Enhanced Antitumor Efficacy by d -Glucosamine-Functionalized and Paclitaxel-Loaded Poly(Ethylene Glycol)-Co-Poly(Trimethylene Carbonate) Polymer Nanoparticles

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Enhanced Antitumor Efficacy by D-Glucosamine-Functionalized and Paclitaxel-Loaded Poly(Ethylene Glycol)-Co-Poly(Trimethylene Carbonate) Polymer Nanoparticles XINYI JIANG,1,2 HONGLIANG XIN,3 JIJIN GU,1 FENGYI DU,2 CHUNLAI FENG,2 YIKE XIE,1 XIAOLING FANG1 1

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai 201203, China Department of Pharmaceutics and Tissue Engineering, School of Pharmacy, Jiangsu University, Zhenjiang 212013, China 3 Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China 2

Received 20 November 2013; revised 23 January 2014; accepted 18 February 2014 Published online 11 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23928 ABSTRACT: The poor selectivity of chemotherapeutics for cancer treatment may lead to dose-limiting side effects that compromise clinical outcomes. To solve the problem, surface-functionalized polymer nanoparticles are regarded as promising tumor-targeting delivery system. On the basis of glucose transporter (GLUT) overexpression on cancer cells, D-glucosamine-conjugated and paclitaxel-loaded poly(ethylene glycol)-co-poly(trimethylene carbonate) copolymer nanoparticles (DGlu-NP/PTX) were developed as potential tumor-targeting drug delivery system in this study. Because of the high affinity between D-glucosamine and GLUT, DGlu-NP/PTX could target to tumor tissue through GLUT-mediated endocytosis to improve the selectivity of PTX. DGlu-NP/PTX was prepared by emulsion/solvent evaporation technique and characterized in terms of morphology, size, and zeta potential. In vitro evaluation of two-dimensional cells and three-dimensional tumor spheroids revealed that DGlu-NP/PTX was more potent than those of plain nanoparticles (NP/PTX) and Taxol. In vivo multispectral fluorescent imaging indicated that DGlu-NP had higher specificity and efficiency on subcutaneous xenografts tumor of mouse. Furthermore, DGlu-NP/PTX showed the greatest tumor growth inhibitory effect on in vivo subcutaneous xenografts model with no evident toxicity. C 2014 Wiley Periodicals, Therefore, these results demonstrated that DGlu-NP/PTX could be used as potential vehicle for cancer treatment.  Inc. and the American Pharmacists Association J Pharm Sci 103:1487–1496, 2014 Keywords: nanoparticles; paclitaxel; D-Glucosamine; GLUT; tumor-targeting delivery system; biomaterials; drug delivery system; biodegradable; polymers; cancer chemotherapy

INTRODUCTION Chemotherapy is the most common method for the treatment of cancer. However, the concentration of chemotherapeutics in tumor tissue is usually limited because of the poor selectivity of antitumor drugs. To overcome this shortcoming, copolymer nanoparticles with core–shell structure have been proposed as targeting drug delivery system with great attentions. The hydrophobic core serves as the reservoir for pharmaceutical compounds with poor solubility and/or low stability in physiological environments, whereas the hydrophilic shell provides the nanocarriers with desirable solubility in aqueous solutions.1,2 Although polymer nanoparticles offer many benefits in drug delivery, conventional nanoparticles suffer from limited in vivoactive targeting ability because of the nonspecific systemic distribution. Surface-functionalized copolymer nanoparticles by tumor-specific targeting moieties (e.g., receptor-binding ligands or antibodies) were considered as active targeting drug delivery system for tumor treatment.3,4 Unlike normal cells, cancerous cells have acquired metabolic ability to survive in harsh microenvironment conditions (e.g., hypoxia and acidity), thus developing a more aggressive phenotype.5 One of the characteristic alterations associated with the increased glycolytic rate of cancer cells is dramatically increased cellular glucose uptake, Correspondence to: Xiaoling Fang (Telephone: +86-21-51980071; Fax: +8621-51980072; E-mail: [email protected]) Xinyi Jiang and Hongliang Xin have contributed equally to this manuscript. Journal of Pharmaceutical Sciences, Vol. 103, 1487–1496 (2014)  C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

which is mediated by glucose transporter (GLUT), a kind of transmembrane proteins. Therefore, GLUT is highly expressed in cancers, including breast, nonsmall cell lung cancer, thyroid, head and neck, colon, and esophagus,6 which suggests that the GLUT might be an efficient target for drug delivery to tumor tissue. Actually, because of GLUT being highly expressive on glioma cells and blood brain barrier (BBB), GLUT showed that it could mediate D-glucosamine-modified nanoparticles across BBB and glioma tissue in intracranial glioma-bearing mice model.7 Poly(trimethylene carbonate) (PTMC) has been widely used in biomedical field because of the tunable biodegradability without formation of acidic compounds and excellent mechanical properties.8,9 In our previous study, we have investigated the synthesis and self-assembly behavior of poly(ethylene glycol) (PEG)–PTMC diblock copolymer. And the PEG–PTMC copolymer nanoparticles have been demonstrated to be an effective carrier for anticancer drug.10 However, the plain PEG– PTMC nanoparticles presented only passive targeting ability to tumor tissue because of the enhanced permeability and retention effect.11 Therefore, it is aspired to conjugate certain tumor-targeting ligands to develop active targeting nanocarrier. The surface-functionalized nanoparticles can specifically target cancerous tissues/cells because of their passive and active targeting abilities, thereby greatly enhancing the therapeutic outcomes while reducing any nonspecific systemic toxicity.12–16 Paclitaxel (PTX) has been demonstrated significant antitumor activity against various solid tumors such as ovarian,

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breast, lung, head, and neck cancer.17 However, its high hydrophobicity and nonselective distribution in vivo seriously restrict the practical use. Thus, there is an urgent demand for targeting drug delivery system for PTX application. In this study, we have constructed a novel PTX-loaded targeting copolymer nanoparticle for tumor-special delivery by employing D-glucosamine as targeting moiety and PEG–PTMC nanoparticle as drug carrier (DGlu-NP/PTX). DGlu-NP/PTX was prepared and characterized in vitro. The targeting efficiency was systematically evaluated by in vitro cell, in vitro three-dimensional (3D) tumor spheroids model and in vivo subcutaneous xenograft nude mice model.

MATERIALS AND METHODS Materials Paclitaxel was obtained from Xi’an San jiang BioEngineering Company Ltd. (Xi’an, China). MPEG3K –PTMC6K and NHS–PEG3.5K –PTMC6K were synthesized as described previously.9 D-Glucosamine (2-amino-2-deoxy-D-glucose) hydrochloride, Hoechst 33342, propidium iodide (PI), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, Missouri). Oregon Green 514 palloidin, antibovine "-tubulin mouse mAb, and Alexa Fluor 633-conjugated goat antimouse IgG antibody were obtained from Molecular Probes Company (California, USA).. 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindotricarbocyanine iodide (DiR) was purchased from Biotium (California, USA). Annexin V-FITC Apoptosis Detection kit and Micro BCA Protein assay kit were purchased from R Biotechnology Company Ltd. (Nantong, China). Beyotime Penicillin–streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (Gaithersberg, Maryland). RG2 cell line was obtained from ATCC. BALB/c mice (20 ± 2 g) were supplied by Department of Experimental Animals, Fudan University (Shanghai, China). All animal experiments were carried out in accordance with guidelines evaluated and approved by the Ethics Committee of the College of Pharmacy, Fudan University. Synthesis and Characterization of D-Glucosamine-Conjugated PEG–PTMC Copolymer NHS–PEG–PTMC (0.0053 mmol) and 2-amino-2-deoxy-Dglucose (0.0106 mmol) were dissolved in 10 mL dimethylsulphoxide with 0.1 mL Et3 N for 48 h reaction. Then, the mixture was dialyzed (MWCO 3500 Da) against deionized water for 48 h. The final solution was lyophilized and stored at −20◦ C until use. The copolymer was characterized by 1 H-NMR spectra in dimethyl sulfoxide (DMSO)-d6. Preparation of D-Glucosamine-Conjugated Targeting Nanoparticles D-Glucosamine-conjugated and paclitaxel-loaded poly(ethylene

glycol)-co-poly(trimethylene carbonate) copolymer nanoparticle was prepared through the emulsion/solvent evaporation technique as previously.18–21 Briefly, 36 mg of MPEG–PTMC, 4 mg of DGlu–PEG–PTMC, and 2.8 mg of PTX in 1 mL dichloromethane (DCM) was added into 5 mL of 0.6% sodium cholate aqueous solution and then sonicated at 200 W on ice. The formed emulsion was added dropwise into 25 mL of Jiang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1487–1496, 2014

0.3% sodium cholate under rapid magnetic stirring. After that, DCM was evaporated by rotary vacuum at 37◦ C. The formed nanoparticle suspension was concentrated by ultrafiltration. After washed twice by deionized water, DGlu-NP/PTX was resuspended in 1 mL saline and kept at 4◦ C for further use. 1,1 -Dioctadecyl-3,3,3 ,3 -tetramethylindotricarbocyanine iodide-labeled DGlu-NP was prepared with the same procedure except that the PTX was prepared by DiR. Characterization of Targeting Nanoparticles The mean particle size, size distribution, and zeta potential of DGlu-NP/PTX were determined by dynamic light scattering (DLS). The morphology of DGlu-NP/PTX was observed using a Carl Zeiss Ultra 55 field emission scanning electron microscope (Oberkochen, Germany). The PTX encapsulated in nanoparticles was quantified via HPLC as described previously.22 Inhibitory Effect Against Tumor Cells As GLUT was overexpressed on brain gliomas,23 we used rat glioma RG-2 cells as cell model to evaluate the inhibitory effect of DGlu-NP/PTX. RG-2 cells were seeded in 96-well plates at the density of 5 × 104 cells/well and cultured at 37◦ C for 24 h. Then, the cells were exposed to various PTX formulations, including Taxol, NP/PTX, and DGlu-NP/PTX with various concentrations. After 72 h incubation, MTT was added into the medium at 0.5 mg/mL for 4 h incubation. Afterwards, 200 :L DMSO was added into each well to dissolve any purple formazan crystals formed. The relative color intensity was measured by Tecan ¨ Safire 2 microplate reader (Mannedorf, Switzerland). Immunofluorescence Analysis RG-2 cells were seeded at a density of 5 × 105 cells/well in sixwell plates. After 24 h incubation, cells were treated with various PTX formulations at equivalent PTX concentration (200 ng/mL) for 24 h. Then, the cells were fixed with 4% formaldehyde for 10 min and permeabilized in 0.1% Triton X-100 phosphate-buffered saline (PBS) solution that contained 1% bovine serum albumin (PBS–BSA) and RNase 100 :g/mL. After washing three times with PBS–BSA, the cells were treated with Oregon Green 514 palloidin (1:100 v/v) in PBS–BSA for 20 min and then incubated for 60 min with antibovine "-tubulin mouse mAb in PBS–BSA. After adding 2 :g/mL Alexa Fluor 633-conjugated goat antimouse IgG antibody, the cells were incubated for another 60 min. The samples were rinsed three times by PBS–BSA and treated with 100 nM PI for 5 in and then viewed by Confocal Laser Scanning Microscopy (CLSM) examination with TCS SP5 of Leica (Solms, Germany). Cell Apoptosis Assay For the quantitative analysis of apoptosis, RG-2 cells were seeded into six-well plate at a density of 5 ×105 cells/well for 24 h incubation. Then, cells were treated with various PTX formulations at equivalent PTX concentration (200 ng/mL). After 24 h incubation, cells were stained using the Annexin V-FITC Apoptosis Detection kit followed by the manufacturer’s instructions. The stained cells were analyzed using a FACSCalibur flow cytometer of BD Biosciences (New York, USA) taking untreated cells as blank control. Data analysis was performed using CellQuest software of Becton Dickinson (New York, USA). DOI 10.1002/jps.23928

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Targeting Effect on Avascular 3D Tumor Spheroids In Vitro RG-2 tumor spheroids made by lipid overlay system were used to mimic the in vivo solid tumor.19,21 Low-melting temperature agarose was heated at 80◦ C for 30 min, and diluted to 2% (w/v) with the serum-free DMEM medium. Each well of 48-cell culture plates was coated with a thin layer (150 :L) of sterilized agarose. RG-2 tumor cells were seeded into each well at the density of 800 cells/well, and incubated at 37◦ C for 7 days. For evaluating the inhibition of glioma, the RG-2 tumor spheroids were incubated with DMEM, Taxol, NP/PTX, and DGlu-NP/PTX at a concentration of 400 ng/mL, respectively. Growth inhibition was monitored by tumor spheroids volume. The major (dmax ) and minor (dmin ) diameters of each spheroid were determined and spheroid volume was calculated using the following formula: V = (B×dmax ×dmin )/6 as described previously.24,25 The RG-2 tumor spheroids volume ratio was calculated with the formula: R = (Vday i /Vday 0 ) × 100%, where Vday i is the RG-2 tumor spheroids volume at the ith day after applying the drug, and Vday 0 is the RG-2 tumor spheroids volume prior to administration. On day 0 and day 5, the treated tumor spheroids were rinsed three times by PBS, fixed by 2.5% glutaraldehyde for 2 h at 4◦ C, then dehydrated and embedded. These tumor spheroids were viewed under Carl Zeiss Ultra 55FE-SEM (Oberkochen, Germany).

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were collected and also visualized using CRi in vivo imaging system. In Vivo Antitumor Efficacy The in vivo antitumor activity of the targeting nanoparticles was evaluated in RG-2 subcutaneous tumor xenograft model. The dose schedule started when the tumor volume reached 40– 80 mm3 . The tumor-bearing mice were randomly divided into four groups (n = 5) and treated with 100 :L of Taxol, DGluNP/PTX, NP/PTX, and physiological saline via tail vein injection on the day 0, 2, and 4 (PTX dosage: 10 mg/kg body weight, Taxol was diluted by physiological saline), respectively. Tumor size was monitored every 2 day and the tumor volume was estimated using the formula: volume = 0.5 × length × (width)2 . On day 14, animals were sacrificed by cervical dislocation, and the tumor mass was harvested, photographed. Finally, the liver and tumor tissues were processed routinely into paraffin, sectioned at a thickness of 5 :m, stained with hematoxylin and eosin (H&E) and then visualized under Leica DMI 4000B fluorescent microscope (Solms, Genmany). Statistical Analysis One-way analysis of variance was used to determine significance among groups, after which post-hoc tests with the Bonferroni correction were used for comparison between individual groups. A value of p < 0.05 was considered to be significant.

In Vivo Pharmacokinetic Study Fifteen male Sprague–Dawley (SD) rats weighting 200 ± 20 g were randomly divided into three groups for pharmacokinetic investigation. Group 1, 2, and 3 received Taxol, NP/PTX, and DGlu-NP/PTX (5 mg/kg PTX vs. the body weight) through the tail vein, respectively. At appropriate time intervals (0, 5, 15, 30 min and 1, 2, 4, 8, 12 h), blood samples were collected from the orbital vein and centrifuged at 1000g for 10 min to obtain plasma. The plasma was stored at −70◦ C prior to analysis by HPLC. Liquid–liquid extraction was performed before analysis. Briefly, 200 :L of plasma was mixed with 3 mL diethyl ether containing 50 :L of 1.0 :g/mL diazepam as an internal standard. The samples were extracted on vortex mixer for 2 min and then centrifuged at 6000g for 10 min. Next, the organic layer was transferred into clean tube and evaporated under gentle stream of nitrogen. The extraction residue was reconstituted in 100 :L acetonitrile and centrifuged at 1500g for 5 min before HPLC analysis. The pharmacokinetic parameters were calculated using the DAS (Drug and Statistic for Windows) software (version 2.0). In Vivo Imaging In vivo real-time fluorescence imaging analysis was used to evaluate the effect of tumor distribution and accumulation ability of the targeting nanoparticles. The subcutaneous tumor xenograft model was established by inoculation of 5 × 105 RG-2 cells (in 100 :L cell culture medium) into the subcutaneous of the right hind leg. When the size of tumor reached 0.5–0.7 cm in diameter, the tumor-bearing mice were injected with DiR-labeled NP and DGlu-NP via tail vein at a dose of 20 :g DiR/mouse, respectively. At the time points of 1, 3, 6, and 12 h, the mice were anesthetized and visualized using CRi in vivo imaging system (Massachusetts, USA). After 12 h of injection, the mice were humanely killed. Then, the tumor and principle organs (including heart, liver, spleen, lung, and kidney) DOI 10.1002/jps.23928

RESULTS AND DISCUSSION Synthesis and Characterization of DGlu–PEG–PTMC Coplymer 2-Amino-2-deoxy-D-glucose was conjugated to the distal end of PEG through a reaction between the NHS group and amino group. Figure 1 included the 1 H-NMR spectrum and the complete peak assignments of the NHS–PEG–PTMC copolymer, 2-amino-2-deoxy-D-glucose and DGlu–PEG–PTMC copolymer. The solvent peak of DMSO-d6 was found at 2.50 ppm. The characteristic peak at 2.59 ppm was assigned to the protons of the NHS unit of NHS–PEG–PTMC copolymer. For DGlu– PEG–PTMC copolymer, the NHS unit peak at 2.59 ppm was disappeared in 1 H-NMR spectrum and a series of characteristic peaks of 2-deoxy-D-glucose were present, which suggested that 2-amino-2-deoxy-D-glucose was successfully conjugated to NHS–PEG–PTMC. Characterization of the Targeting Nanoparticles The drug-loaded core–shell nanoparticle functionalized with Dglucosamine was schematically illustrated in Figure 2a. The hydrophobic drug encapsulated into the core of nanoparticles can protect the drugs from premature degradation and nonspecific interaction, which can improve the bioavailability of the compound. The hydrophilic PEG chains on the surface of nanoparticles can avoid the uptake by reticuloendothelial system to prolong the circulation in vivo. The outer D-glucosamine can actively target to tumor tissue via GLUT-mediated endocytosis. The Z-average particle size of DGlu-NP/PTX was about 71.2 ± 10.6 nm with an acceptably polydispersity index (PDI < 0.16). The Z-average particle size of NP/PTX was about 70.25 ± 2.33 nm with PDI less than 0.2. The zeta-potential of NP/PTX is −7.35 ± 2.82 mV. Therefore, surface modification on nanoparticles with D-glucosamine did not significantly change the physicochemical properties of the nanoparticles. Representative photo Jiang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1487–1496, 2014

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Figure 1.

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

1 H-NMR

spectrum of NHS–PEG–PTMC (a), D-glucosamine (b), and DGlu–PEG–PTMC (c) in DMSO-d6.

Figure 2. Schematic representation of DGlu-NP/PTX (a). The particle size and size distribution of DGlu-NP/PTX determined by DLS using a Malvern Nano ZS (Malvern, UK). Inset: photo of nanoparticle solution at the concentration of 40 mg/mL (b). SEM image of Glu-NP/PTX, the bar is 20 nm (c).

of DGlu-NP/PTX solution at the concentration of 40 mg/mL was shown in Figure 2b. The solution exhibited a slight whitish opalescence. The zeta potential of DGlu-NP/PTX was about −8.12 ± 0.47 mV. The representative SEM of DGlu-NP/PTX is shown in Figure 2c. These nanoparticles exhibited spherical shape of moderate uniform particle size. The PTX loading content and encapsulation ratio of DGlu-NP/PTX were about 6.52 ± 0.89% and 94.8 ± 2.9%, respectively. Jiang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1487–1496, 2014

Inhibitory Effect Against Tumor Cells In vitro cytotoxicity against RG-2 cells of various PTX formulations, including Taxol, NP/PTX, and DGlu-NP/PTX, were evaluated by MTT assay. The results of Figure 3 showed that all PTX-containing formulations exhibited strong inhibitory effect on the proliferation of RG-2 cells. At various concentration points, DGlu-NP/PTX exhibited the strongest inhibitory effect DOI 10.1002/jps.23928

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and late apoptosis because of the elevated PTX intracellular uptake via GLUT-facilitated endocytosis. Effect on In Vitro 3D Tumor Spheroids

Figure 3. In vitro cytotoxicity of various formulations of PTX against RG-2 cells (n = 3). (") p < 0.05 versus Taxol; ($) p < 0.05 versus NP/PTX.

on the proliferation of RG-2 cells among various formulations. The IC50 values were 214.7 ng/mL of Taxol, 225.8 ng/mL of NP/PTX and 143.2 ng/mL of DGlu-NP/PTX. The cytotoxicity of PTX-loaded nanoparticles decorated with D-glucosamine significantly increased, indicating that D-glucosamine could facilitate nanoparticle uptake into the cancer cells and thus produce higher cytotoxicity than NP/PTX and Taxol. Immunofluorescence Analysis Microtubule stabilization of different PTX formulations was visualized utilizing confocal microscopy (Fig. 4). The untreated RG-2 cells demonstrated normal nuclei, centrosomes, and a fine irregular meshwork of microtubules. The obvious assembly of microtubule was observed for all PTX preparations after 24 h treatment, indicating that RG-2 glioma cells were sensitive to PTX. After applying Taxol or NP/PTX, loosely packed microtubule bundles in RG-2 glioma cells were formed and some of microtubules were forming parallel alignments around the nucleus, which is indicative of microtubule stabilization. For RG-2 cells treated with DGlu-NP/PTX, lots of condensations of cytoplasmic microtubules and micronuclears were formed because of the improper mitotic spindle assembly. These results suggested that treatment with DGlu-NP/PTX showed stronger microtubule stabilization during cell cycle blockage at the G2 /M phase as compared with Taxol or its conventional counterpart. Cell Apoptosis Assay To examine whether the encapsulation of PTX in glucosylated nanoparticles modifies cell apoptosis, Annexin V-FITC Apoptosis Detection kit was used to stain the cells and the percentage of cell apoptosis was determined by flow cytometer as shown in Figure 5. The percentage of early and late apoptosis of Taxoltreated RG-2 cells was 15.31 ± 3.25% and 7.01 ± 2.11%, respectively, whereas DGlu-NP/PTX caused 35.82 ± 3.64% and 24.48 ± 3.45% of early and late cell apoptosis. In comparisons between the drug-loaded nanoparticles, the percentage of early and late apoptosis of DGlu-NP/PTX-treated RG-2 cells was evidently higher than that of NP/PTX (17.12 ± 2.27% and 11.21 ± 2.42%). These findings were consistent with the in vitro cytotoxicity and indicated that DGlu-NP/PTX induced more early DOI 10.1002/jps.23928

In contrast to monolayer culture, 3D tumor spheroids may be able to simulate the partial avascular regions of solid tumor, and are more close to the in vivo situation regarding cellular shape and environment.26 Therefore, 3D tumor spheroids have been extensively used to evaluate the penetration ability of drug delivery system.27,28 Figure 6 showed the in vitro tumor spheroid volume ratio after treatment with Taxol, NP/PTX, and DGlu-NP/PTX at the PTX concentration of 400 ng/mL. It was observed that tumor spheroids continued to grow in size and volume in the absence of any drug (151.3% of the primary volume after 7 days). The obvious reduction in volume of tumor spheroids was observed for all PTX formulations after 7 days treatment, indicating that tumor spheroids were sensitive to PTX. The change ratio of tumor spheroid volumes (%) on day 7 was 85.7%, 82.5%, and 41.3% for Taxol, NP/PTX, and DGlu-NP/PTX, respectively. These results indicated that DGluNP/PTX significantly improved the inhibitory effects on the 3D tumor spheroids, which suggests that DGlu-NP/PTX may improve therapeutic effect in vivo. Figure 7 showed the SEM observations of RG-2 tumor spheroids on day 5 after applying various PTX preparations including Taxol, NP/PTX, and DGlu-NP/PTX. As blank control, tumor spheroids were tightly organized and the surface of these spheroids was covered with slice of agarose. After applying Taxol or NP/PTX, the cells on the tumor spheroid surface died and appeared concave-like holes, and the cell leakage and cell membrane lysis occurred. After applying DGluNP/PTX, the spheroids were obviously shrunk and almost lost the 3D structure and the marginal of tumor cells became disintegrated. These results indicated that DGlu-NP/PTX exhibited the strongest penetrating ability, and showed the strongest inhibitory effect on the growth of spheroids. These findings were consistent with the results of the change ratio of tumor spheroids volume. In Vivo Pharmacokinetic The blood clearance curves of various PTX formulations after intravenous administration to SD rats are shown in Figure 8. The DGlu-NP/PTX and NP/PTX showed initial high-blood circulating levels compared with Taxol, whereas PTX formulated in Taxol was quickly removed from the circulating system 6 h after administration. On the contrary, DGlu-NP/PTX and NP/PTX exhibited a markedly delayed blood clearance. Compartmental analysis of the plasma concentrations showed a significant change in pharmacokinetic parameters of PTX in nanoparticles compared with that of commercial formulation (Table 1). It was shown that DGlu-NP/PTX and NP/PTX could extend the elimination half-life (t1/2$ ) of Taxol from 4.19 to 12.29 and 11.02 h, respectively (p < 0.01). Meanwhile, the area under the PTX plasma concentration–time curve (AUC0→∞ ) increased by about sixfold for DGlu-NP/PTX and sevenfold for NP/PTX compared with Taxol, whereas there was no significant difference in AUC0→∞ between DGlu-NP/PTX and NP/PTX (p > 0.05). In addition, Mean Retention Time (MRT) of DGluNP/PTX and NP/PTX was 5.3-fold and 5.8-fold higher than that of Taxol, respectively (p < 0.01). These results suggested that glucosylated modification did not evidently influence the Jiang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1487–1496, 2014

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Figure 4. Confocal Laser Scanning Microscopy images of RG-2 cells treated with distinct PTX formulations (Taxol, NP/PTX, and DGlu-NP/PTX) at equivalent drug concentration of 200 ng/mL for 24 h. Normal RG-2 cells without any treatment served as the control. F-actin, microtubule, and nucleus were labeled by Oregon Green 514 palloidin, PI, and Alexa Fluor 633 stainings, respectively. Original magnification: ×200. White arrow: condensation of cytoplasmic microtubules; yellow arrow: formation of micronuclear. Bar: 6.8 :m.

in vivo long-circulating property of the polymer nanoparticles. In contrast, CL of DGlu-NP/PTX and NP/PTX was significantly lower than that of Taxol implying a longer retention of the drug in blood circulation. This long blood circulation could be illustrated by the stealth behavior of polymeric nanoparticles induced by hydrophilic shell of PEG, which will reduce the absorption by plasma proteins and decrease the rate of mononuclear phagocyte system (MPS) uptake.29 Taking together, these results illustrated the potential utility of DGlu-NP/PTX as longcirculating carrier for cancer treatment. In Vivo Imaging The in vivo distribution and potential tumor-targeting capability of DGlu-NP was determined noninvasively in subcutaneous Jiang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1487–1496, 2014

xenograft-bearing nude mice (Fig. 9). Compared with traditional NP group, the near-infrared fluorescence (NIR-FL) intensity in the tumor region of DGlu-NP group was much higher at any time postinjection ranged from 1 to 12 h (Figs. 9a and 9b), suggesting that glucosylation could facilitate the accumulation of nanoparticles in tumor tissues. The ex vivo organs (heart, liver, spleen, lung, kidney, and tumor) also revealed that the tumor accumulation of DGlu-NP was much more than that of plain NP, whereas there was lower accumulation of nanoparticles in MPS-related organs such as the liver and the spleen (Figs. 9c and 9d). On the basis of organ imaging, mean fluorescence intensities of tumor, heart, liver, spleen, lung, and kidney were calculated. Quantitative analysis of biodistribution of DGlu-NP and NP in tumor-bearing mice revealed that DOI 10.1002/jps.23928

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Figure 5. RG-2 cell apoptosis inducted by Taxol (b), NP/PTX (c), and DGlu-NP/PTX (d) after incubation for 24 h at equivalent PTX concentration (200 ng/mL) by flow cytometry using staining of Annexin V-FITC and PI. Normal RG-2 cells without any treatment served as the control (a). The cells that took up PI but did not bind Annexin V-FITC would most likely be necrotic are shown in the upper left quadrant; late apoptotic cells that bind Annexin V-FITC and PI in are shown upper right quadrant; early apoptotic cells binding Annexin V-FITC are shown in lower right quadrant; viable cells binding neither Annexin V-FITC nor PI are shown in lower left quadrant (n = 3).

cancer tissues in vivo and decrease nonspecific accumulation in reticuloendothelial systems. However, much of the injected targeting nanoparticles were still nonspecific captured by the MPS, reducing nanoparticles being distributed to the target tissue.29 In Vivo Antitumor Efficacy

Figure 6. Inhibition on the growth of tumor spheroids treated with Taxol, NP/PTX, or DGlu-NP/PTX at concentration of 400 ng/mL. Spheroids applying serum-free DMEM culture medium served as the control. The diameter of tumor spheroids was measured using a microscope fitted with an ocular micrometer, and the volume of the spheroids was calculated. Data are mean values ± SD. (") p < 0.01 versus control. ($) p < 0.01 versus Taxol. (() p < 0.01 versus NP/PTX.

the fluorescence intensity in tumor treated with DGlu-NP was 1.94 times higher than that of NP (p < 0.05) (Fig. 9e). These results indicated that DGlu-NP could substantially home to DOI 10.1002/jps.23928

The in vivo antitumor efficacy of Taxol, NP/PTX, and DGluNP/PTX was validated in subcutaneous xenografts mice. As shown in Figures 10a–10c, DGlu-NP/PTX (p < 0.01), NP/PTX (p < 0.05), and Taxol (p < 0.05) exhibited considerable tumor growth-inhibiting efficacy compared with the control saline group. Among these groups, DGlu-NP/PTX exhibited the strongest inhibitory effect on the tumor volume. A combined effect of the passive targeting and enhanced cellular uptake could be the main reason for the significant suppression of tumor growth of DGlu-NP/PTX group. As presented in Figure 10d, it was demonstrated that apoptosis occurred in tumor slices treated with various PTX formulations. It was clear that cell apoptosis of DGlu-NP/PTX group was more severe as compared with those of Taxol injection and NP/PTX, suggesting that D-glucosamine-functionalized nanoparticles could deliver more PTX into tumor tissue and successfully improved antitumor activity of PTX. The in vivo antitumor efficacy of NP/PTX and DGlu-NP/PTX was consistent well with the in vitro cell experiment. The toxicity of drug carrier is always a great concern of nanoparticulate system used in biomedicine. To further Jiang et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1487–1496, 2014

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Figure 7. Morphology of tumor spheroids observed by SEM. Key: spheroids at day 3 after applying serum-free DMEM culture medium, Taxol, NP/PTX, and DGlu-NP/PTX, respectively. Final PTX for all was at 400 ng/mL.

evaluate the preliminary safety of the novel delivery system, the liver tissue histology was carried out to determine cellular infiltration because of the inflammatory response. There was no evidence of acute liver toxicity of any PTX treatment (Fig. 10e). All of these results implied that D-glucosamine-functionalized copolymer nanoparticles were potential drug delivery system for tumor chemotherapy.

CONCLUSIONS D-Glucosamine-functionalized

Figure 8. Plasma concentration–time curves of Taxol, NP/PTX, and DGlu-NP/PTX after i.v. administration to SD rats at the same 5 mg/kg PTX dose (n = 5).

Table 1.

Comparative Pharmacokinetic Parameters of PTX Formulations (n = 5)

Parameters

Formulations Taxol

t1/2 (") (h) t1/2 ($) (h) AUC0→t (:g•L−1 •h−1 ) AUC0→∞ (:g•L−1 •h−1 ) MRT (h) CL (L•h−1 •Kg−1 ) Vd (L•Kg−1 ) a b

and PTX-loaded PEG–PTMC copolymer nanoparticles were developed as tumor-targeting drug delivery system for enhancing cancer chemotherapy as well as circumventing the nonspecial accumulations. The resultant DGlu-NP/PTX presented a size of 71.2 ± 10.6 nm with uniform distribution. D-glucosamine modification on the surface of the nanoparticles could significantly facilitate specific

0.31 4.19 4608.46 6014.49 2.97 0.83 0.87

± ± ± ± ± ± ±

0.11 0.34 1133.21 2363.73 1.04 0.07 0.12

NP/PTX 0.28 12.29 21797.13 43140.96 17.38 0.12 0.82

± ± ± ± ± ± ±

0.08 2.45b 5623.96b 3975.41b 3.93b 0.09a 0.21

DGlu-NP/PTX 0.23 11.02 20625.52 36325.13 15.85 0.12 0.76

± ± ± ± ± ± ±

0.13a 1.78b 4381.22b 5438.33b 3.14b 0.12a 0.67

p < 0.05. p < 0.01, compared with Taxol.

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Figure 9. In vivo fluorescence imaging of subcutaneous tumor-bearing nude mice after injection of DiR-labeled NP (a) or DiR-labeled DGlu-NP (b) via a lateral tail vein. Bright field (BF) was acquired under white light. All NIR-FL images were acquired with a 300-s exposure time at 1, 3, 6, and 12 h. Arrow: the position of tumor. Representative images of dissected organs of mice-bearing subcutaneous tumor sacrificed 12 h after i.v. injection of DiR-labeled NP (c) or DiR-labeled DGlu-NP (d). Ratio of the relative fluorescence intensity in dissected organs mentioned in figures (c) and (d) (e). Key: t, tumor; h, heart; li, liver; s, spleen; lu, lung; and k, kidney.

Figure 10. Tumor growth curves of different groups after treatment indicated. Data are presented as mean ± SD (n = 5) (a). Representative photos of tumors on mice; red arrow: the position of tumor (b). Photographs of tumors harvested from each treatment group after various treatments (c). Images of H&E-stained sections of liver (d) and tumor (e) excised from subcutaneous tumor-bearing mice on 14th day after different treatment. Images were obtained under Leica fluorescence microscope using a 20×objective.

DOI 10.1002/jps.23928

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

uptake by cancer cells via GLUT protein-mediated endocytosis. In comparison with conventional nanoparticles and Taxol, DGlu-NP/PTX displayed the strongest inhibition effect on cancer cells and 3D tumor spheroids, a marked tumor-homing specificity in vivo and the greatest tumor growth inhibitory effect in vivo. And thus, D-glucosamine-functionalized and PTXloaded PEG–PTMC copolymer nanoparticles were potential drug delivery system for cancer therapy.

ACKNOWLEDGMENTS This work was supported from the National Key Basic Research Program of China (2013CB932502) and National Natural Science Foundation of China (81302716, 81302710), National Science and Technology Major Project (2012ZX09304004), Natural Science Foundation of Jiangsu Province (BK2012445), the ordinary university natural science research project of Jiangsu Province (13KJB350004), and Advanced Talent Foundation of Jiangsu University (13JDG013). The authors also acknowledge the support from School of Pharmacy, Fudan University and the Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), and Ministry of Education, China (SDD2012-4).

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DOI 10.1002/jps.23928