Target-Specific Cellular Uptake of Taxol-Loaded Heparin-PEG-Folate

Nov 18, 2010 - Grit Baier , Svenja Winzen , Claudia Messerschmidt , Daniela Frank , Michael Fichter .... Mark E. Keating , Franck Bonnier , Hugh J. By...
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Biomacromolecules 2010, 11, 3531–3538

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Target-Specific Cellular Uptake of Taxol-Loaded Heparin-PEG-Folate Nanoparticles Ying Wang,*,†,‡,§ Yiqing Wang,†,| Jiannan Xiang,§ and Kaitai Yao*,‡ Cancer Research Institute, Southern Medical University, Guangzhou, 510515, China, Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, Atlanta, Georgia 30322, United States, and Biomedical Engineering Center, College of Chemistry and Chemical Engineering, and State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410082, China Received August 28, 2010; Revised Manuscript Received October 16, 2010

To enhance site-specific intracellular delivery against folate receptor, heparin-PEG-folate (H-PEG-F) containing succinylated-heparin conjugated with folate via PEG 1000/3000 spacers has been prepared. Due to covalent strategy, H-PEG-F displays amphiphilic property, which is capable of entrapping a hydrophobic agent, like taxol, to form heparin-PEG-folate-taxol nanoparticles (H-PEG-F-T NPs) in aqueous solution. Hydrophobic agents can be entrapped within the core, while the H-PEG-F conjugates can stabilize the nanoparticles with exposing folate moieties on the surface. The structure of carrier and naoparticles has been characterized by1H NMR, and the content of folate and taxol has been quantitatively analyzed by UV method. The morphology and size of H-PEG-F-T NPs have been measured by field emission scanning electron microscopy (FESEM) and dynamic lighting scatter (DLS). All the NPs are in spherical shape and the sizes are less than 200 nm. The sizes of the NPs increases with increasing PEG segment length. By employing the flow cytomery method, the extent of cellular uptake has been comparatively evaluated under various conditions. The results of cellular uptake demonstrate that the cellular uptake of the carrier and the NPs is exceedingly higher for KB-3-1 cells (folate receptor overexpressing cell line) than for A549 cells (folate receptor deficiency cell line); H-PEG-F-T NPs show far greater extent of cellular uptake than that of H-PEG-F conjugates against A549 cells; when the content of folate is fixed at the same value, the extent of cellular uptake for the carrier and NPs ascends with the increase of PEG chain length against KB3-1 cells. It suggests folate-receptor-mediated endocytosis and formation of nanoparticle and spacer length are considered to coaffect the cellular uptake efficiency of H-PEG-F-T NPs and H-PEG-F conjugates. Flow cytometry analysis depicts that KB-3-1 cells treated with H-PEG-F-T are arrested in the G2/M phase of the cell cycle, which states the similar inhibition mechanism as taxol. The strategy based on the formation of H-PEG-F-T NPs could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

Introduction Recently, nanoparticulate drug delivery systems containing anticancer agents have received much attention due to their unique accumulation behavior at the tumor site.1-3 Enhanced permeation and retention (EPR) effect is now considered as a major mechanism for their unique biodistribution profile in the tumor tissue.4-6 Almost all of these nanomaterials are formed through the association of some polymers, which are utilized to selectively deliver various anticancer agents at the tumor in a passive targeting manner.7-9 In particular, polymeric micelle and polymer nanoparticles are made from amphiphilic polymers, and they have hydrophilic shells and hydrophobic cores in aqueous media. Such nanoparticles with core-shell structure can readily incorporate lipophilic drugs into their cores and permit the controlled release of the drugs for sustained therapeutic effect, and the sizes of these nanoparticles are normally in the range of 10 to 200 nm, which is small enough to avoid filtration by the lung and spleen.10 * To whom correspondence should be addressed. Phone: 86-02061648630. Fax: 86-020-61648225. E-mail: [email protected], [email protected]. † These authors contributed equally to this work and are both considered as first author. ‡ Southern Medical University. § Emory University. | Hunan University.

However, a more effective and active targeting system has been further needed to enhance intracellular uptake of drug within cancerous cells at the tumor site.11-13 Various targeting moieties or ligands against tumor-cell-specific receptors have been immobilized on the surface of carriers to deliver them within cells via receptor-mediated endocytosis. Among them, the receptor for folate is overexpressed in many human cancer cells, including malignancies of the ovary, brain, kidney, breast, myeloid cells, and lung.14 As such, folate has been widely employed as a targeting moiety to facilitate the targeted delivery of anticancer drugs15-18 and folate NPs can be rapidly internalized by receptor-bearing cancer cells19 in a manner that bypasses cancer cell multidrug-efflux pumps.20 Heparin is a biocompatible, biodegradable, and water-soluble natural polysaccharide with a complicated structure. In addition to its well-known anticoagulant activity, heparin has numerous important biological properties associated with its interaction with diverse protein applied to develop heparin-containing systems.21,22 Several recent attempts to explore the formation of PEG functionalized with heparin hydrogels involve developing various biological characteristics.22-24 It also exerts anticancer activities in the processes of tumor progression and metastasis. These distinctive properties have significant implications in the development of a heparin-based nanoparticulate delivery system.24-28 We have previously reported the fabrica-

10.1021/bm101013s  2010 American Chemical Society Published on Web 11/18/2010

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tion of heparin-paclitaxel nanoparticles using succinylatedheparin as carrier together with the corresponding results for the morphology, drug release, in vitro cytotoxicity, and in vivo antitumor activity.29 The nanoparticulate drug delivery systems can increase parent drug solubility as well as enable passive targeting to the solid tumor. However, the development of its cell-specific targeting ability is a growing demand for promoting their intracellular uptake within target cells. It has been well documented that a ligand-mediated nanoparticulate drug carrier composed of heparin-based copolymers was designed to differentially deliver an anticancer drug via folate or folate-PEG as specific ligand. This drug carrier possessed amphiphilic properties by introducing the hydrophobic polymer poly(β-benzyl-L-aspartate) (PBLA) to the hydrophilic heparin backbone, while the specific drug delivery system has not yet investigated.30 To the best of our knowledge, no one has, thus far, prepared heparin-based carrier with amphiphilic capacity by directly introducing small organic groups and specific ligand to heparin segment and further explored physicochemical characteristics for the drug delivery system. In this study, we design PEG as a flexible spacer to connect with folate moieties and succinylated heparin. The amphiphilic property of the prepared H-PEG-F conjugates is investigated owing to the introduction of PEG and many succinlyated groups to heparin. Moreover, a cell-specific targeting H-PEG-F carrier to deliver anticancer drug is developed, wherein the hydrophobic anticancer drugs taxol are entrapped into H-PEG-F conjugate. We focus on investigating their behavior in aqueous solution and particle size change resulting from different side chain length (PEG1000/3000). In particular, it is necessary to examine the influence of folate receptor-mediated endocytosis, nanoparticle effect and side chain length on cellular uptake extend. In addition, the effect on cell cycle progression of H-PEG-F-T NPs will be evaluated in KB-3-1 cells compared with that of free taxol.

Experimental Section Materials. Heparin sodium (Mn ) 1.25 kDa, 189 U/mg) was obtain from Celsus Laboratories (Cincinnati, U.S.A.). Oregon green-488 cadaverine was obtained from Invitrogen Co. (U.S.A.). Boc-PEG-CONHS (Mw ) 1000/3000) was purchased from Rapp Polymere (U.S.A.). All other chemicals and reagents were purchased from Sigma (St. Louis, U.S.A.). Succinylated heparin and folate-NH2 were synthesized in our lab. Spectra/Por 3 Dialysis Membrane (MWCO 3500) was purchased from Spectrum Laboratories (Houston, U.S.A.). Ultrapure water (MilliQ, 18 MΩ) was used in the experiment. Synthesis of Succinylated Heparin. Succinylated heparin was synthesized by the method previously described.28 Briefly, heparin sodium salt (0.25 g) dissolved in 20 mL of water was percolated through a Dowex column (H+ form, 100 mL) at 4 °C. The pH of the solution was adjusted to 6.0 by addition of tributylamine. Excess tributylamine was eliminated by evaporation. The concentrated solution was diluted with water and lyophilized to yield tributylammonium salt (0.5 g). The tributylammonium salt (0.5 g) was dissolved in 10 mL of dry dimenthyl formide (DMF) and was then cooled down to 0 °C under a nitrogen atmosphere. Succinic anhydride (1.3 g, 13 mmol), triethylamine (1.31 g, 13 mmol), and 4-dimethylaminopydine (DMAP; 0.036 g, 0.3 mmol) were successively added and the reaction was allowed to proceed at room temperature for 24 h under nitrogen. Excessive solvent was evaporated, and water was added to the flask, followed by passing through a Dowex column (H+ form, 200 mL) at 4 °C. Then the product was dialysised by a membrane (MWCO 3500). The effluent was neutralized with 1 M NaOH and lyophilized as a white powder (0.20 g). 1H NMR (D2O): δ 2.5 ppm (-CH2CH2COOH, succinylated group). Synthesis of Folate-PEG-NH2. Folate-PEG1000-NH2 was synthesized by reacting Boc-PEG1000-NHS (0.5 g) with an excess quantity

Wang et al. of folate-NH2 in 5 mL of dry dimethylsulfoxide (DMSO) containing 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 10 mL of pyridine. The reaction mixture was stirred overnight in the dark at 35 °C. At this point, excessive DMSO was eliminated by evaporation, and 10 mL of cold acetone was added to remove unreacted folate-NH2. The insoluble byproduct was precipitated and removed by centrifugation, and this process was repeated three times. A total of 1 mL of 90% trifluoroacetic acid (TFA) was added to the flask to deprotect Boc groups for 30 min at room temperature. The prepared folate-PEG1000-NH2 was lyophilized as a yellow powder 1a (0.62 g). Folate-PEG3000-NH2 was synthesized by the same method. Synthesis of Heparin-PEG-Folate Conjugate (H-PEG-F). Succinylated heparin (0.20 g) was dissolved in 20 mL of dry DMF and maintained by gentle heating. Folate-PEG1000-NH2 (0.032 g, 0.05 mmol), EDC (0.019 g, 0.1 mmol), and N-hydroxysuccinimide (NHS; 0.012 g, 0.11 mmol) were successively added to the mixture, and the reaction was allowed to proceed at 35 °C. After overnight stirring, deionized water was gradually added and the solution was dialyzed against deionized water for 48 h in a dialysis membrane (MWCO 3500). After lyophilization, H-PEG1000-F conjugate (0.22 g) was obtained as a yellow powder. H-PEG3000-F conjugate was synthesized in a similar procedure as that of H-PEG1000-F. The folate content was determined by UV measurement. 1H NMR (DMSO-d6): δ 2.5 ppm (-CH2CH2COOH, succinylated group), 3.4-3.5 ppm (-CH2CHO-, PEG). 1H NMR (D2O): δ 2.6 ppm (-CH2CH2COOH, succinylated heparin), 3.4-3.5 ppm (-CH2CHO-, PEG), 6.6 ppm, 7.5 ppm (folate). Preparation of Heparin-PEG-Folate-Taxol Nanoparticles (H-PEG-F-T NPs). Nanoparticles were prepared by a nanoprecipitation method as described previously with minor modifications.6 H-PEG-F (0.2 g) and taxol (0.04 g) in 5 mL of dry DMSO was mixed and added at 35 °C for 6 h, and then cold deionized water was slowly added to the solution. DMSO was removed by a dialysis memberane (MWCO 3500) for 48 h. The excess water was evaporated, and the resulting aqueous solution was filtered through a 600 nm membrane. The nanoparticle suspension was finally lyophilized as a yellow powder (0.24 g). Taxol content was measured by UV method. 1H NMR (DMSO-d6): δ 1.06 ppm (17,16-CH3, taxol), 1.58 ppm (19-CH3, taxol), 1.8 ppm (18-CH3, taxol), 1.9-2.0 ppm (-NHCOCH3, succinylated heparin), 2.1 ppm (C4-OAc, taxol), 2.2 ppm (C10-OAc, taxol), 2.6 ppm (-CH2CH2COOH, succinylated heparin), 3.4-3.5 ppm (-CH2CHO-, PEG), 7.4-8.1 ppm (benzene ring, taxol). 1H NMR (D2O): δ 2.6 ppm (-CH2CH2COOH, succinylated heparin), 3.4-3.5 ppm (-CH2CHO-, PEG), 6.6 ppm, 7.5 ppm (folate). Characterization. The 1H NMR spectra of the product were recorded on a Varian INOVA400 apparatus in D2O and DMSO-d6. The weight of the succinlyated group on the carrier was determined by quantitative analysis of 1H NMR spectroscopy using pyridine as the internal standard, whose proton signals are δ 7.4, 7.9, and 8.4 ppm. The content of folate on the carrier was estimated by UV spectrometer (UV-2401, Shimadzu) based on a standard curve generated with known concentrations of folate in a NaOH solution (0.1 M) at 360 nm. The drug content of the product was calculated by ABST ) ABSH-PEG-F-T-ABSH-PEG-F, determined by UV measurement based on a standard curve generated with known concentrations of taxol in ethanol at 228 nm. The encapsulation efficiency (E.E.) were calculated by equation: E.E. (%) ) (amount of drug in nanoparticles/initial amount of drug) × 100%. Size distributions and zeta potentials of products were measured at 25 °C using a Zetasizer Nano-Zs (Malvern Instruments, U.K.). The concentration of products in distilled water was kept constant at 0.3 mg/mL. The morphology of nanoparticles was observed using a TOPCON DS150 FESEM (field emission scanning electron microscope). The accelerating voltage was set at 5 KV. A 10 µL droplet of nanoparticles in distilled water was placed onto a cleaned Si chip and left to vacuum-dry. The dried sample was coated with 4 nm chromium film using a Denton DV-602 magnetron sputter coater at 2 µm Hg pressure of argon gas.

Target-Specific Cellular Uptake of Nanoparticles Anticoagulant Activity Assay. The anticoagulant activities of products were determined by FXa-dependent coagulant assay using Coatest Heparin according to the manufacturer’s instructions. Briefly, in Antifactor Xa activity assay, all reactions were proceeded at 37 °C. Factor Xa (7.1 nkat/mL, 100 µL) was incubated during 3 min with antithrombin III (0.1 unit/mL, 200 µL) in the presence of products (at various concentrations in 0.05 mM Tris buffer, pH 8.4, 25 µL). The substrate, S-2222 (1 mmol/L, 100 µL), was then added and incubated for 2 min. The reaction was terminated upon the addition of 20% aqueous acetic acid (300 µL). The anticoagulant activity of heparin derivatives was evaluated by comparing its absorbance peak at 405 nm with that of heparin. Cell Culture. A human epidermoid carcinoma cell line, KB-3-1 cells (folate receptor overexpressing cell line), and a human lung epithelial carcinoma cell line, A549 cells (folate receptor deficiency cell line), were provided by Dr. Shin’s lab at the Departments of Biomedical Engineering and Chemistry, Emory University. KB-3-1 cell lines and A549 cell lines were cultured either in folate-free RPMI 1640 or DMEM, with and without folate, in 5% CO2 at 37 °C, respectively. The cell culture media were supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, and 100 units/mL penicillin/streptomycin. Cellular Uptake of H-PEG-F and H-PEG-F-T Nanoparticles. KB3-1 cells and A549 cells were plated in a 6-well plate at a density of 2.5 × 105 cell per well in medium and incubated for 24 h at 37 °C. Oregon green-488 dye-labeled H-PEG-F and H-PEG-F-T NPs were prepared by the similar procedure as that of H-PEG-F. The culture medium were then replaced with 1 mL of medium with Oregon green labeled H-PEG-F and H-PEG-F-T NPs for 30 min at 37 °C under various conditions. In free folate competition studies, 1 mM folic acid was added to the incubation medium. The incubated cells were washed

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three times with cold PBS to eliminate trace product and detached with 0.02% EDTA-PBS and then suspended in PBS containing 0.1% BSA. The suspended cells were directly introduced to a FACSort flow cytometer (Becton Dickinson, U.S.A.) equipped with a 488 nm argon ion laser. Data for 10000 fluorescent events were obtained by recording forward scatter (FSC), side scatter (SSC), and 530/15 nm fluorescence. The autofluorescence of cells was taken as a control. To visualize cellular uptake of H-PEG-F-T NPs via PEG1000/3000, KB-3-1 cells were reseeded in the Lab-Tek chambered slide (Miles Laboratories, U.S.A.). Cells were washed three times with PBS after treatment with Oregon green labeled H-PEG-F-T NPs via PEG1000/ 3000 for 40 min and then fixed by 4% (w/v) para-formaldehyde solution. For confocal microscopy, cells were mounted using DAPIcontaining reagent. The fluorescent images were viewed by confocal microscope (Olympus FV1000, Japan). Flow cytometric detection of cell cycle. KB-3-1 cells were seeded on a 6-well plate and preincubated for 24 h, followed by coincubation with taxol and H-PEG-F-T NPs (an equivalent taxol concentration of 100 µg/mL) for 6 h. The cells were then washed three times with PBS, detached by trypsinization, spun down by centrifugation, and dispersed again in PBS. The cells were fixed in PBS/70% ethanol (1:10) for 1 h and washed three times with PBS, and then stained by PI and RNase for 45 min. The suspended cells were analyzed by a FACSort flow cytometer. Statistical Analysis. Statistical analysis was performed to determine differences between the measured properties of each group. One-way analysis of variance was determined using a statistical program (Statistical Package for the Social Sciences, Version 10.0, SPSS Inc., U.S.A.). All data were performed in triplicate and presented as a mean value with its standard deviation indicated (mean ( SD).

Scheme 1. Designed Strategy of Nanoparticle Delivery System: (a) Synthesis Heparin-PEG-Folate Conjugate (H-PEG-F), (b) Heparin-PEG-Folate-Taxol Nanoparticles (H-PEG-F-T NPs)

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Results and Discussion Although we reported a heparin-paclitaxel nanoparticle delivery system could be accumulated to the solid tumor site in a passive targeting manner by an EPR effect,28 cell-specific targeting ability of heparin-based nanoparticle delivery system is highly desirable. The main goal of this work was to prepare a heparin-based nanoparticle delivery system with folate targeting moieties and examine its target-specific intracellular delivery capacity. Previous study suggested that the hydrophilic PEG shell can increase the solubility of the inner core containing water-insoluble drugs and protect the core from nonspecific interaction with blood serum components, minimizing the clearance rate by reticulo-endotheial system (RES).31-33 Herein, we modified heparin by succinic anhydride via an O-acylation reaction, wherein additional carboxyl groups introduced could increase the content of active groups and, more importantly, improve lipophilic property of heparin. To avoid producing PEG-bis-folate, Boc-PEG-NHS was then selected to conjugate with folate. After a deprotection reaction, the resulting PEGfolate contained a free amino group at the distal end of PEG for subsequent use in a succinylated-heparin conjugate. Due to the necessary chemical functionalization of heparin, the HPEG-F conjugate displayed an amphiphilic property capable of physically entrapping with hydrophobic anticancer drug taxol, thereby forming H-PEG-F-T NPs. The incorporation of folate at the distal end of PEG was expected to improve the specificity toward cancer cells overexpressing a folate receptor as well as enhance the cellular uptake of H-PEG-F-T NPs. We also investigated the influence of the different side chain length on formulation behavior in aqueous solution and cellular uptake extent. The designed strategy of nanoparticle delivery system is shown in Scheme 1. The typical 1H NMR spectra for H-PEG-F and H-PEG-F-T NPs in DMSO-d6 and D2O are presented in Figure 1. In two different solvents for H-PEG-F, 1H NMR spectra showed a peak signal at 2.5 ppm and the broad peaks from 3.4 to 3.5 ppm attributed to the succinylated group in heparin and protons for -CH2CH2O- in PEG, respectively. And the partial proton signals of heparin (3.2-3.3 ppm) could be observed in DMSO-d6, demonstrating the improvement of lipophilic property in carrier. Due to succinylated groups and PEG coeffect, it was noted that the structure of H-PEG-F could be detected in both solvents, indicating the formation of H-PEG-F displayed amphiphilic property. Meanwhile, despite the interference of heparin and PEG, the fraction of folate in low field at different solvents was observed (Figure 1a,b). The results stated the small molecule folate was successfully bonded to the succinylated-heparin via PEG spacer. Direct proofs confirming taxol entrapped into H-PEG-F came from Figure 1c,d. Partial structure of taxol can be identified at peaks from 5 to 9 ppm in the presence of DMSO-d6. Interestingly, in the spectrum taken in D2O as a solvent, the taxol peaks disappeared while the proton peaks at 6.6 and 7.5 ppm corresponded to folate. It suggested carrier was oriented outside with exposing a folate moiety in its distal end in aqueous solution. As a result, the H-PEG-F-T could self-assemble into core/shell type nanoparticle in aqueous solution. The weight percentage of succinylate groups in heparin was about 27% using 1H NMR analysis, implying that sufficient organic groups can raise lipophilic nature of heparin. The content of folate and taxol was quantitative analysis as determined using a UV spectrometric method. Figure 2 presents the UV spectra of H-PEG-F, H-PEG-F-T, and taxol (H-PEG-F-T-H-PEG-F). Obviously, the characterized peak of folate for H-PEG-F and

Figure 1. 1H NMR spectra of (a) H-PEG-F in DMSO-d6, (b) H-PEG-F in D2O, (c) H-PEG-F-T in DMSO-d6, and (d) H-PEG-F-T in D2O.

Figure 2. Quantitative analysis of folate and taxol content as determined by UV method.

H-PEG-F-T appeared at 280 and 360 nm. It was calculated the weight of folate for products via PEG1000 was about 2-fold greater than that of products via PEG3000, which was 1.6 and 0.92%, respectively. The taxol content was calculated by equation ABST ) ABSH-PEG-F-T - ABSH-PEG-F, assuming that the H-PEG-F-T in water and free drug in ethanol has the same molar extinction coefficients and that both followed Lambert-Beer’s law. The calculated weight of H-PEG-F-T NPs via PEG1000/3000 spacers contained 18 and 19% taxol, respectively. The encapsulation efficiency of NPs via PEG 1000/ 3000 was about 95 and 97%, indicating small part of taxol in DMSO was consumed during final treatment (Table 1). According to the amphiphilic property of the carrier, it was found that H-PEG-F conjugates had self-assembled behavior in aqueous solution by DLS, whereas their morphology could not be detected by SEM. Meanwhile, the mean diameters of

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Table 1. Folate and Taxol Loading Weight, Encapsulation Efficiency (w/w %), as Determined by UV Measurement, Size, and Zeta Potential, as Determined by DLS NPs folate loading weight (w/w %) taxol loading weight (w/w %) encapsulation efficiency (w/w %) size (nm; in PBS 7.4) zeta potential (mV)

H-PEG1000-F-T H-PEG3000-F -T 1.6 15.7 95 164 -21.7

0.92 16 97 190 -24

H-PEG-F-T NPs via PEG1000/3000 spacers were 164 ( 9 and 190 ( 5 nm, as measured by DLS, respectively (Figure 3). It might be the presence of the highly mobile and flexible PEG chain and hydrophobic agents introduced to carrier resulting in the formation of self-assembled nanoparticles. Moreover, with the increase of side chain the nanoparticle size ascended. Zeta potential values of H-PEG-F-T NPs were approximately -20 mV, suggesting that negative charge played a critical role in affecting aggregation of nanoparticles. To further investigate the morphology of the core/shell-type nanoparticle, SEM images of H-PEG-F-T NPs were taken (Figure 4). It clearly showed an approximately spherical shape of H-PEG-F-T NPs. Previously, Hashida and co-workers reported that the majority of fenestrates of the liver sinusoid have diameters smaller than 200 nm.34 Thus, large particles hardly reach the parenchymal cells of liver. It was also reported that smaller particles under 200 nm tend to selectively accumulate at tumor sites as a result of the EPR effect.35 Consequently, H-PEG-F-T NPs fall within the range required for cell-specific targeting and passive tumor targeting.

Figure 5. Anticoagulant activity of H-PEG-F conjugates and H-PEGF-T NPs, as determined by FXa assessment. The data represent mean ( SD where n ) 3.

To make sure of the safety and efficiency of the drug delivery system, the anticoagulant activities of H-PEG-F and H-PEGF-T NPs were assessed by FXa-dependent coagulant assay (Figure 5). The anticoagulant activity of H-PEG-F decreased to some extent as compared to that of heparin, while H-PEGF-T NPs decreased to a much greater extent. This might be attributed to the introduction of taxol, which leads to a conformational change in heparin structure, resulting in de-

Figure 3. Size distribution of (a) H-PEG1000-F-T NPs and (b) H-PEG3000-F-T NPs, as measured by DLS.

Figure 4. Scanning electron microscopy photographs of (a) H-PEG1000-F-T NPs, ×20000, and (b) H-PEG3000-F-T NPs, ×20000.

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Figure 6. Cellular uptake extends for KB-3-1 cells and A549 cells, (light blue) control, (burgundy) H-PEG1000-F, (purple) H-PEG3000-F, (red) H-PEG1000-F-T NPs, and (green) H-PEG3000-F-T NPs; (a,b) KB-3-1 cells were treated with H-PEG-F and H-PEG-F-T NPs under conditions 1 and 2; (c,d) A549 cells were treated with H-PEG-F and H-PEG-F-T NPs under conditions 1 and 2; (e,f) KB-3-1 cells were treated with H-PEG-F and H-PEG-F-T NPs under conditions 1 and 2 in the presence of 1 mM folate; control represents the fluorescence emitted from naked KB-3-1 and A549 cells; condition 1: the weight of product was fixed, and the content of folate in the product varied; condition 2: the content of folate in the product was fixed, and the weight of the product varied.

creased affinity to antithrombin III and reducing the effectiveness of the underlying anticoagulant mechanism.36 According to literature reports, cellular uptake could be affected by many factors, such as receptor-mediated endocytosis,37 particle size,38 different cell lines and cell densities,39 spacer length,40 surface properties (surface hydrophobic/hydrophilic balance, surface charge, and zeta potential),39,40 and so on. Although the particle size of H-PEG1000-F-T NPs is smaller than that of H-PEG3000-F-T NPs, PEG3000 has a longer chain length, easily enabling the binding of NPs with folate receptors on KB-3-1 cells. Therefore, in this case, we aimed to test the effects of folate receptor-mediated endocytosis, nanoparticles, and PEG chain length on cellular uptake.

To evaluate the effect of the above factors on cellular uptake extent, KB-3-1 cells and A549 cells were employed as folate receptor overexpressing cancer cells and folate receptor deficiency cancer cells treated with H-PEG-F and H-PEG-F-T NPs at the various experimental conditions: (1) when the weight of H-PEG-F and H-PEG-F-T NPs via PEG1000/3000 spacers was fixed to the same values, the content of folate in products via PEG1000 spacer was higher than that of products via PEG3000 spacer; (2) when the content of folate was set to the same values, the weight of products via PEG1000 spacer was lower than that of products via the PEG3000 spacer. The results are summarized in Figure 6: as a whole, the cellular uptake of H-PEG-F and H-PEG-F-T NPs was exceed-

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Figure 7. Confocal microscopic images of KB-3-1 cells after 40 min incubation with (a,d) Oregon green labeled with H-PEG1000-F-T NPs and H-PEG3000-F-T NPs (green), respectively; (b,e) Colabeled with DAPI (blue); (c,f) Merged confocal image: ×60.

ingly higher for KB-3-1 cells than for A549 cells, and two H-PEG-F conjugates showed similar efficiency for A549 cells due to the lack of folate receptor and nanoparticle effect. Under condition 1 (the folate content of products via PEG1000 spacer was higher than that of products via the PEG3000 spacer), both carrier and NPs via PEG1000 spacer displayed better cellular uptake efficiency than that via PEG3000 spacer for KB-3-1 cells, revealing they were mainly taken up by folate receptor-mediated endocyctosis (Figure 6a). Due to the formation of nanoparticles, H-PEG-F-T NPs had greater cellular uptake than H-PEG-F for A549 cells, and there was no obvious difference between the two H-PEG-F conjugates (Figure 6c). In comparison, at the fixed folate content (condition 2), H-PEG-F-T NPs revealed a far greater extent of cellular uptake than that of H-PEG-F conjugates against both cells, and H-PEG3000-F-T NPs showed better cellular uptake efficiency than any others, which was explained by the result of interaction between longer spacer length and nanoparticle effect (Figure 6b,d). In view of different influent factors, the cellular uptake of NPs via PEG1000 was close to that of H-PEG3000-F for both cells under condition 2. Taken together, the cellular uptake efficiency of H-PEG-F-T NPs and H-PEG-F conjugates relied on a synergistic relationship between folate-receptor mediated endocytosis, formation of the nanoparticle, and spacer length. To further examine the competition experiment in KB-3-1 cells, an excess amount of free folic acid (1 mM) was added to the medium at the two conditions (Figure 6e,f). The cellular uptake of H-PEG-F conjugates and H-PEG-F-T NPs was effectively suppressed in the presence of free folic acid. By comparison, H-PEG-F-T NPs could be easily delivered into KB3-1 cells due to the nanoparticle effect. It was worth noting that no matter what the folate concentration was, both NPs via PEG1000 and via PEG3000 exhibited the same efficiency at the equivalent nanoparticle amount (condition 1). However, cellular uptake of NPs via PEG3000 was higher than that of NPs via PEG1000 because the former had a larger nanoparticle amount (condition 2). According to the data, the factor that was the most influential was the nanoparticle effect on cellular uptake in competition experiment.

Also, cellular uptakes of H-PEG-F-T NPs via PEG1000/3000 by KB-3-1 cells were visualized by a confocal microscope as shown in Figure 7. After 40 min of incubation, it can be visualized the fluorescence of Oregon green labeled H-PEGF-T NPs (green) and the DAPI (blue). The Oregon green labeled H-PEG-F-T NPs were distributed in the cytoplasm more closely located around the nuclei stained by DAPI, which indicated the NPs could be taken up by the cells and located inside the cells.41 It was shown that H-PEG-F-T NPs were taken up by a receptormediated and nanoparticle endocytosis process, in accordance with the FACS results. Taxol is a unique antimicrotubule agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization during the late G2 mitotic phase of the cell cycle. To test the effect of H-PEGF-T NPs on cell cycle compared with that of taxol, KB-3-1 cells were treated for 6 h. A similar experiment was performed with drug-free taxol as a positive control. The cell cycle distribution revealed that only 12.39% of the cells were in the G2/M population increased to 18.11, 20.96, and 23.84% for taxol, H-PEG1000-F-T- and H-PEG3000-F-T-treated KB-3-1 cell, respectively. It showed that H-PEG-F-T block the cell cycle in phase G2/M, which was consistent with previous reports indicating taxol has been shown to cause cell cycle arrest in the G2/M phase and, ultimately, cell death through apoptotic mechanisms (Figure 8).42,43 Hence, it indicated that taxol introduced to H-PEG-F can restrict carcinoma cells in specific cell cycle stages.

Conclusions By the necessary chemical functionalization of heparin, we prepared amphiphilic carrier H-PEG-F, which is capable of entrapping anticancer drug taxol to develop a H-PEG-F-T targeted nanoparticulate delivery system. Flow cytometry analysis demonstrated that the cellular uptake efficiency of H-PEG-F-T NPs and H-PEG-F conjugates relied on a synergistic relationship between folate-receptor-mediated endocytosis, formation of nanoparticle, and spacer length. The present formula-

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Figure 8. Flow cytometric detection of apoptosis of KB-3-1 treated with taxol, H-PEG-F and H-PEG-F-T, for 6 h: (a) control cell, (b) taxol, (c) H-PEG1000-F-T NPs, and (d) H-PEG3000-T NPs. The data represent mean ( SD, where n ) 3.

tion could be used as a promising cancer cell specific delivery system for diverse anticancer agents entrapped with H-PEG-F conjugates. Acknowledgment. The authors thank Dr. Shuming Nie from the Department of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, for his assistance on this project. Meanwhile, flow cytometric assay was supported by the Core Facilities at Emory University.

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