Dual targeted polymeric nanoparticles based on tumor endothelium and tumor cells for enhanced antitumor drug delivery.

Article pubs.acs.org/molecularpharmaceutics

Dual Targeted Polymeric Nanoparticles Based on Tumor Endothelium and Tumor Cells for Enhanced Antitumor Drug Delivery Madhu Gupta,*,† Gousia Chashoo,‡ Parduman Raj Sharma,‡ Ajit Kumar Saxena,‡ Prem Narayan Gupta,§ Govind Prasad Agrawal,∥ and Suresh Prasad Vyas† †

Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar-470003, M.P., India ‡ Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu-180001, India § Formulation & Drug Delivery Division, Indian Institute of Integrative Medicine, Jammu-180001, India ∥ Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar-470003, M.P., India S Supporting Information *

ABSTRACT: Some specific types of tumor cells and tumor endothelial cells represented CD13 proteins and act as receptors for Asn-Gly-Arg (NGR) motifs containing peptide. These CD13 receptors can be specifically recognized and bind through the specific sequence of cyclic NGR (cNGR) peptide and presented more affinity and specificity toward them. The cNGR peptide was conjugated to the poly(ethylene glycol) (PEG) terminal end in the poly(lactic-co-glycolic) acid PLGAPEG block copolymer. Then, the ligand conjugated nanoparticles (cNGR-DNB-NPs) encapsulating docetaxel (DTX) were synthesized from preformed block copolymer by the emulsion/solvent evaporation method and characterized for different parameters. The various studies such as in vitro cytotoxicity, cell apoptosis, and cell cycle analysis presented the enhanced therapeutic potential of cNGR-DNB-NPs. The higher cellular uptake was also found in cNGR peptide anchored NPs into HUVEC and HT-1080 cells. However, free cNGR could inhibit receptor mediated intracellular uptake of NPs into both types of cells at 37 and 4 °C temperatures, revealing the involvement of receptor-mediated endocytosis. The in vivo biodistribution and antitumor efficacy studies indicated that targeted NPs have a higher therapeutic efficacy through targeting the tumor-specific site. Therefore, the study exhibited that cNGRfunctionalized PEG-PLGA-NPs could be a promising approach for therapeutic applications to efficient antitumor drug delivery. KEYWORDS: cyclic NGR, CD13 receptor, nanoparticles, solid tumor, docetaxel

1. INTRODUCTION A chemotherapeutic approach is highly significant in the treatment of solid tumors, but most of the available anticancer drugs damage the normal tissue and create systemic toxicity relating to severe side effects.1 Docetaxel (DTX) is a potent anticancer moiety of the taxanes family, which has broad activity and has been proven efficient in the clinical oncology. The commercial formulation of DTX based on the surfactants is Taxotere; however it has serious limitations, including myelosuppression, hematological, neurotoxicity, and hypersensitivity reactions, which very much limit its applications.2 To overcome these limitations, polymer-based nanoparticles (NPs) were designed. The polymeric NPs are colloidal particles, which are self-assembled in case of amphiphilic block copolymers when exposed to an aqueous media. They offer several benefits such as a higher drug pay load, prolonged blood circulation, and controlled release profiles. In polymeric NPs, the cytotoxic drug can be easily encapsulated into the hydrophobic core. The outer exposed hydrophilic part provided the stable dispersion © 2014 American Chemical Society

by imparting a steric stabilization effect that ultimately enhanced its blood residence time following intravenous injection.3 Poly(lactic-co-glycolic acid) (PLGA) is commonly exploited for drug delivery and the biomedical field owing to its excellent biocompatible, biodegradable nature and well-established safety in clinic applications. However, the block copolymer of PLGA with poly(ethylene glycol) (PEG) as mPEG-PLGA offers an attractive option owing to PEGylated polymeric NPs diminishing the systemic clearance as compared with similar size particles but without PEG and successfully employed for passive targeting. They accumulated the tumor site through enhanced permeability and retention (EPR) effects.4 The continual advancements in drug delivery science and Received: Revised: Accepted: Published: 697

July 13, 2013 January 23, 2014 January 31, 2014 February 11, 2014 dx.doi.org/10.1021/mp400404p | Mol. Pharmaceutics 2014, 11, 697−715

Molecular Pharmaceutics

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Figure 1. Schematic representation of the synthesis of cNGR-PLGA-PEG copolymer.

cNGR peptide was subsequently conjugated to the PEG terminus end of the copolymer. The targeted NPs were designed with this preformed block copolymer and designated as cNGR-DNB-NPs. The dual-targeting effect of engineered nanosystem was investigated, and the targeting potential was systematically explored in vitro using appropriate cell lines (HT1080 and HUVEC cells). The cell uptake, in vitro cytotoxicity, cellular apoptosis, and cell cycle assay were performed in CD13 overexpressed tumor cells and tumor endothelial cells, while in vivo study was carried out on Balb/c mice bearing HT-1080 tumors.

biotechnology both offered various targeting options that specifically identify and bind the receptors that are highly overexpressed on angiogenic vessels within solid tumors and also overexpressed on tumor cells.5 The aminopeptidase N (APN or CD13) is a membranebound, zinc-dependent metalloproteinase and has contributed in tumor invasion and angiogenesis.6 It has also been noted that CD13 is an important biomarker, which is highly overexpressed at endothelial cell surface in the neo-angiogenic blood vessels, and especially in tumor cells that contribute in binding. Their involvement in receptor mediated endocytosis has significant applications in site specific drug delivery.7 Among various targeting moieties, which are presently under investigation, the NGR sequence (asparagine−glycine−arginine) is popular and widely being explored for its implication in delivering the chemotherapeutic drugs, apoptotic peptides,8 liposomes,9 and micelles7,10 to the tumor site. The NGR recognizes a tumorspecific isoform of CD13 and demonstrated a potent tumor targeting potential. A previous study supported that the cyclic NGR (cNGR) peptide with a disulfide bridge constraint is too much critical for stabilization of the bent conformation and significantly improved the targeting efficiency.11 Therefore, it was hypothesized that the NGR-CD13 mediated interaction might be a significant approach for special and effective drug delivery to a solid tumor. Previous reports established that HT1080 cells (fibrosarcoma) overexpressed the higher level of CD13 protein expression, while HUVEC (human umbilical vein endothelial cells) possessed intermediate level of CD13 expression, so HT-1080 and HUVEC cells were selected as model for tumor cells and tumor endothelial cells, respectively.7 The main aim of our investigation was to synthesize an amphiphilic diblock copolymer, HOOC-PEG-b-PLGA; then

2. EXPERIMENTAL SECTION 2.1. Materials. PLGA 502 H (lactide/glycolide ratio 50:50, inherent viscosity 0.22 dL/g) was obtained as a gift sample from Sun Pharma Advanced Research Company Ltd. (SPARC), Vadodara, India. DTX received as a gift sample from M/s Dabur (Franscius Kabi Oncology Lab) New Delhi, India. NH2-PEG-COOH (MW 3000) was purchased from Sigma−Aldrich Chemical (St. Louis, MO) USA. cNGR peptide (CNGRCK) was synthesized by USV Ltd. Mumbai, India. NHS (N-hydroxysuccinimide), N,N′-dicyclohexylcarbodiimide (DCC), and DIPEA (N,N-di-isopropylethylamine) were purchased from HiMedia, India. Dichloromethane (DCM), methanol, DMSO (dimethyl sulfoxide), and diethyl ether were either of analytical grade or high-performance liquid chromatography (HPLC) grade. 2.2. Synthesis of cNGR Decorated PLGA-PEG Copolymer. 2.2.1. Synthesis of PLGA-PEG-COOH Copolymer. Carboxylate-functionalized PLGA-PEG copolymer was synthesized through the conjugation of the COOH-PEG-NH2 to PLGA-COOH part by using the previously reported method 698

dx.doi.org/10.1021/mp400404p | Mol. Pharmaceutics 2014, 11, 697−715


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with slight modification.12 The synthetic scheme of PLGAPEG-COOH diblock copolymers is shown in Figure 1. The synthesized block copolymers (PLGA-PEG-COOH) were characterized by 1HNMR (Avance-300 Bruker, Switzerland) in deutrated chloroform (CDCl3) using tetramethyl silane (TMS) as a standard and also confirmed by FT-IR (Thermo Nicolet Nexus 670, USA) using KBr disc. 2.2.2. Synthesis of cNGR-Decorated PLGA-PEG (cNGRPEG-PLGA). The cNGR peptide was conjugated to PLGA-PEG block copolymer through a NHS group using the previously reported method with slight modification.13 The PLGA-PEGCOOH (50 mg) block copolymer was first dissolved in 4 mL of DCM and stirred at room temperature for 1 h in the presence of NHS and DCC. The formed intermediate was repeatedly washed with an ice-cold mixture of ethyl ether and methanol to remove the residue of NHS and DCC. After that, cNGR peptide (cyclic CNGRCK peptide) (1.2 equiv) and NHS-PEGPLGA (1 equiv) both were dissolved in anhydrous DMSO and stirred for 3 h at room temperature followed by further addition of DMSO. The resulting solution was kept in a dialysis bag and dialyzed against deionized water for 24 h to remove the unconjugated peptides. The final solution was lyophilized and stored at −20 °C until use. The ligand appended synthesized block copolymer (PLGA-PEG-cNGR) structure was confirmed by FT-IR (Thermo Nicolet Nexus 670, USA) using KBr disc. 2.3. Preparation of NPs. The peptide-conjugated DTX loaded NPs (cNGR-DNB-NPs) were developed with an emulsion/solvent evaporation method as described previously with slight modification.14 Briefly, cNGR-PEG-PLGA and PLGA-PEG-COOH copolymer and drug were dissolved in 2 mL of DCM and then added into 5 mL of 0.1% (w/v) pluronic F-68 aqueous solution. The mixture was sonicated using a probe sonicator (Soniweld, Mumbai, India) for 1 min. The emulsion was formed and poured dropwise into 15 mL of 0.1% (w/v) pluronic F-68 containing aqueous solution with stirring by using a mechanical stirrer (Remi Instruments, Mumbai, India) at room temperature for 3 h to obtain a nanoparticulate dispersion. The dispersion was centrifuged (25 000 rpm for 30 min, Hitachi CPMax-100 Japan) to remove the precipitates and then washed three times with distilled water to remove excess surfactant. Considering the requirement of sterility, nanoparticulate suspensions were first prefiltered through a membrane with a pore size of 0.45 μm (Ran Disc Hydrophobic PTFE membrane New Delhi, India), followed by filtering through a 0.22 μm (Ran Disc Hydrophobic PTFE membrane New Delhi, India) filter. 2.4. Characterization of NPs. The shape and morphology of the NPs were observed using scanning electron microscopy (SEM, Leo 435 VP 501B, Philips). The average particle size, PI, and zeta potential were determined by photon correlation spectroscopy (PCS) using the Zetasizer nano ZS90 (ZS 90 Malvern Instruments, UK) at 25 °C. The percent entrapment efficiency (%EE) was estimated by previously reported method.15 The NPs were dissolved in 1 mL of acetonitrile and shaken gently followed by sonication for 5 min. Then, 2 mL of methanol was added to precipitate the polymer. The sample was then centrifuged (25 000 rpm for 30 min, Hitachi CPMax-100, Japan), and the amount of drug in the supernatant was assayed by using a HPLC system. The HPLC system (Shimadzu, Japan) consisting of Hypersil C18 column (250 × 4.6 mm, 5 μ) with a Waters C18 Nova-Pak guard column (20 × 3.9 mm, 5 μ), and the column effluent was measured by using a UV detector at 230 nm. The mobile phase

was a mixture of acetonitrile−methanol−0.02 M ammonium acetate buffer (pH 5.0) in a ratio of 20:50:30 (v/v/v), and it was run at a flow rate of 1.0 mL/min.16 2.5. Estimation of Peptide Conjugation Efficiency and Peptide Density on the NP Surface. The conjugation of cNGR peptide on PEGylated NPs was determined by using the CBQCA Protein Quantitation Kit (Molecular Probes). The conjugation efficiency (CE%) determined the percentage of peptide conjugated on the NP surface using the formula as follows: CE% =

amount of peptide conjugated on the NPs surface × 100 total amount of peptide added

The average number of peptide molecule conjugated per milliliter of NP suspension (N) was calculated using the following equation: N=

6W × 10−3 π (D × 10−7)3 × ρ

where W is the NP concentration (mg/mL), D is the particle size of NPs, and ρ is the NP density (weight per volume unit, 1.1 g/cm3).17 The average peptide molecules conjugated per milliter in the identical NP suspension (M) was calculated as: M=

(Co − C) × 10−3 × 6.02 × 1023 molecular weight of peptide

where Co and C are the peptide concentration of feeding and ultrafiltrate during preparation, respectively. The average number of peptide conjugated per NPs (surface density, P) was calculated by dividing M/N.18 The distance (d) between two neighboring PEG chains linked to peptides can be calculated as the square root of the mean area occupied by each peptide on the particle surface and using a simplified equation.19

d = D(π /P)1/2 2.6. In Vitro Drug Release. The in vitro drug release from NPs was determined with previously reported method by using phosphate buffered solution (PBS, pH 7.4) phthalate buffer (pH 5.0) and mice plasma as the release media.20 Briefly, 2 mL of NPs was placed in a dialysis bag and dialyzed against the 30 mL of release media containing 0.1% Tween-80 under slow, however continuous, magnetic stirring at 100 rpm and at 37 ± 1 °C. At appropriate time intervals, the aliquots from release media were taken and replaced with fresh media. The samples for each data point were collected and measured for drug contents in triplicate. 2.7. Protein Adsorption to the NP Surface. The protein adsorption on the NPs surface was determined by using a previously reported method with slight modifications.21 A fixed amount of NPs was incubated with 5 mL of either 5% fetal bovine serum (FBS) or 2% bovine serum albumin (BSA) at 37 °C for 24 h, and samples were analyzed by PCS using the Zetasizer nano ZS90 (ZS 90 Malvern Instruments, UK) (n = 3). 2.8. Hemolysis Toxicity. Hemolysis toxicity was studied following the previously reported procedure with minor modifications.22 The RBC suspension (5% hematocrit) was collected in HiAnticlot blood collection vials from the human blood (Himedia Laboratories, Mumbai, India). A known 699



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medium and incubated for 24 h. The medium was replaced with 100 μL of fresh medium containing various formulations at a concentration of 0.25−10 μg/mL and coincubated for 24, 48, and 72 h. At designated time periods, the medium was replaced with DMEM containing MTT (5 mg/mL), and then cells were further incubated at 37 °C for 4 h. After that, the MTT solution was removed, and 100 μL of DMSO was added into each well to dissolve any formazan crystals precipitated. The plate was vigorously shaken for 15 min before the determination of optical absorbance at 540 nm using a microplate reader (Biorad, 680, America). 2.9.5. Evaluation of Cell Apoptosis Activity. The cell apoptosis was detected by assessment of nuclear morphology staining with 4,6-diamidino-2-phenylindole (DAPI) staining was conducted as per the method reported previously with slight modification.26 Briefly, HT-1080 cells and HUVEC cells were seeded in 6-well plates containing a coverslip with 2 × 105 cells per well and cultured at 37 °C for 24 h. Cells were further incubated for another 36 h with formulations and culture medium as control. Samples were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and stained with 0.2 μg/mL DAPI in PBS at room temperature for 15 min and washed twice with ice-cold PBS. Coverslips were mounted onto glass slides. Slides were then examined under inverted fluorescence microscope (Olympus 1 × 70). 2.9.6. Cell Cycle Analysis. 5 × 106 HT-1080 cells were seeded on 6-well plate, allowed to adhered and grow for 24 h, then incubated with 1 mL media containing 0.5 μg/mL formulations and kept for 36 h. After that, media was removed; cells washed with PBS and treated with 200 μL of trypsin− EDTA. The EDTA treated cell was collected and centrifuged at 1500 rpm for 5 min, and the cell pellet was washed twice with PBS and centrifuged. Cells were resusupended in 1 mL of cold PBS and vortexed at slow speed, and 2 mL of absolute ethanol was added dropwise to make the final concentration 70% (v/v). After 15 min of incubation at 4 °C, cells were resusupended in 250 μL of staining PBS solution composed of RNase A (0.1 mg/mL), PI (10 μg/mL), and Triton-X 100 (0.05% v/v). After incubation for 1 h at room temperature in dark, the cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences, USA); the median number of events analyzed for each sample was 10 000, and data were analyzed with Cell-Quest software. 2.9.7. Cellular Uptake of NPs and Competition Assay. For cell uptake examination, HT-1080 cells or HUVEC cells were seeded at a density of 2 × 104 cells/well in 6-well plates over glass coverslips and incubated for 24 h. The medium was replaced with FITC-loaded various polymeric NPs in medium for 4 h at 37 °C with 5% CO2 influx in an incubator. The medium was removed, and cells were washed with cold PBS followed by fixing with 4% paraformaldehyde in PBS for 30 min at room temperature. Cells on coverslip were mounted in Vectashieldanti-fade mounting medium with DAPI (Vector Laboratories) for 5 min, and the fluorescent images of cells were captured using a confocal laser scanning microscope (CLSM, Leica, TCS SP2, Germany). To observe if cNGR peptide could hinder the cNGR-DNB-NPs mediated endocytosis, HT-1080 and HUVEC cells were preincubated with 1 mg/mL of free cNGR peptide for 1 h at 37 °C to saturate CD13 receptors, before they were exposed to FITC-labeled cNGR-DNB-NPs. For quantitative analysis of the of the cellular uptake of cNGR-DNB-NPs, HUVEC and HT-1080 cells were seeded at a density of 5 × 106 cells in 6-well plates for 24 h. After this, cells

volume (0.5 mL) of suitably diluted free DTX solution and nanoparticulate dispersions were added separately to 4.5 mL of normal saline and incubated with RBC suspension. The tubes were allowed to stand for 1 h with gentle intermittent shaking and then centrifuged for 15 min at 2500 rpm (Spinwin, Microcentrifuge India). After centrifugation, supernatants were taken and diluted with an appropriate volume of normal saline. The absorbance was measured at λmax 540 nm (Shimadzu 160A UV−visible spectrophotometer, Japan), and the degree of hemolysis was determined. 2.9. Cell Culture. A mouse macrophage cell line (RAW 264.7), human fibrosarcoma cell line (HT-1080), and human umbilical vein endothelial cells (HUVEC) were selected for this purpose. Cells were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM, SigmaAldrich) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), penicillin (100 U/mL), and streptomycin (100 U/mL). The cells were routinely passaged at 90−95% confluence. 2.9.1. Phagocytosis Uptake. RAW 264.7 cells (1 × 105 cells) were plated in 12-well flat bottom plates and allowed to adhere overnight, after 48 h cells were incubated with 100 μL of specified fluorescein isothiocyanate (FITC) loaded formulations. Then plates were incubated for 1, 2, and 4 h at 37 °C for phagocytosis. Then cells were washed three times with cold PBS (pH 7.4) and then treated with sodium azide (10 mM) and harvested using trypsin−ethylenediaminetetraacetic acid (EDTA) treatment followed by centrifugation at 1500 rpm for 5 min. Samples were kept at 4 °C under protection from light, and FACS analysis was conducted within 1 h of sample preparation.23 FACS analysis was conducted using a BD FACS Calibur flow cytometer for the acquisition of samples, and data were analyzed using the CellQuest software. 2.9.2. Trypan Blue Exclusion Assay. The method for trypan blue exclusion has been previously described.24 Both types of cells (HUVEC and HT-1080 cells) were kept in a 24-well microplate (at 1 × 105 cells/mL), with 1 mL of DMEM medium/well and incubated overnight at 37 °C, 5% CO2 and 95% air. After that, cells were treated with various DTX based formulations and transferred into sterile polypropylene tubes, and samples of cells were drawn after 8, 12, 24, and 48 h incubation and treated with 0.4% trypan blue in 0.9% saline for 5 min. The stained, nonviable cells and unstained, viable cells were then respectively counted using a hemocytometer. 2.9.3. Anticancer Activities of Various Formulations Determined by Clonogenic Assay. A clonogenic assay was performed to measure the effectiveness of the tested formulations against long-term cancer cell proliferation.25 Approximately 5 × 105 HT-1080 and HUVEC cells were trypsinized and seeded after 24 h, and drug loaded nanoparticulate formulations in equivalent amounts were incubated with the cell cultures for 48 h. Then, cells were trypsinized, plated on petri dishes (10 000 cells per dish), and incubated in a growth medium under cell culture conditions. Macroscopic colonies formed from viable cells after 10−14 days; then they were fixed and stained with 0.5% solution of methylene blue in ethanol, and their numbers were counted. The plating efficiency (PE) of each treatment was calculated as (number of colonies formed × 100%)/(number of cells plated). For every concentration point, six samples were prepared. The experiments were repeated at least three times. 2.9.4. Cytotoxicity Assay. HT-1080 and HUVEC cells were seeded (5000 cells per well) in 96-well plate in DMEM 700



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intravenously administered with various formulations at a dose of 10 mg/kg DTX, respectively. After i.v. injection at designated time points (0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h), the mice were sacrificed, and blood samples had been withdrawn from the retro-orbital plexus, followed by collection of different organs (heart, liver, spleen, lung, kidney, brain, and tumor). The organs were subsequently washed with normal saline and stored at −20 °C until analyzed by a HPLC system. The organs were washed with Ringer’s solution to remove any adhered debris and dried using a tissue paper. Briefly, 1 g of the tissue sample was homogenized with 5 times of normal saline, using the tissue homogenizer (Ultrasonic cell crusher noise isolating thamber, Lark, Chennai, India) for 5 min. The tissue homogenate was extracted with 800 μL of ethyl acetate and subsequently centrifuged at 15 000 rpm (Hitachi CPMax-100, Japan) for 20 min. Thereafter, the organic layer was evaporated at room temperature. The leftover residue was dissolved in 200 μL of acetonitrile, centrifuged at 15 000 rpm (Hitachi CPMax100, Japan) for 20 min; supernatant was collected and assayed for DTX content by the HPLC method. For the assay of drug in plasma, serum was separated from the collected blood samples by centrifugation at 3500 rpm (Spinwin, Microcentrifuge India) for 20 min, and the serum was collected. Thereafter, 200 μL of plasma samples was extracted by adding 250 μL of methanol and 250 μL of acetonitrile by vortex-mixing the samples for 30 s. The mixture was then processed as done in organ homogenates. 2.11. In Vivo Toxicological Parameters. 2.11.1. Hematological Study. Balb/c mice were divided in various treatment groups with three mice in each group. Each group received an intravenous injection of 10 mg/kg equivalent of DTX loaded various formulations or saline respectively, once for two days for a week. Blood sample was collected after 24 h of the last administration from the animals of all of the groups and analyzed at a pathology laboratory for RBC count, WBC count, platelets count, and hemoglobin (Hb) content. 2.11.2. Evaluation of Nephrotoxic and Hepatotoxic Effect. Balb/c mice were divided in various treatment groups of three mice in each group. Each group received an intravenous injection of various formulations at a dose of 10 mg/kg body weight. Blood samples were collected through retro-orbital plexus of the eye after 72 h of administration. The samples were subjected to centrifugation at 3500 rpm for 20 min, and the serum was collected and analyzed for serum level of urea, creatinine, SGOT (serum glutamic oxaloacetic transaminase) or AST (aspartate aminotransferase), SGPT (Serum glutamic pyruvic transaminase) or ALT (alanine aminotransferase), and alkaline phosphatase (ALP). 2.12. Data Analysis. The data were expressed as the mean ± standard deviation. The treated groups were compared with control by using the Student t-test and analysis of variance (ANOVA). Differences were considered to be statistically significant at a level of P < 0.05.

were incubated with FITC labeled DNB-NPs, cNGR-DNBNPs, and preincubation with 1 mg/mL of free cNGR for 1 h at 4 and 37 °C to saturate CD13 receptors before the cells were exposed and incubated with FITC-labeled cNGR-DNB-NPs. After this, cells were washed three times with PBS, trypsinized, and then centrifuged at 1500 rpm for 5 min to obtain a cell pellet, which was subsequently resuspended in PBS and analyzed using a flow cytometer (FACS Calibur, BD Biosciences, USA). 2.9.8. Tracking of Uptake Pathways Using Various Endocytic Inhibitors. To investigate the effect of various inhibitors on the uptake pathway of the NPs, both cells 5 × 106 cells/well were seeded in 6-well plates and adhered for 24 h, and then, cells were preincubated with the following inhibitors individually at concentrations which were not toxic to the cells.27 Sodium azide (0.1% w/v for 1 h, energy-dependent mechanisms), chlorpromazine (10 μg/mL for 30 min, inhibition of clathrin-mediated uptake), genistein (1 μg/mL for 30 min, caveolae-mediated endocytosis), and cytochalasin D (30 μM for 30 min, macropinocytosis inhibitor) were added into each well. After incubation, the treated agents were withdrawn from the wells, and cells were further treated with various formulations for 4 h, respectively. Subsequently, the cells were washed three times with PBS, trypsinized, collected, again washed, and resuspended in PBS. The fluorescence signal was analyzed using a flow cytometer (FACS Calibur, BD Biosciences, USA). 2.10. In Vivo Studies. The in vivo studies were performed as per the guidelines compiled by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Ministry of Culture, Government of India). All of the animal studies were conducted in accordance with the protocol approved by the Institutional Animal Ethical Committee of Dr. H.S. Gour University, Sagar (MP) India (vide letter no. Animal Eths. Comm./10/87, dated 22/05/ 2010). 2.10.1. In Vivo Antitumor Activity. The antitumor efficacy was investigated in HT-1080 cells bearing Balb/c mice (6−8 weeks, weighted 18−22 g). HT-1080 cells were injected subcutaneously at 2 × 106 cells in the flanks of mice. When the tumor volume reached about 100 mm3, this day was designated as “day 0”. Then, the mice were categorized into various groups (n = 9) and treated using with one of the following dosing regimens. Each group of mice was treated every fourth days for a total four times by tail vein injection of the different formulations. Weight loss and tumor growth were measured. V = πab2/6, here a = major axis, b = minor axis. After 28 days, three mice per group were sacrificed for the measurement of tumor weight, and the remaining animals were used for studying the survival period. The tumor volume growth percentage inhibition (VI) was calculated as follows: VI (%) = [1 − (mean tumor volume at the end of therapy in the treatment group)/(mean tumor volume at the end of therapy in the control group)] × 100. The tumor weight growth percentage inhibition (WI) was calculated as follows: WI (%) = [1 − (mean tumor weight in the treatment group at the end of therapy)/(mean tumor weight in the control group at the end of therapy)] × 100.28 2.10.2. Biodistribution Studies. The plasma and tissue biodistribution studies of various DTX loaded formulations were performed on tumor induced Balb/c mice.29 Tumor induced mice were randomly divided into various groups, each group comprising of 27 animals. The various groups were

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of HOOC-PEGPLGA. The block copolymer PLGA-PEG-COOH can be synthesized by direct conjugation of PLGA-COOH with NH2-PEG-COOH, to form PLGA-PEG-COOH (Figure 1), and characterized by FT-IR and 1H NMR. After synthesis, the cNGR ligand moiety was attached to the carboxyl group of copolymer, and the synthesized copolymer was then characterized by FT-IR analysis. 701



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Figure 2. (a) SEM photograph of cNGR-DNB-NPs; (b) in vitro drug release profiles of DTX from the NPs at 37 ± 1 °C in PBS solution at pH 7.4 (c) phthalate buffer at pH 5.0 and (d) mice plasma. Data presented as the mean ± SD (n = 3). DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

PEG-COOH, a prominent peak at 1746.14 cm −1 is corresponding to −CO stretching in the polymers. The presence of a prominent peak at 1626.71 cm−1 could be attributed to the formation of an amide bond between amino group and carboxyl group. The band around 3527.89 cm−1 could be attributed to the characteristic absorption band of the −OH stretching of synthesized polymer, while the bands at 2929.46 cm−1 and 2850.97 cm−1 could be due to C−H stretch. 3.2. Synthesis and Characterization of cNGR-PEGPLGA. The conjugated structure of synthesized PLGA-PEGcNGR was confirmed by FT-IR spectroscopy (Figure 1s,e). The cNGR-PEG-PLGA part represented the greatly weakened peak at 3300−3500 cm−1 due to involvement of free carboxylic group of PLGA-PEG in reaction with amino groups of cNGR. An additional, peak appeared at 1626.82 cm−1 could be assigned to the amide groups. As compared with PLGA-PEGCOOH, the enhanced strength of the amide groups in cNGRPLGA-PEG was further suggesting the increase in amide groups. The other characteristic peak at 1243.96 cm−1 may be due to the C−S stretching and indication of cNGR peptide conjugation and synthesis of the formed PLGA-PEG-cNGR copolymers. The other peak such as 1745.40 cm−1 is assigned

The synthesis of PLGA-PEG-COOH was accomplished through the NHS/DCC covalent coupling method. The chemical composition of the synthesized copolymer was confirmed by 1H NMR (Figure 1s). The PLGA block represents the characteristic peaks at 1.55, 4.8, and 5.2 ppm which belong to the methyl (d, −CH3), methylene (m, −CH2), and methine (m, −CH) protons, respectively (Figure 1s,a), while the peak at 3.6 ppm is mainly due to the methylene (s, −CH2) proton of the PEG chain (Figure 1s,b). In 1H NMR of PLGA-PEG-COOH (Figure 1s,c) the large peak at 3.6 ppm (s, −CH2 of PEG) was mainly due to the methylene protons of PEG, while the integrated signals at or around 1.55 ppm could be assigned to the methyl protons (d, −CH3) of the D- and Llactic acid repeat units. The characteristic peaks at 5.2 ppm (−CH) are mainly due to lactic acid and the glycolic acid associated CH2 protons at 4.8 ppm. The anticipated peaks confirmed thus the successful synthesis of PLGA-PEG-COOH copolymer. The FT-IR spectra further affirmed the synthesis of PLGAPEG-COOH as shown in Figure 1s,d. The principal chemical reactions involved in synthesis of PLGA-PEG-COOH are the formation of amide bonds. To the synthesized polymer PLGA702



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Table 1. Physicochemical Characteristics of Various DTX Loaded NPs

a

formulation codea

size (nm)

PI

zeta potential (mV)

% EE

CE %

surface density (P)

D (nm)

DN DNB cNGR-DNB

167.4 ± 5.12 192.3 ± 7.81 217.5 ± 9.4

0.123 ± 0.011 0.112 ± 0.013 0.114 ± 0.012

−23.9 ± 1.6 −30.2 ± 1.9 −32.8 ± 2.1

74.93 ± 3.93 76.78 ± 2.62 80.56 ± 3.25

35.2

262 ± 7.6

23 ± 1.2

DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

to the −CO stretch, while the peak at 1536.28 cm−1 is due to the C−N stretch of imines. 3.3. Characterization of the NPs. The morphology of the optimized system was examined by using SEM as shown in Figure 2a. It appeared that the NPs were regular and spherical in shape, with a smooth surface morphology and nanometeric in size. The functionalized NPs prepared by blending PLGAPEG-COOH and cNGR-PEG-PLGA had an average diameter of about 217.5 ± 9.4 nm for cNGR-DNB-NPs, with a PI of below 0.1 suggesting a uniform particle size distribution. The physicochemical characteristics of the various NPs are summarized in Table 1. The ligand conjugated system revealed larger in size owing to the aqueous bulk facing orientation of the peptidic ligand moiety. The NPs with carboxyl-modified PEG showed negative surface charge (−30.2 ± 1.9 mV), and this may be due to that hydrophilic PEG group facilitated the appearance of carboxylic acid on the NPs surface.30 The zeta potential of ligand conjugated NPs was slightly more negative than PEGylated ones that can be attributed to peptidic moiety. The percent entrapment efficiency (% EE) of the DN (PLGANPs), DNB-NPs (PLGA-PEG-NPs), and cNGR-DNB-NPs (PLGA-PEG-cNGR-NPs) formulation was 74.93 ± 3.93%, 76.78 ± 2.62%, and 80.56 ± 3.25%, respectively. The cNGR peptide conjugation efficiency was measured to be 35.2%, while the surface density on the NPs surface was 262 respectively. The distance between two neighboring PEG chains linked to the cNGR peptide moiety based NPs was 23 nm (shown in Table 1). This suggests that the multivalent array of cNGR peptidic on NPs surface would tender higher a binding efficacy to the CD13 receptors. 3.4. In Vitro Release Study. The in vitro drug release profile from DN, DNB, and cNGR-DNB-NPs formulation was the same, that is, an initial fast release followed by slower and sustained release recorded in all of the cases. A fast release of DTX from NPs was recorded during initial 24 h. After that 74.4%, 81.5%, and 87.9% was released within 10 days in PBS pH 7.4 and 73.5%, 79.3%, and 89.5% in the next 4 days in phthalate buffer at pH 5.0 from cNGR-DNB-NPs, DNB-NPs, and DN-NPs respectively (Figure 2b and c). The initial faster release might be due to release of surface associated drug.31 On the other hand, the slow and sustained release was mainly depends on the diffusion of the drug from within the matrix of the NPs or by means of the polymeric matrix erosion.32 Further, the slower release in targeted NPs is due to the interactions between peptidic-NH2 groups and carboxylate groups of the DTX that slow down the release rate; also, it may be due to the structural integrity of peptide coupling and produce a double barrier effect for drug diffusion.15 However, the release behavior obviously presented the pH-dependent release, in which the faster release in pH 5.0 than in pH 7.4. The mechanism, however, remains unknown. In the medium containing 50% mice plasma, DTX was released from all tested NPs at a constant rate after first 24 h burst release phase. All tested formulations exhibited similar release patterns as recorded in PBS buffer media (Figure 2d).

The release rate recorded was faster in dissolution media that additionally contained plasma. This characteristic could be ascribed to the hydrolysis of polymer by plasma enzymes.4 3.5. Plasma Protein Adsorption. The plasma protein adsorption to the particle surface facilitated the phagocytosis.33 Therefore, PCS was used to assess the possibility of plasma protein adsorption onto the NP surface. The fate of various NPs was evaluated in the presence of 5% FBS first, subsequently in the presence of 2% BSA. At incubation with 5% FBS the average size of DNB and cNGR-DNB-NPs was remained nearly unchanged (Figure 2s, i and ii). In contrast, the plain PLGA based NPs (DN) aggregated resulting in larger size and the broader PI obtained after 24 h incubation with 5% FBS. Similar results were recorded in case of coincubated NPs (Figure 2s, iii and iv). The study supported the fact that PEGylated and targeted NPs can remain stable in serum environment since due to steric hindrance of crown polymer (PEG); the adsorption of plasma protein is not allowed, and hence aggregation could be prevented.29 Earlier study supported that serum protein adsorption on particles critically influenced their phagocytosis uptake34 and also confirmed by other parameters as phagocytosis study that significantly justified their role in receptor-mediated recognition. 3.6. Hemolytic Toxicity. The hemolytic behavior is considered to be most critical for an intravenously administered formulation because hemolytic effect can restrict their applicability. Free DTX solution caused higher hemolysis approximately more than 30% with greater 0.4% (w/v) concentration. On the other hand, in PEGylated-NPs (DNBNPs), the hemolytic toxicity was low as compared to plain NPs. However, cNGR anchored NPs resulted in considerably decreased hemolysis at all concentrations. The hemolytic toxicity was almost 7-fold and 4.6-fold low in case of targeted NPs as compared to DN and DNB-NPs, respectively, at higher concentrations (Figure 3s). Interestingly, the cNGR-peptide conjugation on the surface of NPs drastically reduced the RBC hemolysis due to reduced interaction between RBC. The lower hemolytic effect might be attributed in case of PEGylated NPs and target oriented NPs, due to stealth PEG layer and presence of ligand moiety on the nanoparticulate surface. 3.7. Phagocytosis Uptake. The plasma protein adsorption studies were confirmed using the macrophage cell line at ex vivo level. In the presence of RAW 264.7 cells, various NPs were internalized at different extent for various time periods at 37 °C was investigated. The results suggested that plain NPs showed higher macrophage uptake due to the higher hydrophobicity of DN-NPs. It is interesting to note that PEGylated and ligand conjugated formulations resulted in relating lower extent of internalization than plain NPs in the macrophage cell line. The uptake percentage of DN, DNB, and cNGR-DNB-NPs was 65.63%, 10.67%, and 11.72%, respectively, at 4 h (Figure 4s). The data showed that the PEGylation of the surface of the NPs offers significant steric repulsion at the in vitro level. The surface property of NPs is changed after PEG conjugation; hence macrophage uptake is reduced due to inhibition of interaction 703



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Figure 3. In vitro cytotoxicity of various formulations of DTX against HUVEC cells after (a) 24 h, (b) 48 h, (c) 72 h, and in HT-1080 cells after (d) 24 h, (e) 48 h, and (f) 72 h. (n = 6). DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGRPLGA-PEG copolymer based NPs.

drug solution (DS) did not significantly affect the normalized membrane integrity even on 48 h of exposure. The normalized membrane integrity was affected prominently only in the case of cNGR-DNB-NPs on prolonged incubation for both the cell lines. Among the DTX formulations, cNGR-DNB-NPs are comparatively most toxic and showed higher diminishment of the membrane integrity. This study concluded that drug

with serum proteins. The results are in accordance with other authors.35 3.8. Influence of Drug-Loaded NP Formulations on Cell Membrane Integrity. The normalized membrane integrity of HT-1080 and HUVEC cells after treatment with DTX based various formulations at different time points is recorded and shown in Figure 5s. The results showed that free 704



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Figure 4. Influence of DTX and DTX loaded NPs on nuclear morphology and apoptotic bodies formation in (A) HT-1080 cells and (B) HUVEC cells with (a) untreated control cells, (b) cells treated with free DTX solution (DS), (c) DN, (d) DNB, and (e) cNGR-DNB. Bar 50 μM. (C) Percent of apoptotic cells in HT-1080 and HUVEC cell line after 36 h treated with various formulations in the section. Data represent the mean ± SD (n = 3). DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

assay on both cell lines. In HT-1080 cells, the cNGR-DNB-NPs based treatments exhibited significantly higher toxicity, and cell killing results when used in the concentration range (2.5 μg/ mL). On the other hand, in HUVEC cells the cNGR-DNB-NPs based treatment presented 1.86-fold greater cells killed as compared to DN based NPs, while in DNB based treatments 1.27-fold cells were killed. All of the facts supported that targeted NPs were efficiently more toxic toward both types of cells. In agreement with the trypan blue assay results, the

solution has transient cell inhibition effect as compared to nanoparticulate systems. This can be easily explained on the cellular uptake mechanism. The more toxic effects of nanoparticulate system are likely to be due to NGR (ligand)CD13 (receptor) mediated cellular uptake with sustained and controlled drug release from these NPs.7,36 3.9. Anticancer Activities of Various Formulations Determined by Clonogenic Assay. Figure 6s displayed the longer-term cancer growth suppressive effect of various formulations treated group as measured by the clonogenic 705



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Figure 5. (A) Cell cycle analysis of HT-1080 cells after being treated with various DTX loaded formulations at equivalent drug concentration of 0.5 μg/mL for 36 h and analyzed by flow cytometry. Data are representative of three individual experiments (n = 3). Areas P2, P3, P4, and P5 represent sub-G0/G1, G0/G1, S, and G2/M phases, respectively, of the cell cycle. (a) Control, (b) DS, (c) DN, (d) DNB, and (e) cNGR-DNB. (B) Kinetics of distribution of the G2/M and sub-G0/G1 population induced by DTX based formulations. Points, mean of three individual experiments (n = 3). DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

cNGR-DNB-NPs treated cell group were more therapeutically effective and exhibited a higher anticancer effect. 3.10. Cytotoxicity Assay. In vitro cytotoxicity of various DTX formulations, including DS, DNB, and cNGR-DNB-NPs to HUVEC (Figure 3a−c) and HT-1080 cells (Figure 3d−f) were examined after 24, 48, and 72 h incubation. The

angiogenic tumor vessels play an important role in tumor growth; hence the cytotoxicity of DTX based formulations investigated by using HUVEC cell line. As showed by IC50 value after 48 and 72 h, the antiproliferation ability of the various formulations followed the order: cNGR-DNB-NPs > DNB > DN > DS. Significantly enhanced cytotoxicity of various 706



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Figure 6. Cellular association of various formulations in HUVEC cells (a, b, and e) and HT-1080 cells (c, d, and f) as showed CLSM using FITC as the fluorescence probe. (a) PEGylated (DNB) NPs in HUVEC cells, (b) targeted cNGR-DNB-NPs in HUVEC cells, (c) PEGylated (DNB) NPs in HT-1080 cells, (d) targeted cNGR-DNB-NPs in HT-1080 cells, (e) cNGR-DNB-NPs in the presence of excess cNGR in HUVEC cells, (f) cNGRDNB-NPs in the presence of excess cNGR in HT-1080 cells. DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs. 707



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dense nuclear parts and further distributed into apoptotic bodies. All of these results exhibited that, after 36 h, drug treated formulations decreased the number of cells, and chromatin condensation attributed to apoptosis. The percentage of apoptotic cells induced by cNGR conjugated polymeric DTX-loaded NPs were significantly higher as compared to other formulations (Figure 4C) for both types of cells. These results indicated that ligand functionalized NPs induced more apoptosis in tumor cell in case of HT-1080, but somehow lesser apoptotic activity on the tumor endothelial cells HUVEC. The MTT and cell apoptosis assay further confirmed that targeted NPs presented the more inhibitory effects to HT-1080 cells. 3.12. Cell Cycle Analysis. Cell cycle analysis was performed to determine the growth phase due to inhibition of cell cycle progression. DTX induced microtubule damage resulted in a marked and prolonged G2/M phase arrest and causing apoptosis. The control group (without treatment) presented no evidence on the cell cycle and did not induce significant apoptosis as shown in Figure 5. Treatment with free DTX solution (DS) and different DTX-NPs caused the G2/M phase arrest, which was accompanied by increase in the subG0/G1 phase, showing increasing extent of apoptotic cells. In sub-G0/G1 phase, the DNA content in DN, DNB, and cNGRDNB-NPs is observed to be 31.71%, 37.49%, and 47.58%, respectively, at 36 h after treatment with various DTX based NPs, respectively. The targeted NPs exhibited higher arrest in the G2/M phase in regard with plain PLGA or PEGylated NPs or free drug solution, which proved the superior antitumor activity of conjugated NPs than other one. The observed results inferred that cell cycle arrest was formulation-dependent, and DTX was released into the cytosol and caused cell cycle arrest in the G2/M phase. The effectiveness of NPs as a potential carrier for DTX among free drug solution can be illustrated that in case of NP mediated therapy more drug is present at the site of action for a significantly higher time period than drug in solution, ultimately greater therapeutic efficacy of the NPs in arresting cell growth. However, the ligand conjugated targeted NPs have higher cell cycle arrest in the G2/M phase may be due to receptor mediated endocytosis, thus allowing more apoptosis and higher cells arrest in the G2/M phase which is consistent with the data obtained by other studies. The NPs system exerted higher apoptosis that was more related with effective counter effects against cellular antiapoptotic factors that prevent the translation of cell damage to apoptosis. However, the apoptosis caused by DTX-NPs could be proceeding with the different molecular mechanisms from DTX.40 3.13. Cellular Uptake of NPs and Competition Assay. FITC-labeled NPs were used to investigate cellular uptake, the results of which are shown using CLSM fluorescent images (Figure 6). HUVEC and HT-1080 cells treated with various NPs exhibited fluorescent intensity corresponding to type of formulation. Here, the blue fluorescence of nuclei was employed to localize the position of NPs (green fluorescence) and representing that nanoparticulate systems accumulated in membrane or cytoplasm or nuclei. The results showed that, in both cells, cNGR-DNB-NPs exhibited higher fluorescence intensity (Figure 6a−d), suggesting that cNGR functionalized particle could considerably facilitate the NPs uptake by HUVEC and HT-1080 cells. However, the fluorescent intensity in HT-1080 cells (Figure 6c and d) was more intense than HUVEC cells (Figure 6a and b) in targeted-NPs at the same condition. In the competition binding assay, after adding free

nanoparticulate systems of DN, DNB, and cNGR-DNB-NPs (IC50 values 1.8, 3.2, and 4.8 times lesser than that of free drug) after 48 h which were recorded to be (1.58, 4.27, and 9.3 times lower as compared to free drug solution after 72 h treatment, respectively) was achieved for the HUVEC cell. The IC50 value for HUVEC cells treated with cNGR-DNB-NPs was found to be 3.4 μg/mL after 24 h, that is, 2.1 times lower than DN and 1.4 times lower than DNB based NPs, respectively. The IC50 value of DN, DNB, and cNGR-DNB-NPs for the HT-1080 cell at designated time periods are presented in Figure 4d, e, and f, respectively. All formulations showed a clear dose-dependent, time-dependent, and formulation-based cytotoxicity against respective cells with equivalent dose from 0.25 to 10 μg/mL of DTX. But, a significant difference was recorded for IC50 values in the case of targeted NPs as compared to other formulations, implying that ligand anchored NPs represented higher cytotoxicity against HT-1080 cells at all concentrations for 24, 48, and 72 h. The results suggested that DTX loaded NPs expressed higher cytotoxicity compared to free drug solution especially after a long incubation period (48 and 72 h) in both cell lines. On the other hand, the cell viability was low in case of free drug solution (DS) than DN-NPs at 24 h. It may be due to that drug enters inside the cells through passive diffusion across cell membrane and instantly becomes available to the cells. After a time point, drug attained a steady phase after which further drug internalization was limited. It was investigated that the antiproliferative effect of drug is linked with its intracellular residence time, and a small extent of free drug is accessible in the cytoplasmic compartment that is responsible for exhibiting the antiproliferative effect of native DTX.29 However, the antiproliferative effect of unconjugated or conjugated PLGA based NPs can be justified on the basis of their internalization mode. It may be noted here that targeted nanoparticulate system presented a higher cytotoxicity in HT1080 cells owing to the presence of higher density of CD13 receptor, hence greater interaction between cells and nanosystem, while HUVEC cells represent only an intermediate level of CD13 receptor expression.37,38 It was also observed that drug encapsulated peptide conjugated NPs were more toxic for HT-1080 cells compared with HUVEC. One of the differences between both the cell lines is their population rate. The HUVEC cells have 30 h of population doubling time, whereas HT-1080 doubles in 18 h of population doubling time. On the basis of cell repair mechanism, it was believed that more active cells having a longer population doubling time due to every cell cycle phase is longer than the usual39 and may be responsible for drug based NPs that is more sensitive with HT-1080 as compared with HUVECs cells. The study concluded that peptide conjugated targeted NPs enhanced the intracellular delivery of drug, thus allowing more cell apoptosis and higher cytotoxicity which is consistent with the data obtained by other studies such as normalized membrane integrity and anticancer activity by clonogenic assay. 3.11. Evaluation of Cell Apoptosis Activity. The cell apoptosis activity was examined to evaluate that drug encapsulation in NPs modifies their DTX induced apoptotic cell death or not. The results showed that nuclei of untreated HT-1080 cells (Figure 4Aa) and HUVEC cells (Figure 4Ba) illustrated homogeneous fluorescence with not possessed any type of segmentation and fragmentation after DAPI staining. Meanwhile, different DTX formulations treated groups evidenced the severely fragmented cell nuclei in both cells at 36 h. These results suggested that nuclei were segmentated into 708



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uptake of the ligand anchored NPs was effectively inhibited by CPZ and genistein, which specified that these NPs were internalized by HUVEC cells, primarily through caveolae and the clathrin mediated pathway. Alternatively, when cultured HUVEC cells were preincubated with cytochalasin D (30 μM), a specific inhibitor of macropinocytosis inhibits the formation of actin filaments and membrane ruffling.43 It was observed that cells treated with cytochalasin D caused a significant decrease in cellular uptake of DN and DNB-NPs by about 36−42%, suggesting that macropinocytosis might be used in the uptake of unconjugated NPs. However, there was no obvious effect of cytochalasin D on cellular uptake of targeted NPs. Pretreatment of sodium azide (0.1% w/v) depleted cellular ATP and less so the uptake of various NPs as compared to the control, indicating the internalization of the NPs by HUVEC cells occurred through energy-dependent endocytosis. Energy depletion by sodium azide significantly decreased the cellular uptake process for various NPs (Figure 8s,a). To elucidate the internalization mechanism of various FITC labeled NPs, the effects of ATP depletion and endocytosis inhibitors on cellular uptake were also evaluated quantitatively in HT-1080 cells. Incubation with CPZ reduced the cellular uptake of cNGR-DNB-NPs to 35.6%. It exhibited the involvement of clathrin-mediated endocytosis in cellular uptake of cNGR conjugated NPs (Figure 8s,b). When cells were preincubated with genistein, the cell uptake of cNGR-DNBNPs was found to increase significantly by 48.5% for HT-1080 cells, indicating that caveolae-mediated endocytosis was involved in targeted-NPs for internalization (Figure 8s,b). Moreover, when both CPZ and genistein were added to the cultured cells for preincubation, a synergistic inhibitory effect was observed for the cNGR-DNB-NPs uptake up to 16.4% (Figure 8s,b). These results suggested that the maximal contribution of clathrin and caveolae-mediated endocytosis on the internalization of targeted NPs by HT-1080 cells. The cell uptake of unconjugated NPs into HT-1080 cells was significantly reduced by 42.2% and 51.4%, for DN and DNBNPs, respectively, after treating with cytochalasin D. However, there was no obvious effect of cytochalasin D on cellular uptake of targeted NPs. Preincubation with sodium-azide depleted cellular ATP and decreased the cellular uptake of different polymeric NPs up to 40−60% of the control. It indicated that internalization of the NPs by HT-1080 cells occurred through energy-dependent endocytosis. These inhibition studies presented herein suggest for both cells that the internalization of polymeric NPs is mediated by more than one cellular uptake mechanisms. It was noted that the cellular internalization of targeted NPs into HUVEC cells mainly observed through the caveolae pathway, yet clathrin-coated vesicles were also involved. For HT-1080 cells, the internalization of ligand anchored-NPs was mainly depend on the both clathrin and caveolae-mediated endocytosis, but clathrin mediated uptake is slightly more than caveolae. The differences between targeted NPs and nonmodified NPs illustrated the presence of peptide ligand on NPs had direct influence on the cellular internalization mechanism. 3.15. In Vivo Antitumor Activity. The antitumor efficacies of different types of formulations were evaluated in Balb/c mice bearing HT-1080 tumor cells. The tumor inhibition effect was evaluated by accessing tumor volume changes. As compared to a control group, all treated groups suppressed the tumor growth after i.v. injection of various formulations; however, tumor volumes of each group changed differently (Figure 7a).

cNGR ligand (Figure 6e and f), the uptake of FITC-labeled cNGR-DNB-NPs was evidently reduced. The results suggested that decreased intensity of green fluorescence may be due to that free cNGR peptide and the cNGR motif on the surface of conjugated NPs competed with the same CD13 receptor. Therefore, the cellular uptake of targeted NPs could be competitively inhibited by cNGR ligand. For quantitative analysis of the cellular uptake of the NPs by HT-1080 or HUVEC at 37 and 4 °C were shown in Figure 7s. It was reported that cellular uptake of NPs, compared with cells treated with nonmodified NPs and the plain PLGA-NPs, the percent of FITC-positive cells increased from 38.4% to 73.2% (Figure 7s,a) at 8 h with 37 °C in HT-1080 cells. However, with free cNGR peptide preincubation, cellular uptake of cNGR-DNB-NPs was significantly reduced to 54.2% at 37 °C. The results showed that the cellular uptake of cNGR-DNB-NPs was significantly inhibited by pretreatment with free cNGR peptide compared to other tested formulations. On the contrary, at 4 °C for HT-1080 cells, the cell uptake of cNGR-DNB-NPs reduced to 46.9%. Meanwhile, in the presence of free cNGR, the cellular uptake of targeted-NPs decreased to 30.7% at 4 °C (Figure 7s,b). The more reduced uptake of FITC loaded NPs was observed at 4 °C compared to that at 37 °C, suggesting an energy-dependent uptake.41 In case of HUVEC cells, the uptake of cNGR-DNB-NPs showed the 57.8% at 37 °C in the absence of free cNGR at 8 h. While cells were preincubated with free cNGR, cellular uptake was reduced to 41.9% at 8 h (Figure 7s,c). Similar results were also found via incubation of various FITC labeled formulations with HUVEC cells at 4 °C (Figure 7s,d). However, addition of free cNGR-peptide in the media, the % uptake was inhibited more intensely of cNGR-DNB-NPs as compared to other tested formulations. The results demonstrated that free cNGR peptide and cNGR-DNB-NPs competed with the CD13 receptors, which were present on the surface of both type of cells, and free cNGR peptide could hinder the uptake of targeted NPs. Additionally, remarkable decreases in the association of FITC-loaded NPs with both type cells were observed at 4 °C compared to that at 37 °C, suggesting an energy-dependent uptake. These phenomena confirmed that cellular uptake and internalization depended on the certain degree of temperature, since endocytosis is generally inhibited at 4 °C.42 3.14. Tracking of Uptake Pathways Using Various Endocytic Inhibitors. To recognize, which uptake mechanisms are involved in the cellular entry of NPs, various endocytic inhibitors are employed, and each is specific for a particular endocytic pathway. When HUVEC cell cultures were preincubated with CPZ (clathrin mediated endocytosis) or genistein (caveolae-mediated endocytosis) and then treated with cNGR-DNB-NPs, the uptake of targeted NPs were 63.1% and 69.5%, respectively, compared to that of the cells not treated with CPZ and genistein (Figure 8s,a). On the other hand, the plain and PEGylated NPs have not inhibited cellular uptake more remarkably. Interestingly, when both CPZ (10 μg/ mL) and genistein (1 μg/mL) were coincubated to the cultured HUVEC cells, an additive inhibitory effect was observed for the NPs uptake, and 83.6% reduction in targeted NPs was observed. The targeted NPs uptake was blocked by using both inhibitors simultaneously but not individually; this confers that targeted NPs have uptake by both caveolae and the clathrin mediated route where one pathway compensates NPs uptake, while the second pathway gets arrested. Hence, the cellular 709



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Figure 7. (a) In vivo antitumor efficacy of the different DTX formulations in tumor induced Balb/c mice by changes of tumor volumes. (b) % Body weight changes of Balb/c mice by bearing HT-1080 tumor cells. (c) The effect of free DTX and DTX loaded formulations on volume growth percentage inhibition (% VI) of different treatments. (d) The effect of free DTX and DTX loaded formulations on tumor weight growth percentage inhibition (%WI) of different treatments on growth of established HT-1080 tumor induced Balb c/mice at the end of therapy DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

curve of Figure 7b). But in the case of the DN, DNB, and cNGR-DNB-NPs groups, the body weight was increased by 8.2%, 11.3%, and 17.6%, respectively (Figure 7b). These results lead to a conclusion that targeted NPs presented less toxicity to mice when administered i.v. under the present experimental conditions. After the experimental therapy after 28 days, the tumor volumes of the DTX-treated groups were all notably smaller than that of the saline group (Figure 7c) and followed the same order: cNGR-DNB-NPs < DNB < DN < DS < saline. Based on tumor volume and weight, the %VI (tumor volume growth percentage inhibition) of cNGR-DNB-NPs, DNB, DN, and DS were calculated to be 71.1%, 51%, 43.5%, and 31.1% (Figure 7c), while the %WI (tumor weight growth percentage inhibition) was 62.3%, 39.2%, 27.4%, and 18.5%, respectively (Figure 7d). Among all of the groups of mice, DTX-loaded targeted polymeric NPs exhibited delaying tumor growth with the least tumor volume and weight among all of the treatment groups. The antitumor activity of the formulations was also evaluated by the survival experiment assay performed on tumor induced

Owing to sustained release behavior of DN and DNB-NPs, the tumor volume in the case of DN and DNB-NPs treatment was measured to be smaller than free DTX solution (DS) treatment. For the newer designed system, a significant antitumor efficacy was recorded with a higher magnitude of regression of the established tumor volume. The final mean tumor load was 226.5 ± 18.4 mm3 for cNGR-DNB-NPs remarkably smaller than other treated groups. It was hypothesized that delivery of DTX to the tumor cells had been enhanced due to the binding of NPs to target cells through NGR peptidic. To evaluate the safety of these DTX formulations, body weight was monitored as a marker of overall toxicity. The superior antitumor effect can be easily observed, while the toxicity remained minimal by the increasing body weight of the animals during the experimental period. It was observed that the average body weight of the saline group and free DTX treated group decreased progressively and reduced 14.6% and 8.8% of their initial weight after the experimental terminal. The free DTX treated group presented a serious body weight decrease due to its toxic side effects44 (as shown in the bottom 710



711

*** (P < 0.001) *** (P < 0.001)

DNB DN

10

10

DNB

cNGR-DNB

a

117.9 59.606−62.060 0.477 61 62.3 ± 2.2 71.1 ± 2.1

60.8

53.6 40.652−44.348 0.719 42.5 43 39.2 ± 1.4 51 ± 2.0

10 DN

DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

< 0.001)

< 0.001)

< 0.001)

< 0.001)

27.4 ± 1.1 43.5 ± 1.3

10

37

18.5 ± 0.8 31.1 ± 1.8

36.5

0.5

35.215−37.786

32.1

*** (P *** (P *** (P *** (P 17.9 27.791−28.875 31.606−34.060 0.516 0.477 28.3 32.8 28 33 saline DS

formulation codea

dose (mg/kg)

%VI

%WI

median survival time (days)

mean survival time (days)

standard error

95% confidence in interval

increase in survival time (% IST)

saline

DS

*** (P < 0.001) *** (P < 0.001) *** (P < 0.001)

log rank test

Table 2. Effect of Free DTX and DTX Loaded Formulations on Tumor Volume Growth Inhibition (%VI), Tumor Weight Growth Percentage Inhibition (%WI), and Survival Time of Different Treatments on Growth against the HT-1080 Tumor in Balb/c Mice at the End of Therapy

Balb/c mice. The median survival time of the animals treated with saline, DS, DN, DNB, and cNGR-DNB-NPs were 28, 33, 37, 43, and 61 days, respectively (Table 2). The cNGR-DNBNPs treated group presented significantly prolonged animal survival time as compared with DNB-NP (41.9%), DN (64.9%), DS (84.8%), and saline (117.9%). In conclusion, targeted NGR ligand anchored formulations enhanced antitumor effects and prolonged survival time as compared with the DS and DN and DNB-NPs. 3.16. Biodistribution Studies. The time course of DTX levels in mice plasma after i.v. administration of 10 mg/kg dose of free DTX solution and equivalent DTX doses of nanoparticulate systems such as DN, DNB, and cNGR-DNB-NPs are shown in Figure 8a. In plasma, the percentage of recovered dose after 48 h for cNGR-DNB-NPs was approximately 1.75 times higher than DNB-NPs. However, no drug was detected after 24 h in the case of DN-NPs. The data presented that, in case of PEGylated PLGA-NPs and targeted NPs blood clearance delayed, the blood concentration remained much higher after 24 h. Furthermore, PEGylated NPs and ligand anchored formulations had impeded the drug release and almost sustained manner as compared to DS and DN. The obtained results can be easily interpreted with results obtained for previous studies such as plasma protein adsorption and phagocytosis uptake. The delayed blood clearance effect could be illustrated by the stealth nature of polymer NPs induced by hydrophilic shell of PEG, which lowered the plasma protein absorption and reduce their phagocytosis uptake. Therefore, these results pointed out the potential utility of PLGA-PEG and cNGR-PLGA-PEG polymer based NPs as a long circulating reservoir and targeting property for hydrophobic anticancer agents. Organ biodistribution of DTX following i.v. administration of DS and various nanoparticulate systems was assessed in HT1080 cells bearing tumor induced Balb/c mice. The distribution profiles of the various formulations in different organs up to 48 h post administration period is illustrated in Figure 8. Drug was distributed into various organs (heart, liver, spleen, kidney, lung and tumor) after intravenous administration of the various DTX based formulations. In tumor tissue, the higher drug concentration was found and followed the order: cNGR-DNBNPs > DNB > DN > DS. The higher drug retention was estimated in the case of DN, DNB, and cNGR-DNB-NPs within the tumor to be 4.5, 15.2, and 24.7 times greater than that of the DS treated group at 24 h postinjection, respectively (Figure 8b). It was recorded that, in tumor tissue, the drug concentration retained a higher extent when treated with the cNGR-DNB-NPs group that led to accumulation specifically in the tumor via ligand−receptor interaction.7,36 The results supported that, after 48 h, the DTX content with cNGR-DNBNPs tumor tissue was enhanced by 2.5- and 16.2-fold when compared to DNB and DN-NPs, respectively. All of the results suggest that functionalization of NPs with cNGR peptide might be facilitated the greater accumulation of NPs in tumor tissue and proved that it can be used as an active targeting module in a delivery system. The liver was the major cumulative organ for all types of nanoparticulate systems. In detail, DS (from 34.5% of the injected dose at the first 30 min to 23.8% at 48 h) and DN-NPs (from 31.2% of the injected dose at the first 30 min to 20.2% at 48 h) were accumulated in the liver. On the contrary, DNBNPs presented 21.1% and 6.3% of the initial dose were recovered from the liver at the first 30 min and 48 h later,

*** (P < 0.001)

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Figure 8. Biodistribution of DTX after intravenous administration of various formulations in tumor induced Balb/c mice at the dose of 10 mg/kg. (a) Plasma, (b) tumor, (c) liver, (d) spleen, (e) lung, (f) heart, and (g) kidney. DS = free drug solution, DN = PLGA based NPs, DNB = PLGA-PEG based NPs, cNGR-DNB-NPs = cNGR-PLGA-PEG copolymer based NPs.

respectively. The targeted formulation cNGR-DNB-NPs showed the 18.6% at the first 30 min and 6.9%, respectively, at 48 h after being intravenously injected (Figure.8 c). The

observed data exhibited that PEGylated and targeted NPs were accumulated in liver to a limited extent, which was significantly lower compared to other formulations. The plain NPs have a 712



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sustained release pattern and shielding effect that could significantly reduce hepato and nephrotoxicity.

hydrophobic surface and are taken up by the liver after few minutes due to the opsonization process.45 The drug concentrations in the spleen and lung, for mice in the DS and other nanoparticulate systems, gradually decreased after 4 h (Figure 8d and e). However, cNGR-decorated PEGylated NPs tended to accumulate in the spleen at a higher extent than PEGylated-NPs. Previous study showed that liposomes modified with cNGR accumulated in the spleen after intravenous injection.46,47 As shown in Figure 8f, at 4 h post injection, the DTX concentration within the heart of mice in the free drug solution was mainly distributed to the heart, which might cause the acute toxicity. In contrast, the drug concentration present in the heart was a much smaller amount for all of the other nanoparticulate system. 3.17. Hematological Study. Hematological parameters were determined to analyze the toxic manifestations of the prepared formulations on the blood components. The blood cell count such as WBC count, RBC count, Hb content, and platelet count decreased in a remarkable manner after administration of free DTX solution for a designated time period and might be possibly due to the direct exposure of free drug with blood cells (Table 2s). The same amount of drug entrapped in various nanoparticulate formulations (such as DN, DNB, and cNGR-DNB-NPs) decreased hematological toxicity. The data showed that the PEGylated-NPs caused a smaller decrease in the blood cell count compared with plain PLGANPs, possibly due to the long-circulation effect. The cNGRDNB-NPs group had a relatively minor effect on the hematological parameters, due to the fact that ligand conjugated NPs directly released their contents into the target sites; hence a negligible amount of drug was leached out in blood, and this was also significantly contributed through active targeting with a higher accumulation in the tumor site, correlating with the distribution results in in vivo. 3.18. Evaluation of Nephrotoxic and Hepatotoxic Effects. To evaluate the renal and liver function, various markers are used such as the serum urea/creatinine level and SGOT/SGPT/ALP level, respectively. The data indicated that the serum urea and creatinine level was significantly present to a higher extent upon administration of plain DTX solution (Table 3s). However, the urea and creatinine level was also elevated in the serum, but to the smallest extent in the case of targeted formulation, indicating that the formulation evades damage to renal functions. This may be attributed to the small quantity of ligand anchored formulations that gained access in the kidney. In vivo hepatotoxicity induced by the anticancer drug administered in free/entrapped form was calculated in terms of SGOT, SGPT, and ALP concentration. Free drug induced toxicity in animals that was reflected by the increased activity of SGOT (21.2 ± 3.5 IU/L), SGPT (25.3 ± 4.6 IU/L), and ALP (87.2 ± 7.4 IU/L) concentration. On the other hand, plain PLGA-NPs of DTX represented a lesser value in the activity of SGOT (18.5 ± 3.3 IU/L), SGPT (20.6 ± 4.2 IU/L), and ALP (81.4 ± 7.3 IU/L). PEGylated NPs (DNB-NPs) exhibited a smaller amount in the activity of all biochemical parameters as compared to plain PLGA-NPs. cNGR-DNB-NPs (SGOT 14.2 ± 2.7 IU/L; SGPT 15.3 ± 2.8 IU/L; ALP 72.4 ± 6.3 IU/L) revealed only a slight change in activity of these enzymes as compared with control animals. The results showed that the free drug solution showed liver and kidney cell cytotoxicity, while the PEGylated and targeted formulation exhibited a

4. CONCLUSION In summary, we prepared the dual targeted nanoparticulate drug delivery system functionalized with cNGR peptide as the ligand for CD13 receptor overexpressed in tumor cells and tumor endothelial cells. First, the amphiphilic diblock copolymer (HOOC-PEG-PLGA) was synthesized, and cNGR peptide was attached to the NHS-activated PEG block of the copolymer. The resulting cNGR-DNB-NPs appeared to be uniformly spherical in shape with a particle size of 217 nm. These cNGR functionalized NPs may be able to significantly enhance their specific uptake by tumor cells (HT-1080 cells) and tumor endothelial cells (HUVEC cells) via a CD13 receptor-mediated endocytosis. The enhanced intracellular uptake of targeted NPs directly results in a stronger induction of apoptosis and greater cell cycle arrest at the G2/M phase. The enhanced accumulation of cNGR-DNB-NPs in tumor tissue in vivo tumor-bearing Balb/c mice was also observed by a biodistribution study. These properties strongly suggest that cNGR peptide-conjugated PLGA-PEG-NPs can be successfully explored for the design of dual targeted delivery systems for targeting the tumor cells and vasculature and have a potential approach for CD13-overexpressed specific tumors.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra, plasma adsorption studies, hemolytic toxicity, phagocytic update, results of trypan blue exclusion assay, clonogenic assay, cellular uptake data, and effects of various endocytic inhibitors (Figures 1s−8s), as well as the IC50 value of DTX loaded various formulations, various hematological parameters, and effect on kidney and liver function parameters (Tables 1s−3s). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91-7582-265525. Fax:07582-265525. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are thankful to Sun Pharma Advanced Research Company Ltd. (SPARC), Vadodara, and Dabur (Franscius Kabi Oncology Lab) New Delhi, India for providing the gift sample of PLGA and DTX, respectively. The authors are also thankful to AIIMS (New Delhi, India) for carrying out the SEM of the formulations. Financial support provided by the AICTE in the form of a senior Research Fellowship (SRF) to Madhu Gupta is duly acknowledged.

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REFERENCES

(1) Gupta, M.; Agrawal, G. P.; Vyas, S. P. Polymeric Nanomedicines as Promising Vehicle for Solid Tumor Therapy and Targeting. Curr. Mol. Med. 2013, 113, 179−204. (2) Zhigaltsev, I. V.; Winters, G.; Srinivasulu, M.; Crawford, J.; Wong, M.; Amankwa, L.; Waterhouse, D.; Masin, D.; Webb, M.; Harasym, N.; Heller, L.; Bally, M. B.; Ciufolini, M. A.; Cullis, P. R.; Maurer, N. Development of a Weak-Base Docetaxel Derivative that can be Loaded into Lipid Nanoparticles. J. Controlled Release 2010, 144, 332−40.

713



Article

(3) Yang, X.; Zhang, Q.; Wang, Y.; Chen, H.; Zhang, H.; Gao, F.; Liu, L. Self-aggregated Nanoparticles from Methoxy Poly(ethylene glycol)modified Chitosan: Synthesis; Characterization; Aggregation and Methotrexate Release in Vitro. Colloids Surf., B: Biointerfaces 2008, 61, 125−31. (4) Guo, J.; Gao, X.; Su, L.; Xia, H.; Gu, G.; Pang, Z.; Jiang, X.; Yao, L.; Chen, J.; Chen, H. Aptamer-functionalized PEG-PLGA Nanoparticles for Enhanced Anti-glioma Drug Delivery. Biomaterials 2011, 32, 8010−20. (5) Jiang, X.; Sha, X.; Xin, H.; Chen, L.; Gao, X.; Wang, X.; Law, K.; Gu, J.; Chen, Y.; Jiang, Y.; Ren, X.; Ren, Q.; Fang, X. Self-aggregated PEGylated Poly (Trimethylene Carbonate) Nanoparticles Decorated with c(RGDyk) Peptide for Targeted Paclitaxel Delivery to IntegrinRich Tumors. Biomaterials 2011, 32, 9457−69. (6) Shim, J. S.; Kim, J. H.; Cho, H. Y.; Yum, Y. N.; Kim, S. H.; Park, H. J.; Shim, B. S.; Choi, S. H.; Kwon, H. J. Irreversible Inhibition of CD13/ Aminopeptidase N by the Antiangiogenic Agent Curcumin. Chem. Biol. 2003, 10, 695−704. (7) Wang, X.; Wang, Y.; Chen, X.; Wang, J.; Zhang, X.; Zhang, Q. NGR-modified Micelles Enhance their Interaction with CD13Overexpressing Tumor and Endothelial Cells. J. Controlled Release 2009, 139, 56−62. (8) Ellerby, H. M.; Arap, W.; Ellerby, L. M.; Kain, R.; Andrusiak, R.; Rio, G. D.; Krajewski, S.; Lombardo, C. R.; Rao, R.; Ruoslahti, E.; Bredesen, D. E.; Pasqualini, R. Anti-cancer Activity of Targeted Proapoptotic Peptides. Nat. Med. 1999, 5, 1032−8. (9) Garde, S. V.; Forte, A. J.; Ge, M.; Lepekhin, E. A.; Panchal, C. J.; Rabbani, S. A.; Wu, J. J. Binding and Internalization of NGR-PeptideTargeted Liposomal Doxorubicin (Tvt−Dox) In CD13-Expressing Cells and its Antitumor Effects. Anti-cancer Drugs 2007, 18, 1189−200. (10) Zhao, B. J.; Ke, X. Y.; Huang, Y.; Chen, X. M.; Zhao, X.; Zhao, B. X.; Lu, W.; Lou, J. N.; Zhang, X.; Zhang, Q. The Antiangiogenic Efficacy of NGR-Modified PEG-DSPE Micelles containing Paclitaxel (NGR-M-PTX) for the Treatment of Glioma in Rats. J. Drug Target. 2011, 19, 382−90. (11) Colombo, G.; Curnis, F.; De Mori, G. M.; Gasparri, A.; Longoni, C.; Sacchi, A.; Longhi, R.; Corti, A. Structure−activity Relationships of Linear and Cyclic Peptides containing the NGR Tumor-homing Motif. J. Biol. Chem. 2002, 277, 47891−7. (12) Cheng, J.; Teply, B. A.; Sherif, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C. Formulation of Functionalized PLGA−PEG Nanoparticles for In Vivo Targeted Drug Delivery. Biomaterials 2007, 28, 869−76. (13) Son, S.; Singh, K.; Kim, W. J. Bioreducible BPEI-SS-PEG-cNGR Polymer as a Tumor Targeted Nonviral Gene Carrier. Biomaterials 2010, 31, 6344−54. (14) Gao, X.; Tao, W.; Lu, W.; Zhang, Q.; Zhang, Y.; Jiang, X.; Fu, S. Lectin-conjugated PEG-PLA Nanoparticles: Preparation and Brain Delivery after Intranasal Administration. Biomaterials 2006, 27, 3482− 90. (15) Esmaeili, F.; Ghahremani, M. H.; Ostad, S. N.; Atyabi, F.; Seyedabadi, M.; Malekshahi, M. R.; Amini, M.; Dinarvand, R. Folatereceptor-Targeted Delivery of Docetaxel Nanoparticles Prepared by PLGA−PEG−Folate Conjugate. J. Drug Target. 2008, 16, 415−23. (16) Zhao, X.; Chen, L. J.; Wei, Y. Q.; Qian, Z. Y. Poly(εcaprolactone)-poly(ethylene glycol)−poly(ε-caprolactone) (PCL− PEG−PCL) Nanoparticles for Honokiol Delivery In Vitro. Int. J. Pharmaceutics 2009, 375, 170−6. (17) Olivier, J. C.; Huertas, R.; Lee, H. J.; Calon, F.; Pardridge, W. M. Synthesis of PEGylated Immunonanoparticles. Pharm. Res. 2002, 19, 1137−43. (18) Xin, H.; Sha, X.; Jiang, X.; Chen, L.; Law, K.; Gu, J.; Chen, Y.; Wang, X.; Fang, X. The Brain Targeting Mechanism of Angiopepconjugated Poly(ethylene glycol)-co-poly(ε-caprolactone) Nanoparticles. Biomaterials 2012, 33, 1673−81. (19) Yu, D. H.; Lu, Q.; Xie, J.; Fang, C.; Chen, H. Z. Peptideconjugated Biodegradable Nanoparticles as a Carrier to Target Paclitaxel to Tumor Neovasculature. Biomaterials 2010, 31, 2278−92.

(20) Wang, H.; Zhao, Y.; Wu, Y.; Hu, Y.; Nan, K.; Nie, G.; Chen, H. Enhanced Anti-tumor Efficacy by Co-delivery of Doxorubicin and Paclitaxel with Amphiphilic Methoxy PEG-PLGA Copolymer Nanoparticles. Biomaterials 2011, 32, 8281−90. (21) Essa, S.; Rabanel, J. M.; Hildgen, P. Characterization of Rhodamine Loaded PEG-g-PLA Nanoparticles (NPs): Effect of Poly(ethylene glycol) Grafting Density. Int. J. Pharmaceutics 2011, 411, 178−87. (22) Hou, L.; Fan, Y.; Yao, J.; Zhou, J.; Li, C.; Fang, Z.; Zhang, Q. Low Molecular Weight Heparin-All-Trans-Retinoid Acid Conjugate as a Drug Carrier for Combination Cancer Chemotherapy of Paclitaxel and All-Trans-Retinoid Acid. Carbohydr. Polym. 2011, 86, 1157−66. (23) Chaudhari, K. R.; Kumar, A.; Khandelwal, V. K. M.; Ukawala, M.; Manjappa, A. S.; Mishra, A. K.; Monkkonen, J.; Murthy, R. S. R. Bone Metastasis Targeting: a Novel Approach to Reach Bone using Zoledronate Anchored PLGA Nanoparticle as Carrier System Loaded with Docetaxel. J. Controlled Release 2012, 158, 470−8. (24) Liu, D.; Liu, Z.; Wang, L.; Zhang, C.; Zhang, N. Nanostructured Lipid Carriers as Novel Carrier for Parenteral Delivery of Docetaxel. Colloids Surf., B: Biointerfaces 2011, 85, 262−9. (25) Wong, H. L.; Rauth, A. M.; Bendayan, R.; Manias, J. L.; Ramaswamy, M.; Liu, Z.; Erhan, S. Z.; Wu, X. Y. A New Polymer-Lipid Hybrid Nanoparticle System Increases Cytotoxicity of Doxorubicin against Multidrug-Resistant Human Breast Cancer Cells. Pharm. Res. 2006, 23, 1574−85. (26) Danhier, F.; Lecouturier, N.; Vroman, B.; Jérôme, C.; Marchand-Brynaert, J.; Feron, O.; Préat, V. Paclitaxel-loaded PEGylated PLGA-based Nanoparticles: In vitro and In vivo evaluation. J. Controlled Release 2009, 133, 11−7. (27) Nam, H. Y.; Kwon, S. M.; Chung, H.; Lee, S. Y.; Kwon, S. H.; Jeon, H.; Kim, Y.; Park, J. H.; Kim, J.; Her, S.; Oh, Y. K.; Kwon, I. C.; Kim, K.; Jeong, S. Y. Cellular Uptake Mechanism and Intracellular Fate of Hydrophobically Modified Glycol Chitosan Nanoparticles. J. Controlled Release 2009, 135, 259−67. (28) Liu, B.; Yang, M.; Li, X.; Qian, X.; Shen, Z.; Ding, Y.; Yu, L. Enhanced Efficiency of Thermally Targeted Taxanes Delivery in a Human Xenograft Model of Gastric Cancer. J. Pharm. Sci. 2008, 97, 3170−81. (29) Parveen, S.; Sahoo, S. K. Long Circulating Chitosan/PEG blended PLGA Nanoparticle for Tumor Drug Delivery. Eur. J. Pharmacol. 2011, 670, 372−83. (30) Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J. P.; Langer, R. Targeted NanoparticleAptamer Bioconjugates for Cancer Chemotherapy In vivo. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 6315−20. (31) Magenheim, B.; Levy, M. Y.; Benita, S. A New In Vitro Technique for Evaluation of Drug Release Profile from Colloidal Carriers Ultrafiltration Technique at Low Pressure. Int. J. Pharmaceutics 1993, 94, 115−23. (32) Gu, F.; Zhang, L.; Teply, B. A.; Mann, N.; Wang, A.; RadovicMoreno, A. F.; Langer, R.; Farokhzad, O. C. Precise Engineering of Targeted Nanoparticles by using Self-assembled Biointegrated Block Copolymers. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586−91. (33) Moghimi, S. M.; Patel, H. M. Serum-mediated Recognition of Liposomes by Phagocytic Cells of the Reticuloendothelial System-the Concept of Tissue Specificity. Adv. Drug Delivery Rev. 1998, 32, 45− 60. (34) Thiele, L.; Diederichs, J. E.; Reszka, R.; Merkle, H. P.; Walter, E. Competitive Adsorption of Serum Proteins at Microparticles Affects Phagocytosis by Dendritic Cells. Biomaterials 2003, 24, 1409−18. (35) Prior, S.; Gander, B.; Blarer, N.; Merkle, H. P.; Subirá, M. L.; Irache, J. M.; Gamazo, C. In vitro Phagocytosis and MonocyteMacrophage Activation with Poly(Lactide) and Poly(Lactide-CoGlycolide) Microspheres. Eur. J. Pharm. Sci. 2002, 15, 197−207. (36) Dunne, M.; Zheng, J.; Rosenblat, J.; Jaffray, D. A.; Allen, C. APN/CD13-Targeting as a Strategy to Alter the Tumor Accumulation of Liposomes. J. Controlled Release 2011, 154, 298−305. (37) Hensbergen, Y.; Broxterman, H. J.; Elderkamp, Y. W.; Lankelma, J.; Beers, J. C.; Heijn, M.; Boven, E.; Hoekman, K.; 714



Article

Pinedo, H. M. A doxorubicin−CNGRC−Peptide Conjugate with Prodrug Properties. Biochem. Pharmacol. 2002, 63, 897−908. (38) Yokoyama, Y.; Ramakrishnan, S. Addition of an Aminopeptidase N-Binding Sequence to Human Endostatin Improves Inhibition of Ovarian Carcinoma Growth. Cancer 2005, 104, 321−31. (39) Naik, S.; Patel, D.; Chuttani, K.; Mishra, A. K.; Misra, A. In vitro Mechanistic Study of Cell Death and In Vivo Performance Evaluation of RGD Grafted PEGylated Docetaxel Liposomes in Breast Cancer. Nanomed. Nanotechnol. Biol. Med. 2012, 8, 951−62. (40) Luo, G.; Yu, X.; Jin, C.; Yang, F.; Fu, D.; Long, J.; Xu, J.; Zhan, C.; Lu, W. LyP-1-Conjugated Nanoparticles for Targeting Drug Delivery to Lymphatic Metastatic Tumors. Int. J. Pharmaceutics 2010, 385, 150−6. (41) Knudsen, N. Ø.; Schiffelers, R. M.; Jorgensen, L.; Hansen, J.; Frokjaer, S.; Foged, C. Design of Cyclic RKKH Peptide-Conjugated PEG Liposomes Targeting the Integrin α2β1 Receptor. Int. J. Pharmaceutics 2012, 428, 171−7. (42) McKeown, M.; Knowles, G.; McCulloch, C. A. Role of the Cellular Attachment Domain of Fibronectin in the Phagocytosis of Beads by Human Gingival Fibroblasts In Vitro. Cell Tissue Res. 1990, 262, 523−30. (43) Huth, U. S.; Schubert, R.; Peschka-Suss, R. Investigating the Uptake and Intracellular Fate of pH-sensitive Liposomes by Flow cytometry and Spectral Bio-imaging. J. Controlled Release 2006, 110, 490−504. (44) Chu, C. Y.; Yang, C. H.; Yang, C. Y.; Hsiao, G. H.; Chiu, H. C. Fixed Erythrodysaesthesia Plaque due to Intravenous Injection of Docetaxel. Br. J. Dermatol. 2010, 142, 808−11. (45) Grislain, L.; Couvreur, P.; Lenaerts, V.; Roland, M.; Deprez Decampeneere, D.; Speiser, P. Pharmacokinetics and Distribution of a Biodegradable Drug-Carrier. Int. J. Pharmaceutics 1993, 15, 335−45. (46) Murase, Y.; Asai, T.; Katanasaka, Y.; Sugiyama, T.; Shimizu, K.; Maeda, N.; Oku, N. A Novel DDS Strategy, ‘‘Dual-Targeting”, and its Application for Antineovascular Therapy. Cancer Lett. 2009, 28, 165− 71. (47) Pastorino, F.; Brignole, C.; Marimpietri, D.; Cilli, M.; Gambini, C.; Ribatti, D.; Longhi, R.; Allen, T. M.; Corti, A.; Ponzoni, M. Vascular Damage and Anti-Angiogenic Effects of Tumor VesselTargeted Liposomal Chemotherapy. Cancer Res. 2003, 63, 7400−9.

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