB-231 cells treated with control, free DTX, Taxotere, docetaxel loaded nanoparticles (DNP) and Bombesin conjugated-DTX loaded nanoparticles (BDNP) after 48 h.

136 ± 3.95 nm (Fig. 1a) with 0.247 polydispersity index while the zeta potential was decreased to −11.5 mV (Table 1). The change in surface potential confirmed the conjugation between BBN and PLGA nanoparticles. AFM analysis showed that BDNP were spherical in shape (Fig. 1b). The average particle size observed with AFM analysis was 142.35 ± 8.91 nm (n = 5).

formulation showed only glass transition temperature (Tg ) peak of PLGA at 50.4 ◦ C. It suggested two important things: first, the pure DTX was present as crystalline form but in nanoparticles it was transformed to amorphous or disordered crystalline or to the solid solution state. Second, the nanoparticle preparation method used in this study, does not affects the properties of the PLGA.

3.4.2. FTIR analysis FTIR analysis was carried out to confirm the surface group interaction between NH2 group of BBN and COOH group of DNP (Fig. 2). FTIR spectra of pure BBN showed two characteristic peaks at 3299 cm−1 (free amine group) and 1647 cm−1 (carbonyl group of peptide bond). Unconjugated drug loaded nanoparticles showed characteristic carbonyl peak of carboxylic group at 1761 cm−1 . BDNP showed amide bond peaks at 1646 cm−1 and 1566 cm−1 confirming the conjugation between BBN and DNP.

3.4.4. TGA The thermal behavior of pure drug, polymer and nanoparticle formulation was studied using thermogravimetric analyzer. Fig. 3b shows the thermal degradation behavior of DTX, BNP, DNP and BDNP. The weight loss in pure DTX was started about 200 ◦ C while in case of BNP and DNP it occurred after 300 ◦ C. BDNP formulation showed an early degradation at 220 ◦ C.

3.4.3. DSC studies The physical state of DTX as pure compound and inside the nanoparticles was determined by DSC analysis (Fig. 3a). The melting endothermic peak of pure DTX was appeared at 178.7 ◦ C. However this peak was not observed in nanoparticle formulation. DNP

3.4.5. XRD analysis XRD spectra of pure DTX showed characteristic sharp peaks between 2◦ and 20◦ , confirming the crystalline state of DTX (Fig. 4). In DTX loaded nanoparticle spectra, the sharp peaks of DTX were disappeared and a hump was observed from 10 to 22◦ which indicated that in nanoparticle formulation DTX was present in its amorphous form rather than crystalline. In BDNP spectra, the some

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Table 1 Physicochemical characterization of nanoparticles: particle size, size distribution, zeta potential and drug encapsulation efficiency. Data represent mean ± SD, n = 6. Formulation

Particle diameter (nm)

Polydispersity index (PDI)

Zeta potential (mV)

Drug loading (%)

Encapsulation efficiency (%)

BNP DNP BDNP

92.71 ± 1.46 111.35 ± 2.19 136 ± 3.95

0.083 ± 0.029 0.152 ± 0.046 0.247 ± 0.051

−28.7 ± 2.76 −26.9 ± 2.55 −11.5 ± 1.34

– 8.03 ± 1.4 7.52 ± 0.63

– 81.38 ± 2.45 83.27 ± 1.74

sharp peaks were appeared which may be because of BBN present on nanoparticle surface. 3.5. In vitro drug release In vitro release of DTX from pure drug suspension, Taxotere and nanoparticles were performed in phosphate buffer saline, pH 7.4 (PBS) (Fig. 5a) and in sodium acetate buffer, pH 5.0 (SAB) (Fig. 5b). From pure drug suspension and Taxotere, DTX were released completely within 10 h in both PBS and SAB. The nanoparticle formulations showed biphasic pattern of DTX release. About 21% of drug was released in first 24 h but after that only 11% and 15.3% DTX was released in next 96 h from DNP and BDNP formulations, respectively. The initial faster drug release may be due to the release of drug present on the surface or near to the periphery of the nanoparticles [29]. The drug release was slightly higher in BDNP than from DNP which may be because of hydrophilic nature of BBN present on the surface. A similar kind of pattern was observed in SAB. But the overall release of DTX from nanoparticles was more in SAB (36.2%) compared to PBS (31.57%). It can be explained by the faster degradation of PLGA at acidic pH [30]. This faster degradation of nanoparticles and hence increased DTX release in acidic pH than PBS would provide the advantage of maximum drug release in cancer cells. 3.6. Cytotoxicity studies The anticancer activity of docetaxel, Taxotere and nanoparticle formulations was studied by MTT assay in MDA MB231 (breast cancer) cells (Figs. 6 and 7). MDA MB231 cells were selected for in vitro cytotoxicity study for three reasons. First, these cells are reported to over express GRP receptors [31]. Second, MDA MB231 cells are triple negative cells – estrogen receptor negative, progesterone receptor negative and human epidermal growth factor 2 (HER 2) receptor negative. So the hormonal and trastuzumab therapy may not be useful for the treatment of these cells. Third, MDAMB 231 cells show intermediate response to the chemotherapy [32]. So the effect of peptide conjugated formulation can be clearly observed from the effect of unconjugated formulation. The observed half maximal inhibitory concentration (IC50 ) for both free DTX and Taxotere was more than 375 ng/ml. Both DNP and BDNP showed more cellular toxicity than free DTX and Taxotere. The IC50 value for DNP and BDNP was 142.23 ± 18.95 and 35.53 ± 5.91. At low concentration of 3.125 ng/ml, both DNP and BDNP showed significant cytotoxicity compared to either pure DTX or Taxotere. However, the difference of cytotoxic potential of DTX was insignificant between DNP and BDNP. Significant improvement in cytotoxicity was observed with BDNP compared to DNP in a dose-dependent manner from 6.25 to 100 ng/ml. At higher concentration of 200 ng/ml, BDNP could not enhance the cytotoxicity over DNP. This may be due to saturation of GRP receptors on the cells in the conditions employed with the number of cells taken. The significant increase in toxicity of DNP compared to free DTX and Taxotere can be attributed to passive targeting of nanoparticles. Several anticancer drugs including dexamethasone, paclitaxel, vincristine, curcumin, etoposide, campatothecin, 5-fluorouracil, doxorubicin and cisplatin have been successfully delivered and passively targeted by encapsulation in PLGA nanoparticles [33].

Bombesin conjugated nanoparticles (BDNP) were 4 times more toxic (p < 0.001) than unconjugated nanoparticles (DNP) which may be due to receptor-mediated targeting of nanoparticles. The enhanced cellular internalization leads to increase in cytotoxicity of actively targeted BDNP nanoparticles. Further, Koziara et al. [34] reported that nanoparticles internalized through receptor mediated endocytosis does not trigger the P-glycoprotein pump. It will help to overcome the efflux of drug by multidrug resistance, a common problem of conventional chemotherapy. 4. Conclusion Targeted drug delivery systems have shown a great advance in delivery of anticancer drugs exclusively to cancer cells. In this study, Bombesin peptide was first time conjugated with biodegradable polymeric nanoparticles for the delivery of an anti-cancer drug, docetaxel, to breast cancer cells. The breast cancer cell lines used in this study were low to intermediate chemotherapy responsive. The peptide conjugated nanoparticles showed significantly higher in vitro cytotoxicity to the targeted cells than free docetaxel. As per the generalized and well accepted concept, nanoparticles of <200 nm size with hydrophilic surface exhibits improved EPR effect due to longer retention of nanocarriers in the blood stream. Because of small size, sustained drug release and enhanced and targeted delivery, the developed drug delivery system has great potential to deliver anticancer drugs to GRP receptor over-expressing and to low chemotherapy-responsive cells. Conflict of interest The authors report no conflict of interest for this work. Acknowledgements The authors are grateful to the Director, CSIR-Indian Institute of Chemical Technology, Hyderabad for providing the necessary facilities. The generous help of Therdose Pharma is greatly acknowledged. Hitesh Kulhari is thankful to the Head of IICT-RMIT Research Centre for providing the Junior research fellowship. Deep Pooja thank to Council of Scientific and Industrial Research (CSIR), New Delhi for awarding Senior Research fellowship. This work was partially supported by CSIR Grant under project ADD (CSC 0302). References [1] J.D. Byrne, T. Betancourt, L. Brannon-Peppas, Adv. Drug Deliv. Rev. 60 (2008) 1615. [2] F. Danhier, O. Feron, V. Préat, J. Control. Release 148 (2010) 135. [3] R. Oliveira, P. Zhao, N. Li, L.C.S. Maria, J. Vergnaud, J. Ruiz, D. Astruc, G. Barratt, Int. J. Pharm. (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.031. [4] V.K. Venishetty, R. Samala, R. Komuravelli, M. Kuncha, R. Sistla, P.V. Diwan, Nanomed. Nanotechnol. Biol. Med. 9 (2013) 388. [5] J. Prakash, L. Beljaars, A.K. Harapanahalli, M. Zeinstra-Smith, A. de JagerKrikken, M. Hessing, H. Steen, K. Poelstra, Int. J. Cancer 126 (2010) 1966. [6] M. Pimm, A. Perkins, R. Duncan, K. Ulbrich, J. Drug Target. 1 (1993) 125. [7] A. David, P. Kopeˇcková, T. Minko, A. Rubinstein, J. Kopeˇcek, Eur. J. Cancer 40 (2004) 148. [8] J.H. Kim, S.M. Bae, M.H. Na, H. Shin, Y.J. Yang, K.H. Min, K.Y. Choi, K. Kim, R.-W. Park, I.C. Kwon, J. Control. Release 157 (2012) 493. [9] Y. Mo, L.Y. Lim, J. Control. Release 108 (2005) 244. [10] C. Yu, Y. Hu, J. Duan, W. Yuan, C. Wang, H. Xu, X.D. Yang, PLoS ONE 6 (2011) e24077.

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