RESEARCH ARTICLE Copyright © 2012 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Biomedical Nanotechnology Vol. 9, 1–8, 2012 Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Tu Uyen Ly 1† , Ngoc Quyen Tran 1 3† , Thi Kim Dung Hoang 1 , Kim Ngoc Phan 2 , Hai Nhung Truong 2 , and Cuu Khoa Nguyen 1 3 ∗ 1 Institute of Chemical Technology, Vietnam Academy of Science and Technology, HCMC 70000, Vietnam 2 Stem Cell Research and Application Laboratory, University of Science HoChiMinh City, HCMC 70000, Vietnam 3 Institute of Applied Materials Science, Vietnam Academy of Science and Technology, HCMC 70000, Vietnam Dendrimer, a new class of hyper-branched polymer with predetermined molecular weight, is being received much attention in nano biomedical applications such as anticancer drug delivery, gene therapy, disease diagnosis and etc. In this study, polyamidoamine (PAMAM)-based dendrimer gen- eration 3.0 (G 3.0) was synthesized and subsequently pegylated. Obtained results showed that pegylation degree of the dendrimer was around 31% for its external amine groups. TEM image of the pegylated dendrimer exhibited spherical shape and nano sizes ranging from 30 to 40 nm. The fluorouracil (5-FU)-loaded pegylated dendrimer showed a slow release profile of the drug. In vitro study, at the primary screening concentration of 100 g/mL, the PAMAM dendrimer pre- sented higher toxicity in MCF-7 cells as compared to its pegylated counterpart. Meanwhile, the (5-FU)-loaded pegylated dendrimer exhibited the antiproliferative activity against the cell line with the IC50 of 992 ± 019 g/mL. In vivo tumor xenograft study, we succeeded in generating MCF-7 cells-derived cancer tumors on mice that was well-confirmed by using flow cytometer assay. The 5-FU encapsulated pegylated dendrimer exhibited a significant decrement in volume of the tumors which was generated by MCF-7 cancer cells. Keywords: Fluorouracil, Pegylated Dendrimer, Anticancer, Drug Delivery. 1. INTRODUCTION Dendrimer is a family of hyper-branched polymers that was introduced in 1980’s by Donald A. Tomalia. Synthe- sis of the dendrimers involves a molecular growth process which occurs through step-wise polyester formation or Michael addition and amidation of multifunctional groups to issue layer of branches around the core. The reac- tions result in producing a precisely defined chemical structure of PAMAM or polyester dendrimer. 1 2 In the family, PAMAM is one of the most studied dendritic poly- mers. For the high-generation dendrimers (G3, G4, G5), these structures possess internal cavities which can be ultilized as a novel nanocarrier for anticancer drug delivery because drugs can be encapsulated via non-covalent inter- actions. Moreover, its externally exposed amine or car- boxylic groups could be decorated with targeting or/and ∗ Author to whom correspondence should be addressed. † These authors contributed equally to the work. drug molecules. 3 4 Several reports indicated that anticancer drugs (camptothecin, 6-mercaptopurin, methotrexate, adri- amycin, 5-fluorouracil, and paclitaxel) were encapsu- lated into the PAMAM dendrimer exhibiting a significant enhancement of its water solubility, storage stability, and anti-tumor activity. 5–10 However, there are a few disadvantages accompanied with PAMAM dendrimer drug-delivery system includ- ing hemolytic toxicity and cell lysis due to a strong interaction of the positively charged dendrimer and the negatively charged cell membrane resulting in mem- brane disruption. 9 11–13 Like other cationic polymers such as polylysine and poly(ethyleneimine), these disadvan- tages have been solved by conjugating biocompatible and hydrophilic polymers into amine group-terminated dendrimer. Up to now, polyethylene glycol (PEG) has been one of the best choices for the research. PEG is well-known that is highly water soluble, nontoxic and nonimmunogenic. The pegylation can help in reducing J. Biomed. Nanotechnol. 2012, Vol. 9, No. 2 1550-7033/2012/9/001/008 doi:10.1166/jbn.2012.1479 1 RESEARCH ARTICLE Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Ly et al. toxicity by preventing the contact between terminal protonated amine groups with cell membranes, resulting in improving their biocompatibility. The conjugation may lead to increase in the inner cavity space of dendrimers that contribute to increment of drug-loading capacity. 14 Moreover, pegylation of the drug nanocarriers can increase the residence time of the drug in blood circulation by its stealth properties in the blood plasma. These improving effects of pegylated nanocarriers were well-confirmed in several studies, both in vitro and in vivo. 8 914 15 Despite this, little effort has been made to evaluate treatment effi- cacy on implanted tumor tissue. In this article, we prepared the pegylated PAMAM den- drimer G3.0 for loading 5-fluorouracil anticancer drug. The in vitro and in vivo effectiveness against human breast cancer MCF-7 cell line of this complex was investigated using Sulforhodamine B colorimetric assay and xenograft technique, respectively. 2. MATERIALS AND METHODS 2.1. Materials Ethylenediamine (EDA), methyl methacrylate, and 5-fluorouracil were purchased from Merck Chemicals. Monomethoxy polyethylene glycol 5000 (MPEG-5000) was obtained from Sigma-Aldrich Co. p-nitrophenyl chlo- roformate (NPC) was purchased from Acros Organics. Fig. 1. Synthetic scheme of Pegylated PAMAM dendrimer G3.0. PAMAM dendrimers G3.0 were prepared in Organic Chemistry and Polymer laboratory 16 (Institute of Chemical Technology, Vietnam Academy of Science and Tech- nology) following the procedure reported by Tomalia 1 Regenerated Cellulose MWCO 3500-5000D and Cellulose Ester MWCO 10000D dialysis bags were purchased from Spectrum Laboratories Inc. All other chemicals were used without further modification. 2.2. Synthesis of Pegylated PAMAM Dendrimer G3.0 Activated MPEG-5000 was prepared similarly to the method described by Tran et al. Five grams of dried MPEG 5000 (1.0 mmol) was completely dissolved in dimethyl- formamide at 40 C and then reacted with p-nitrophenyl chloroformate (2.0 mmol) in the presence of triethylene amine under nitrogen atmosphere. The mixture was stirred overnight. The product was precipitated in excess diethyl ether to obtain a white powder of the activated MPEG. The product was then dried and used further synthesis. 17 A mixture of PAMAM G3.0 dendrimer (155.0 mg, 22.4 mol) and activated MPEG-5000 (4.63 g, 896.0 mol) in 30 mL of DMF was stirred under nitrogen atmosphere for 48 h (Fig. 1). The crude product was dialyzed (MWCO 10,000D) against water under strict sink conditions in 48 h. The product was then lyophilized and used for drug loading preparation. 2 J. Biomed. Nanotechnol. 9, 1–8, 2012 RESEARCH ARTICLE Ly et al. Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity 2.3. Drug Loading and In Vitro Release Evaluation 5-FU was loaded in pegylated PAMAM dendrimers fol- lowing the equilibrium dialysis method reported earlier. 9 The pegylated PAMAM dendrimer G3.0 (428.3 mg, 7.4 mol) was added to a nearly saturated solution con- taining 100 molar times of 5-FU (97.0 mg, 746 mol). The mixed solution was incubated under slow stirring (50 rpm) for 24 h. This solution was twice dialyzed under strict sink conditions in 20 min to remove free drug from the formulation, which was then estimated spectrophotometri- cally (265.5 nm) to determine indirectly the amount of drug loaded within the system. Optimal drug loading in the nanocarrier was determined on the same method. Briefly, pegylated dendrimer was dissolved in water at 0.75 mM polymer concentration. And then, an excess 5-FU was added to the pegylated dendrimer solution under slow stir- ring (50 rpm) for 24 h. The mixture was centrifugated (5000 rpm) to remove the amount of insoluble 5-FU. Fol- lowing the above method, the optimal drug loading could be determined within the system. The dialyzed formulation was lyophilized and used for further studies. The drug- loading (DL%) and entrapment efficiency (EE%) of 5-FU in pegylated dendrimer were calculated from the following equations. 18 19 DL% = Weight of 5-FU in nanocarrier Weight of 5-FU in nanocarrier × 100% EE% = Weight of 5-FU in nanocarrier Weight of feeding 5-FU × 100% For in vitro release study, the 5-FU-loaded pegylated den- drimer (260.6 mg) and 10 mL deionized water were added to dialyzer membrane (MWCO 3,500D). The aqueous con- taining membrane was dialyzed against 1000 mL deion- ized water. At a predetermined time interval, 10 mL of dialyzed solution was drawn to determine 5-FU release by absorbance measurement at wavelength 265.5 nm and another 10 mL of deionized water was added to the dia- lyzed solution to compensate for the withdrawn volume. The similar concentration of dendritic polymer solution without drug loading was dialyzed in the same condition to serve as control. 2.4. Cytotoxicity Assays The cell proliferation was measured using Sulforhodamine B (SRB) colorimetric assay. 20 The inhibition capability of cell growth of PAMAM, pegylated PAMAM, free 5-FU and pegylated PAMAM dendrimer 5-FU complex was esti- mated at the screening concentration of 100 g/mL. MCF- 7 cells (Frederick, MD, U.S.A) were seeded at a density of 10 4 cells per well in 96 well plates and allowed to grow in culture medium (DMEM/F12 containing 10% FBS, 1% antibiotics and 5% CO 2 atmosphere) overnight. These wells were then incubated with the medium containing tested compound for 48 h. A negative control was culture medium. Blank sample was culture medium containing compound but without cells. After the incubation period, the cells were fixed with 50% (wt/vol) trichloroacetic acid and stained with 0.2% (wt/vol) SRB for 20 min. The excess dye was removed by washing repeatedly with 1% (vol/vol) acetic acid. Finally, the protein-bound dye was dissolved in 10 mM Tris-base solution for optical density (OD) determination at 492 and 620 nm using a Multiskan Ascent Reader (Thermo Electron Corporation). The OD at certain wavelength was defined as the mean absorbance of tested wells minus the blank value. The OD value of each sample was subsequently calculated as the OD at 492 nm subtracting from the background measure- ment at 620 nm. The percentages of cell growth inhibition were calculated using the formula below. For IC50 estimation, cell viability was analyzed at different material concentrations using sulforho- damine colorimetric assay. Based on the dose–response curve between the compound concentration and growth inhibition percent, the IC50 values were subsequently determined using regression analysis. Experiments were performed in triplicates for each compound and each experiment was carried out at least twice. The values were expressed as means ± standard deviation (STD). 2.5. In Vivo Tumor Xenograft The study was conducted at Stem Cell Research and Application Laboratory, University of Science - Vietnam National University HCM City. Briefly, twenty Swiss mice of similar weights and sizes were used in this experi- ment. Before injecting MCF-7 human breast cancer cells, the mice were administered immunosuppressive drugs (20 mg/kg of Busulfan and 200 mg/kg of Cyclophos- phamide per day for five days) to suppress the immune system. The tumor bearing mice were created by sub- cutaneous injection of MCF-7 into mouse’s thigh at a same cell density (10 7 cells/animal). After two weeks, the animals (n = 12) that their tumors reached required and less changed volumes were divided into three individual groups. The first group served as control, the second group was given a dose of 10 mg/kg 5-FU per day, the other was given a dose of pegylated PAMAM dendrimer-drug complex with an equivalent amount of 10 mg/kg 5-FU per day. The animals were treated in 15 days and the changes of the tumor volumes were recorded during the treatment. The existence of MCF-7 cells in tumors was confirmed by flow cytometry using anti-HLA monoclonal antibody. All experimental procedures and manipulations were approved by our Institutional Ethical Committee (Laboratory of Stem cell Research and Application, University of Science, VNU-HCM, VN). J. Biomed. Nanotechnol. 9, 1–8, 2012 3 RESEARCH ARTICLE Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Ly et al. 2.6. Characterizations Nuclear magnetic resonance (NMR) data was collected using CDCl 3 as solvent on a Bruker AC 500 MHz spec- trometer. The average molecular weights of pegylated polymer was calculated from Gel Permeation Chromatog- raphy (GPC) technique using Agilent 1100-GPC system. Deionized water was used as an eluent at a flow rate of 1 mL/min through a Ultrahydrogel column. Peak analy- sis was performed basing on a universal calibration curve generated by a pulullan polysaccharide standard of nar- row polydispersity. For Transmission Electron Microscopy, pegylated dendrimer was dissolved in methanol at a certain concentration, placed on 300 mesh carbon-coated copper grid, and then air-dried for several hours. TEM images (TEM) were obtained at 100 kV with a JEM-1400 (JEOL) with magnifications up to 100,000×. 3. RESULTS AND DISCUSSION 3.1. Synthesis of Pegylated PAMAM G3.0 Dendrimer PEGylation was a common method to reduce immuno- genicity of proteins or drug nanocarriers. The hydroxyl group of MPEG chain could be activated by p-nitrophenyl chloroformate (NPC). The NPC-activated MPEG could be well confirmed by 1 H NMR, in which there were two dou- blets at 8.29–8.27 ppm and 7.41–7.39 ppm corresponding to the protons of the phenyl. The activation was also well- proved by the chemical shift of the protons (H c ) of the ter- minal CH 2 group that was originally attached to hydroxyl group towards lower frequency region, represented by a multiplet at 4.45–4.44 ppm (Fig. 2). Calculation from the integral ratio of the proton signals of the benzene ring (in the p-nitrophenyl group) and the signal of the protons of the terminal methoxy group in MPEG chain (at 3.40 ppm) showed that the conversion rate of MPEG to its activated form was nearly complete with a yield of about 92%. 21 The activated MPEG was utilized for pegylation of PAMAM dendrimer (Fig. 1). As conjugated to PAMAM, the signal of MPEG methylene protons next to the pre- viously activated group shifted from 4.45 to 4.18 ppm Fig. 2. 1 H NMR spectrum of the NPC-activated MPEG. Fig. 3. 1 H NMR spectrum of the pegylated PAMAM dendrimer G3.0. (Fig. 3). Besides appearance of typical peaks for MPEG methylene and methyl protons, PAMAM methylene pro- tons could be also presented in the Figure 3. The obtained GPC result demonstrated (Fig. 4) that the PEGylation degree was about 30% as estimated between Mw of PAMAM (6,909 g/mol) and its pegylation (57,800 g/mol; PDI = 14). Ten amine groups had been pegylated among total thirty two amine groups. It was said that the pegylated product could help in reducing toxic- ity by preventing the contact between terminal protonated amine groups with cell membranes, resulting in improv- ing its biocompatibility. Moreover, it can contribute to the stealth properties in the blood plasma and improve the specificity of the pharmacodynamic action. 22 23 The pegylation of dendrimer could be well-defined by TEM in which its morphology is spherical shaped and diameter ranging from 30 nm to 40 nm (Fig. 5). There is a significant size increment of the pegylated PAMAM dendrimer as compared to the original PAMAM dendrimer G3.0 (diameter under 5 nm; data not shown here) Structural characterization of the pegylated dendrimer showed an increment of nanoparticles size that can lead to increase in the inner cavity space of the dendrimers and contribute to increment of drug-loading capacity. Fig. 4. GPC result of the pegylated PAMAM dendrimer G3.0. 4 J. Biomed. Nanotechnol. 9, 1–8, 2012 RESEARCH ARTICLE Ly et al. Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Fig. 5. TEM image of the pegylated PAMAM dendrimer G3.0. 3.2. Drug Loading and In Vitro Release The drug formulation was prepared by incubating the pegylated PAMAM dendrimer with a saturated 5-FU solu- tion. The amount of drug that had been loaded was calcu- lated indirectly from the amount of unbound drug which was determined spectrophotometrically at the wavelength of 265.5 nm. In this study, the loading efficiency was about 30% (as shown in Table I), calculating approxi- mately 30 drug molecules that were found to be encapsu- lated within each pegylated PAMAM dendrimer molecule structure. Moreover, the optimal drug loading was deter- mined around 35% as shown in Table I. The table also showed that entrapment efficiency of 5-FU in the pegylated dendrimer didn’t significantly increase as used of excess 5-FU. According to drug loading (DL%) and entrapment efficiency (EE), we thought that use of pegylated den- drimer for loading a saturated 5-FU solution may be more effect than loading 5-FU saturated in the pegylated den- drimer solution. Signal of 5-FU proton (7.61–7.62 ppm) could be observed in 1 H NMR spectra of drug loaded dendrimer after the free drug was removed using dialysis mem- brane (data not shown). Therefore, this could be concluded that 5-FU was simply physically entrapped inside pegy- lated PAMAM dendrimer cavities. Release profile of the encapsulated drug molecules under strict sink conditions is shown in Figure 6. The release profile shows an initial burst release of 40% 5-FU from the drug-loaded dendrimer within the first hour of the experiment. After that time, the drug slowly releases Table I. Drug-loading (DL%) and entrapment efficiency (EE%) in pegylated dendrimer (n = 3). Samples EE% DL% PAMAM + saturated 5-FU solution 30.15 ± 1.27 5.54 ± 0.32 Saturated 5-FU in PAMAM solution 34.75 ± 3.71 10.65 ± 0.78 Fig. 6. Release profile of 5-FU from the drug-loaded dendrimer. from the system and reaches to more than 84% released at 24 hours. This behavior is very significant to prolong drug bioavailability because 5-FU anticancer drug was reported to have a short remaining time in blood circulation. The drug can be excreted or metabolized 95% out of blood plasma after one hour of administration. 9 24 3.3. Cytotoxicity Assay and IC50 The antiproliferative activities of PAMAM, pegylated PAMAM, free 5-FU drug and 5-FU-loaded pegylated PAMAM on MCF-7 cells were expressed indirectly via the cellular protein content that could electrostatically bound to sulforhodamine B dye molecules in the assay. The obtained results showed cytotoxicity of PAMAM and its pegylated derivative were negligible at the experimental condition (shown in Table II). However, the result obvi- ously showed pegylation can reduce the cytotoxic ability of PAMAM. Meanwhile, the percentages of cell growth inhibition of the two free and entrapped 5-FU samples at the primary screening concentration were too high. There- fore, IC50 estimation assays were conducted to assess accurately the cytotoxicity. The IC50 values were deter- mined at 105 ± 0 19 g/mL and 992 ± 019 g/mL for 5-FU free drug and entrapped 5-FU samples, respectively. It was not surprising that the free drug seemed to be nine Table II. In vitro cytotoxicity of PAMAM, pegylated PAMAM, free 5-FU drug and 5-FU encapsulated pegylated PAMAM on MCF-7 cancer cell. Sample Concentration Antiproliferative activity PAMAM 100 g/ mL Inhibited 14.06 ± 1.42% cell growth Pegylated PAMAM 100 g/ mL Inhibited 6.36 ± 1.42% cell growth Free 5-FU 1.05 ± 0.19 g/ mL Inhibited 50% cell growth 5-FU encapsulated 9.92 ± 0.19 g/ mL Inhibited 50% cell growth pegylated PAMAM J. Biomed. Nanotechnol. 9, 1–8, 2012 5 RESEARCH ARTICLE Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Ly et al. times more effective than the encapsulated drug because the drug content in the pegylated carrier was quite low. The total thirty drug molecules per pegylated PAMAM den- drimer molecule werewas only equivalent to around 6.5% (wt/wt). Therefore, it could be concluded that 5-FU loaded inside the pegylated dendrimer still maintained an signifi- cantly antiproliferative activity on the MCF-7 cancer cell. The result could be attractive to studies on drug nanocar- rier and its utilization for many toxic anticancer drugs. 3.4. In Vivo Tumor Xenograft Results Before using in vivo experiments, the cell suspension was analyzed with flow cytometer which confirmed being MCF-7 breast cancer cell using anti-HLA monoclonal anti- body. Figures 7(a) and (b) showed that the cells suspension was predominantly MCF-7 cell with 97.51% of popula- tion. The cells in the generated cancer tumor via xenograft assay were re-collected and determined integrin expres- sion again by flow cytometer to confirm that they were predominantly MCF-7 cells (Fig. 7(c)). After two week of implantation, the MCF-7 cells easily generated cancer tumors on each mouse’s thigh (Fig. 8(a)). Those cancer tumors were then exploited to evaluate tumor-killing effi- cacy of the pegylated dendrimer loading 5-FU. To remove the errors caused by the inconsistence in initial tumor vol- umes, and the variation in physiological reponses from the treatment, the in vivo efficacy was evaluated on the aver- age percentage decrease in tumor volume. The obtained results showed that the tumor volume on mice decreased as treated 5-FU and the 5-FU loaded in the pegylated den- drimer (Figs. 8(b and c)). In comparison among the three studied groups, group treated 5-FU encapsulated pegylated PAMAM dendrimer gave the best treatment results. The tumor volumes were decreased gradually over the time, and the maximum decreasing percentage was found after 15 days and it was 82.87% that was far exceeding result of the free 5-FU treated group, which was decrement in tumor vol- umes being 42.29%. Meanwhile, the control group had increment in tumor volumes approximately 21.58%. The decrement of tumor volumes is a result from anticancer activity of 5-FU, a commercial anticancer drug that is administrated for long-term treatment of cancer patients. The tumor-killing efficacy in the three groups over the study time is illustrated in Figure 8(d). 5-FU encapsulated the pegylated PAMAM dendrimer and showed a higher efficacy of tumor killing in comparison with free 5-FU treatment. This could be explained that 5-FU encapsulated in pegylated PAMAM dendrimer prolonged drug bioavail- ability due to a slow drug release. It is well-known that 5-FU anticancer drug being a short remaining time in blood circulation, 95% out of blood plasma after one hour of administration. 9 22 Figure 8(c) also shows that a high standard deviation can be seen in all studied groups. This Fig. 7. Expression of HLA-DR on MCF-7 cells. Flow cytometer analy- sis of the MCF-7 cells at SSC versus FSC histogram (a), HLA-DR FITC versus count (b) and the tumor-isolated MCF-7 cells were determined by flow cytometer using anti-HLA antibody (c) SSC: Side Scatter, FSC: Forward Scatter. 6 J. Biomed. Nanotechnol. 9, 1–8, 2012 RESEARCH ARTICLE Ly et al. Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Fig. 8. Results of xenograft assay: mouse with generated cancer tumor (a), mouse with tumor after treated with the 5-FU loaded dendrimer (b) and decrement percentage of tumor volumes (c). (n = 4 ± SD). is due to an initial difference in tumor volumes among mice that could be improved by increment in amount of the studied mice. To further clarify the tumor-killing effect of the drug-loaded pegylated dendrimer, administrative treat- ments at various dose levels and time interval for the treated doses as well as a larger amount of the studied mice are going on study. 4. CONCLUSION The pegylated PAMAM dendrimer was prepared and well- defined in structure. Drug-loaded dendrimer was formu- lated, wherein the 5-FU drug molecules were physically entrapped in the cavities of the structure of pegylated PAMAM dendrimer. The drug-loaded pegylated dendrimer maintained subtantial antiproliferative activity of free drug on the MCF-7 cell line in vitro, and furthermore showed a significant improvement in the anti-tumor activity as com- pared to controls without drug and with free drug treatment in vivo. The obtained results may contribute to further studies and applications in killing cancer cell of tumors by using the nanocarrier. Acknowledgments: This work was financially sup- ported by Material Science and Technology project of Vietnam Academy of Science and Technology. References and Notes 1. D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, and P. Smith, A new class of polymers: starburst- dendritic macromolecules. Polym. 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Revised/Accepted: 25 June 2012. 8 J. Biomed. Nanotechnol. 9, 1–8, 2012 . al. Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor Activity Fig. 5. TEM image of the pegylated PAMAM dendrimer G3.0. 3.2. Drug Loading and In Vitro. lyophilized and used for drug loading preparation. 2 J. Biomed. Nanotechnol. 9, 1–8, 2012 RESEARCH ARTICLE Ly et al. Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing. reserved Printed in the United States of America Journal of Biomedical Nanotechnology Vol. 9, 1–8, 2012 Pegylated Dendrimer and Its Effect in Fluorouracil Loading and Release for Enhancing Antitumor