Bioconjugate Chem. 2005, 16, 391−396
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Polymer-DNA Hybrid Nanoparticles Based on Folate-Polyethylenimine-block-poly(L-lactide) Chau-Hui Wang†,‡ and Ging-Ho Hsiue*,† Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 300 Taiwan, ROC, and Polymer Technology Division, Union Chemical Laboratories, Industrial Technology Research Institute, Hsinchu, 300 Taiwan, ROC. Received October 13, 2004; Revised Manuscript Received January 25, 2005
The ability of amphiphilic block copolymers that consist of polyethylenimine (PEI) and poly(L-lactide) (PLLA) to modulate the delivery of plasmid DNA was evaluated. Folate-polyethylenimine-blockpoly(L-lactide) (folate-PEI-PLLA) was synthesized by linking folic acid and PLLA to PEI diamine. Water-soluble polycation PEI provides gene-loading capability. Additionally, PEI is considered to exhibit high transfection efficiency and endosomal disrupting capacity. Hydrophobic PLLA that is incorporated into the gene delivery vector is believed to enhance the cell interactions and tissue permeability of the delivery system. Polymeric carrier containing folic acid is expected to be able to identify tumor surface receptors and transfect cells by receptor-mediated endocytosis. The results of agarose retardation assay indicated that the folate-PEI-PLLA began to form polyplexes at a polymer/DNA weight ratio (P/D) of over 10, whereas branched polyethylenimine (B-PEI) formed polyplexes with DNA at a ratio of above 1. The spherical particle morphology was supplemented with a particle size of approximately 100 nm at 10 P/D ratio. The results indicated that folate-PEI-PLLA with proper PEI/PLLA ratio effectively reduced cytotoxicity and maintained acceptable transfection efficiency. Low cytotoxicity of the folate-PEI-PLLA gives an advantage to high-dose administration.
INTRODUCTION
Nonviral gene carriers are considered to be safer to administer and to be able to be designed with more versatility than viral-gene carriers. Nonviral gene vectors such as cationic lipids and cationic polymers have been employed to deliver DNA, RNA, and oligonucleotides (ODNs) into cells (1-3). Branched polyethylenimine (BPEI) is a polycation often used to condense DNA through electrostatic interactions. B-PEI exhibited high transfection efficiency, probably because it has a high buffering capacity. The proton sponge effect of B-PEI gives the polyplexes an endosomal disrupting capability, thus enhancing gene expression (4). On the other hand, linear polyethylenimine (L-PEI), which is synthesized by hydrolyzing poly(2-substituted-2-oxazoline), has been shown to have lower cytotoxicity but higher transfection efficacy than B-PEI (5). Fully hydrolyzed L-PEI is water-insoluble because it is crystalline. The water-soluble L-PEI can be prepared by the controlled acid hydrolysis of poly(2-ethyl2-oxazoline) (6). PEI was also modified to be multifunctional by introducing polymer segments or targeting moieties to extend and enhance the release of the gene (7, 8). Biodegradable polyesters such as poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) have been demonstrated to encapsulate DNA for sustained gene expression (9-11). Park et al. conjugated antisense ODN to biodegradable PLGA to yield an amphiphilic structure, which could form micelles in the aqueous phase (12). Furthermore, hydrophobic moieties are considered to improve the cell interactions and increase the tissue permeability of * To whom correspondence should be addressed. E-mail:
[email protected]. † National Tsing Hua University. ‡ Industrial Technology Research Institute.
delivery systems. Kissel et al. prepared hyperbranched polyethylenimine-g-poly(-caprolactone)-b-poly(ethylene glycol) (hy-PEI-g-PCL-b-PEG) as gene vectors, and a high gene transfection efficiency was obtained by properly controlling the compositions of copolymers (13). Pluronics could also be incorporated into cell membranes as a result of the presence of the hydrophobic poly(propylene oxide) (PPO) chain (14, 15). In this study, folate-polyethylenimine-block-poly(Llactide) (folate-PEI-PLLA) was prepared as a new gene carrier. Partial hydrolysis of poly(2-ethyl-2-oxazoline) (PEOz) yielded water-soluble L-PEI with a high buffering capacity. PLLA and folic acid were conjugated to L-PEI to increase the transfection efficiency and reduce the cytotoxicity. The amphiphilic folate-PEI-PLLA copolymers condensed plasmid DNA and formed micelles in aqueous solution. The micelle core comprised PLLA segments and hydrophobic complexes. Moreover, the folate receptors were overexpressed on carcinomas (16, 17). Polymeric carrier that contained folic acid was expected to be able to identify tumor surface receptors and transfect cells by receptor-mediated endocytosis (18). Receptor targeting, endosomal disruption, improved cell permeability, and reduced cytotoxicity were combined for designing this nonviral gene vector. Synthesis, electrophoresis, surface morphology, and luciferase gene expression of folate-PEI-PLLA polyplexes were investigated in this work. EXPERIMENTAL SECTION
Materials. L-Lactide (Aldrich) and 1,4-dibromo-2butene (Aldrich) were recrystallized from acetone and n-hexane, respectively. 2-Ethyl-2-oxazoline (Aldrich) was purified by vacuum distillation over CaH2. Acetonitrile and benzyl alcohol were dried over CaH2 and distilled under dry nitrogen. Folic acid and 3-(4,5-dimethylthiazol-
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2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from ICN Biomedicals, Inc. N,N′-Dicyclohexylcarbodiimide (DCC) was purchased from Lancaster Synthesis Ltd. Succinic anhydride and 4-(dimethylamino)pyridine (DMAP) were purchased from TCI Co., Ltd. pUHC 13-3 encoding for luciferase as a reporter gene was a gift from Prof. Jia-Ling Yang of National Tsing Hua University, Taiwan. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were the products of Gibco-BRL. Folate-free DMEM was purchased from Sigma. HeLa human adenocarcinoma cells were provided by Prof. Yu-Chen Hu of National Tsing Hua University, Taiwan. Dialysis tubing (MWCO 3500) was purchased from Spectrum Medical Industries, Inc. Synthesis of Polyethylenimine (PEI) Diamine. Poly(2-ethyl-2-oxazoline) (PEOz) was synthesized by cationic ring-opening polymerization of 2-ethyl-2-oxazoline using 1,4-dibromo-2-butene as an initiator. A solution of 1,4-dibromo-2-butene (210 mg, 0.982 mmol) and 2-ethyl2-oxazoline (10 mL, 98.86 mmol) in dry acetonitrile (30 mL) was heated to 100 °C and stirred for 16 h in an atmosphere of nitrogen. The living PEOz polymer chains were terminated by adding acetonitrile solution that contained NH3 (0.12 N) at 0 °C; the solution was then stirred for 30 min. The polymer solution was filtered through the silica gel and was purified by precipitation in diethyl ether. After it had been completely dried, a powdery product was obtained. The molecular weight and polydispersity of PEOz diamine were analyzed by taking gel permeation chromatography (GPC) measurements. PEOz diamine (2 g) was heated with a mixture of concentrated hydrochloric acid (11 mL, 35%) and water (16 mL) at 110 °C for 3 h. The pH of the polymer solution was adjusted to 9-10 by adding NaOH pellets. The product was then purified by dialysis for 3 days using cutoff dialysis tubing with a molecular weight of 3500. After it had been vacuum-dried, the product was stored at -20 °C. 1H NMR (D2O): δ 0.92 (N(COCH2CH3)CH2CH2), 2.25 (N(COCH2CH3)CH2CH2), 2.69 (NHCH2CH2), 3.40-3.50 (N(COCH2CH3)CH2CH2). Synthesis of Monocarboxypoly(L-lactide) (PLLACOOH). L-Lactide (4 g, 27.75 mmol) and benzyl alcohol (216 mg, 2 mmol) were polymerized with 1 wt % stannous octoate in dry toluene (2 mL) at 140 °C for 16 h under nitrogen. After being allowed to cool, the product was filtered using a syringe filter (0.22 µm) and then precipitated in n-hexane. The yielded PLLA-OH was vacuumdried for 24 h. PLLA-OH (1 g, 0.4 mmol), succinic anhydride (80 mg, 0.8 mmol), and DMAP (97.6 mg, 0.8 mmol) were dissolved in 1,4-dioxane (7 mL) and reacted in an atmosphere of nitrogen for 24 h at room temperature. The product was precipitated in n-hexane and reconstituted using tetrahydrofuran. After 0.1 N of HCl had been added, the polymer solution was poured into a large amount of distilled water. The precipitates were dissolved in tetrahydrofuran and then purified by precipitation in nhexane. 1H NMR (CDCl3): δ 1.42 (COCH(CH3)O), 3.75 (COCH2CH2COOH), 4.35 (COCH2CH2COOH) 5.19 (COCH(CH3)O), 7.38 (C6H5). Synthesis of Folate-Polyethylenimine-block-poly(L-lactide) (folate-PEI-PLLA). PEI diamine (500 mg, 0.167 mmol), PLLA-COOH (208 mg, 0.083 mmol), DMAP (40.8, 0.33 mmol), and DCC (69 mg, 0.33 mmol) were dissolved in DMSO (10 mL). The reaction was carried out in a nitrogen atmosphere at room temperature. After 24 h, folic acid (7.4 mg, 0.017 mmol), DMAP (40.8, 0.33 mmol), and DCC (69 mg, 0.33 mmol) were added into the
Wang and Hsiue
mixture under nitrogen, and reacted at room temperature for a further 24 h. The unreacted PLLA-COOH and folic acid were purified by dialysis (with a molecular weight cutoff of 3500) in DMSO and distilled water for 3 days, respectively. Following lyophilization, the obtained folatePEI-PLLA was stored at -20 °C. Preparation of Polyplexes. A 5 µg amount of luciferase encoding plasmid pUHC-13-3 and the desired amount of polymer were each diluted in 100 µL of distilled water and gently mixed. The polyplex formulation was incubated at room temperature for 30 min before it was used. The particle size was analyzed by dynamic light scattering (DLS, Malvern Instrument Series 3000). Gel Retardation Assay. Various formulations of polyplexes were prepared as previously described, before loading into the gel. The samples were electrophoresed on 1% agarose gel in 1X TRIS-boric acid-EDTA (TBE) buffer at 100 mV until the 1X loading dye ran through 80% of the gel. A 1 kb DNA marker (Violet) used for comparing the size of DNA was also run. The gel was stained with 0.5 µg/mL ethidium bromide for 45 min and analyzed using an UV transilluminator. Atomic Force Microscopy. The surface morphology of polyplexes was analyzed by atomic force microscopy (AFM). The polyplex solution was diluted in 1 mL of distilled water and dropped on a silica wafer. After it was spin-coated with a spin coater, the sample was dried in the dry box for 2 days and in vacuo for 30 min before use. Luciferase Transfection and Total Protein Assay. Human cervix carcinoma HeLa cells (2 × 105 cells/well) plated in six-well plates were incubated in DMEM media supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. Transfection was conducted after the cells had become about 80% confluent. A 2 mL amount of growth medium that contained the DNA or polyplexes with a final amount of 5 µg of plasmid was incubated for 48 h at 37 °C. Following transfection, the cells were washed with cold PBS and then lysed with 400 µL of 1X cell lysis buffer. The cell lysate was then transferred into eppendorf tubes and centrifuged for 1 min at 12 000 rpm. Luciferase activity was measured in terms of relative light unit (RLU) using a 96-well plate luminometer (Wallac 1420 Multilabel Counter, Perkin-Elmer) and a luciferase assay kit (Promega). Total protein was measured at 595 nm using a BioRad protein assay. In Vitro Cell Viability. Human cervix carcinoma HeLa cells (1 × 104 cells/well) were cultured onto a plate with 96 wells in DMEM media supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. The growth medium was replaced with a medium that contained the desired amount of polymers. Cells were incubated for 24 h, and cell viability was assayed by adding 100 µL of medium that contained 10 µL of MTT PBS solution (5 mg/mL). After incubation for 4 h, the formazan crystals were dissolved in 100 µL of DMSO. The absorbance of each well was measured using a microplate reader (Stat Fax 2100, Awareness) at a test wavelength of 570 nm and a reference wavelength of 630 nm. RESULTS AND DISCUSSION
Synthesis of Folate-PEI-PLLA. The PEOz was synthesized by cationic living polymerization with a bifunctional initiator 1,4-dibromo-2-butene (19). The reaction was terminated by adding NH3 acetonitrile solution to introduce NH2 functional groups to both ends
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Figure 1.
1H
NMR spectrum of folate-PEI-PLLA.
Scheme 1. Synthesis of Folate-PEI-PLLA by the DCC-Mediated Polycondensation Reaction
of polymer chains (20). The number average molecular weight (Mn) and polydispersity (PDI) determined by GPC were 9800 and 1.18, respectively. Acid hydrolysis of the PEOz diamine was performed using hydrochloric acid. The percentage hydrolysis was estimated by comparing the integral peak area of the ethylenimine groups to N-propionylethylenimine groups. The molecular weight following hydrolysis was calculated from the percentage hydrolysis. The L-lactide and benzyl alcohol were polymerized with stannous octoate. PLLA-OH was allowed to react with succinic anhydride in the presence of DMAP to prepare PLLA with a carboxyl group at the end of the chain (21). The Mn determined by GPC was 2500, and the PDI was 1.3. The amphiphilic folate-PEI-PLLA copolymers were synthesized by the DCC-mediated polycondensation reaction of two oligomers: PEI diamine and PLLA-COOH (Scheme 1). The composition of folate-PEI-PLLA was varied to optimize the gene expression. Figure 1 presents the 1H NMR spectrum of folate-PEI-PLLA. The molar ratio of PLLA to PEI was determined by the ratio between of the area under the peak of the methyl group in the PLLA block to that of the ethylene groups in the PEI block. The folic acid content in folate-PEI-PLLA was measured using a UV/vis spectrophotometer. Folate-PEI-
Table 1. Characterizations of B-PEI, L-PEI, and Folate-PEI-PLLA code
Mn (PEI)
B-PEI 10000 L-PEI 5200 FEA1 5600 FEA2 12600 FEA3 3600
hydrolysis PEI/PLLA folic acid particle size (%) (mol) (mmol/g) (nm) 84 75 66 85
0.90 0.93 0.41
0.1100 0.0750 0.0421
68 154 156 166 110
PLLA was dissolved in DMSO, and the absorbance at a wavelength of 289 nm was determined. The concentration of folic acid was calculated according to the calibration curve made from free folic acid. Both R- and γ-carboxyl groups on the folic acid have been reported to undergo coupling reactions, and only the γ-conjugate can bind to the folate receptor. The DCC-mediated polycondensation reaction involves about 80% γ-linked and 20% R-linked conjugates (22). Table 1 characterizes B-PEI, L-PEI, and folate-PEI-PLLA. The DLS analyses of the folate-PEIPLLA and the DNA showed that no particle was formed before mixing them in solution. Gel Retardation Assay. Agarose gel electrophoresis was performed to assess the influence of B-PEI, L-PEI, and folate-PEI-PLLA on DNA condensation. Figure 2 presents the gel retardation results for the polyplexes. Increasing the concentrations of the polymers reduced the electrophoretic mobility of the plasmid, by increasing
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Figure 2. Gel retardation assay. Lane 1: DNA ladder, Lane 2: plasmid DNA, Lane 3: B-PEI/DNA polyplex, Lane 4: L-PEI/ DNA polyplex, Lane 5: FEA1/DNA polyplex, Lane 6: FEA2/ DNA polyplex, Lane 7: FEA3/DNA polyplex.
Figure 4. Luciferase gene expressions of FEA1, FEA2, FEA3 polyplexes at various P/D ratios (n ) 3).
Figure 3. Atomic force microscopy (AFM) image of the folatePEI-PLLA polyplexes (FEA3, P/D: 10).
DNA condensation and compaction. The B-PEI and L-PEI are completely retarded as can be seen as the polymer/ DNA (P/D) ratio (w/w) approaches 1 and 6, respectively. The results of agarose retardation assay indicated that the DNA condensing capacity of folate-PEI-PLLA was slightly decreased. The folate-PEI-PLLA began to form polyplexes at a P/D ratio of above 10. Atomic Force Microscopy. The surface morphology of folate-PEI-PLLA polyplex was determined by atomic force microscopy (AFM). Figure 3 shows an AFM image of folate-PEI-PLLA polyplexes (FEA3) spread on the silica wafer. The spherelike particles were around 100 nm in diameter at a P/D ratio of 10. In Vitro Transfection. The transfection efficiencies of B-PEI, L-PEI, and folate-PEI-PLLA polyplexes prepared at various P/D ratios on HeLa human cervix carcinoma were evaluated. Transfection efficiency was determined by luciferase assay for the luciferase encoding plasmid. The total protein production, which was an indirect measure of induced toxicity, was analyzed to normalize the RLU. Figure 4 shows the luciferase gene expression of folate-PEI-PLLA at various P/D ratios on HeLa cells. The change in RLU is directly proportional to the amount of luciferase produced in the transfected cells. For a given amount of plasmid DNA (5 µg), increasing the amount of polymers increased the transfection efficiency. This result indicated that only FEA3 exhibited extensive luciferase gene expression. FEA1 and FEA2, which have relatively high PLLA contents, did not exhibit improved transfection efficiency. Figure 5 com-
Figure 5. Luciferase gene expressions of the B-PEI, L-PEI, and FEA3 polyplexes at various P/D ratios (n ) 3).
Figure 6. The highest level of luciferase expression in naked DNA, B-PEI, L-PEI, and folate-PEI-PLLA and their total protein productions (n ) 3). B-PEI (P/D: 3), L-PEI (P/D: 3), FEA3 (P/D: 7).
pares the luciferase gene expressions of B-PEI, L-PEI, and FEA3 polyplexes at various P/D ratios. For B-PEI and L-PEI polyplexes, luciferase expression in HeLa cells is stronger at a P/D ratio of above 3. FEA3 polyplex showed greater transfection at a P/D ratio of 7. Figure 6 shows the highest level of luciferase expression in naked DNA, B-PEI, L-PEI, and folate-PEI-PLLA. The results reveal that the appropriate gene carrier exhibited greater transfection efficacy than naked DNA. B-PEI and L-PEI
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is depends partly on the corresponding toxicity of the polymer. The low cytotoxicity of folate-PEI-PLLA allows it to be safely administered. CONCLUSION
Figure 7. The luciferase gene expression of FEA3 polyplexes at different concentrations of free folic acid (n )3).
In this work, a new polymeric gene carrier, folate-PEIPLLA, was successfully synthesized. The introduction of PLLA and folic acid was expected to increase cell permeability and biocompatibility. However, polymers that contain relatively large amounts of high PLLA mediated gene transfer less efficiently than did B-PEI and L-PEI. Folate-PEI-PLLA effectively reduced toxicity on HeLa cells but also reduced the luciferase expression. Additionally, the morphology of folate-PEI-PLLA polyplex was spherical, thus unlike that obtained from the B-PEI polyplex. B-PEI formed a polyplex with DNA as an oblique sphere, which has been suggested to be able to enter the cell easily (24). Although biodegradable polyesters are commonly used to deliver plasmid DNA, their transfection efficiency compared with PEI has been rarely discussed. The results herein reveal that folate-PEIPLLA with a proper PEI/PLLA ratio effectively reduced cytotoxicity and maintained an acceptable transfection efficiency. Low cytotoxicity of the folate-PEI-PLLA gives the advantage for the high dose amount of administration. ACKNOWLEDGMENT
The authors thank Prof. Jia-Ling Yang at the Institute of Biotechnology of National Tsing Hua University for the assistance with luciferase expression assay and the Industrial Technology Research Institute of Taiwan (ITRI) for financial support. LITERATURE CITED Figure 8. The cytotoxicity of B-PEI, L-PEI, and FEA3 on HeLa cells at various polymer concentrations (n ) 6).
showed better gene expression than that of FEA3. In contrast, the protein production of FEA3 was similar to that of naked DNA, indicating that FEA3 had low cytotoxicity. Folate-PEI-PLLA exhibited less transfection than B-PEI and L-PEI, so determining whether free folic acid suppresses transfection is important. It is not certain that HeLa cells have folate receptors. However, a number of publications have indicated that the folate receptors on HeLa cells are up-regulated (>15 × 106 folate binding sites/HeLa cell) (23). As shown in Figure 7, free folic acid competed with FEA3 polyplexes in transfection. The common DMEM media contains 0.004 mg/mL of folic acid. The luciferase expression increased as the free folic acid concentration declined. The total protein production was maintained at about 22 µg, revealing that free folic acid did not affect cytotoxicity, and considerably inhibiting the endocytosis of FEA3. Cell Viability. The in vitro cytotoxicity of B-PEI, L-PEI, and FEA3 at various concentrations was determined in the HeLa cell line by MTT assay. The wells that contained only the media and untreated polymer were regarded as positive controls, with a cell viability of 100%. The relative cell viability was determined as [Abs]sample/ [Abs]control × 100. Figure 8 reveals that B-PEI exhibited high toxicity, resulting in cell death. In contrast to B-PEI and L-PEI, the cell viability of FEA3 exceeded 80% at a concentration of 0.5 mg/mL. The efficacy of a gene carrier
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