ARTICLE pubs.acs.org/bc
Galactosylated N-2-Hydroxypropyl Methacrylamide-b-N-3Guanidinopropyl Methacrylamide Block Copolymers as Hepatocyte-Targeting Gene Carriers Zhu Qin, Wei Liu, Ling Li, Liang Guo, Chen Yao, and Xinsong Li* Biomaterials and Drug Delivery Laboratories, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China ABSTRACT: As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may provide flexible chemistry for the attachment of targeting moieties. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) were prepared in order to develop hepatocyte targeting gene transfection carriers. The block copolymers were synthesized by aqueous reversible additionfragmentation chain transfer (RAFT) polymerization of N-2-hydroxypropyl methacrylamide (HPMA) and N-3-aminopropyl methacrylamide (APMA), followed by galactosylation and guanidinylation. The molecular weight of GHG copolymers determined by static light scattering method was in the range from 48 600 to 76 240 g/mol. In addition, the galactose content in the GPMA block in the copolymers was determined to be 6.58.0 mol % according to the sulfuric acid method. The GHG copolymers complexed completely with plasmid DNA (pDNA) to show positive zeta-potential values with diameter 100250 nm from charge ratio of 4, which demonstrated the excellent DNA condensing ability of guanidino groups. Furthermore, the MTT assay data of GHG/pDNA complexes on HepG2 cells and HeLa cells indicated that GHG copolymers had significantly lower cytotoxicity than PEI. In addition, the copolymers with GPMA component from 30.23% showed higher transfection efficiency than PEI at charge ratio of 12 in HepG2 cells. The result revealed that the conjugation of galactose groups in the copolymers brought asialoglycoprotein-receptor (ASGP-R) mediated transfection. The employing of HPMA component decreased the aggregation of protein in transfection presence of serum. The GHG copolymers combined the advantages of galactose moieties, guanidino groups, and HPMA component might show potential in safe hepatocyte targeting gene therapy.
’ INTRODUCTION Nonviral gene delivery carriers based on cationic polymers have attracted much attention facilitating the entry of plasmid DNA (pDNA) or siRNA into cells in the past decade. As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may have advantages such as safety and stability, avoidance of immunogenicity, and ease of structure modification and manufacturing practice.13 N-(3-Aminopropyl)methacrylamide hydrochloride (APMA), a primary amine containing monomer with the backbone of methacrylate, was potentially useful in the development of gene delivery carriers of cationic polymers.4 N-(2-Hydroxypropyl) methacrylamide (HPMA), a permanently hydrophilic monomer with the similar methacrylamide backbone, can be copolymerized with APMA to shield the surface charge of the latter and create a steric barrier against aggregation. It is shown that APMA-HPMA copolymers were efficient carriers for delivery of anticancer agents in the literature.57 Aqueous reversible additionfragmentation chain transfer (RAFT) technology is a living radical polymerization process, which is a powerful method for the synthesis of well-defined polymers for biomedical applications. Compared with traditional radical polymerization, recent advances in controlled radical r 2011 American Chemical Society
polymerization (CRP) especially RAFT polymerization have facilitated the synthesis of narrowly dispersed copolymers with controlled molecular weight, polymer architecture, and reactive structopendant or structoterminal functionality for bioconjugation.812 Furthermore, Narain and McCormick have reported the aqueous RAFT polymerization method for the direct preparation of well-defined primary amine-based homopolymers, block and statistical copolymers, without protecting group chemistry.13,14 It may be a significant step toward the tailored construction of well-defined copolymers for targeted gene delivery.15 Gene delivery to a very specific set of cells might be highly demanded in cancer therapies. Cationic polymers generally do not have the capacity for cell-specific targeting but provide flexible chemistry for the attachment of targeting moieties that allow both increased cell uptake and, often, cell specificity. A wide range of targeting moieties, such as folate,16 epidermal growth factor,17 transferrin,18 or antibodies 19 have been conjugated to the residues of side chains or the backbone of cationic polymers. It is reported that Received: November 24, 2010 Revised: May 18, 2011 Published: June 20, 2011 1503
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Table 1. Composition for the Preparation of HPMA-b-APMA Block Copolymer (HA) by Aqueous RAFT Polymerization, and Galactosylated HPMA-b-GPMA Copolymer (GHG) by Galactosylation and Guanidinylation HPMA-b-APMA block copolymer (HA)
galactosylated HPMA-b-GPMA block copolymer (GHG)
target DPa content of
actual content
sample
HPMA
APMA
of primary amino groupsb, mol %
actual content sample
galactosec in feed, mol %
of galactosec,d, mol %
MWe, g/mol
HA1
60
30
16.75
GHG1
10
6.5
76 240
HA2 HA3
60 60
60 120
30.23 41.20
GHG2 GHG3
10 10
7.2 6.5
68 000 69 600
HA4
30
150
55.60
GHG4
10
8.0
48 600
a
The [M]0/[CTA]0 ratio was equal to the target degree of polymerization (DP), while the [CTA]0/[ACVA]0 ratio was kept at 2/1. b As determined by the Ninhydrin assay. c Divided by the content of GPMA block in copolymers. d As determined by sulfuric acid micromethod. e As determined by a static light scattering instrument, dn/dc = 0.17 mL/g.
the asialoglycoprotein receptors (ASGP-R) of hepatocytes could recognize and internalize glycoproteins bearing terminal galactose or N-acetylglucosamine residues via clathrincoated pits.20 Therefore, the employments of lactose21,22 and galactose2325 as conjugation partners with cationic polymers have been studied to deliver gene into liver parenchymal cells. Zanta et al. and Cook et al.’s reports revealed that 5% galactose conjugated to PEI could show high transfection efficiency in hepatocyte-derived cell lines.26,27 Cell-penetrating peptides (CPPs) such as Tat and other arginine-rich peptides has been reported that they can be taken up by cells via other receptor- or protein-independent transmembrane pathway than endocytosis and localize in the nucleus28,29 in the past several years. A common feature of these peptides is that they consist of a large number of positively charged amino acids, especially arginine.3032 Compared with primary amino groups (pKa = 10.8), the stronger basic guanidino group of arginine with pKa of 12.5 may provide greater DNA condensing capability. The guanidino groups conjugated to linear poly(L-lysine) (PLL),33 chitosan,34 poly(propylene imine) (PEI),35 and poly(amido amine) (PAMAM) dendrimer,36 have showed greatly enhanced transfection efficiency than unmodified amino ones. Furthermore, Koo et al. reported that poly(L-arginine) (PArg) possessed both higher transfection efficiency and lower cytotoxicity than PLL, and PArg could be promising as an efficient gene delivery carrier.37 Therefore, it is supposed that changing the primary amino groups of cationic polymer to guanidino groups should be beneficial for enhancing cellular uptake and transfection efficiency of gene delivery. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) was developed as a novel nonviral gene transfection vector. They were synthesized by aqueous RAFT polymerization of HPMA and APMA, followed by galactosylation for hepatocyte targeting and guanidinylation for cell penetrating. The complexes of galactosylated HPMA-b-GPMA block copolymers with plasmid DNA were investigated by agarose gel electrophoresis, particle size, and zeta potential measurements. In addition, the cytotoxicity, and transfection experiment of GHG/ pDNA complexes were examined in HepG2 cells and HeLa cells.
’ EXPERIMENTAL PROCEDURE Materials. N-3-Aminopropyl methacrylamide hydrochloride (APMA) and N-2-hydroxypropyl methacrylamide (HPMA) were purchased from PolySciences. 4,40 -Azobis(4-cyanovaleric acid) (ACVA), β-lactose, sodium cyanoborohydride, 1-H-pyrazole-1-carboxamidine hydrochloride, and branched PEI (MW 25 000) were purchased from Sigma-Aldrich. 1,4-Dioxane, acetone, boric acid, sodium borate, and Na2CO3 were purchased from Sinopharm (China). 4-Cyanopentanoic acid dithiobenzoate (CTP) was synthesized in our lab. The Endo-Free Plasmid Maxi Kit was purchased from Omega (China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, trypsin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Gibco. The plasmid pGL3-control luciferase reporter vector and Luciferase assay system were purchased from Promega. BCA protein assay kit was purchased from Biocolor (China). Syntheses of HPMA-b-APMA Block Copolymers (HA) (13, 15). A series of N-2-hydroxypropyl methacrylamide-b-N-3-aminopropyl methacrylamide (HPMA-b-APMA) block copolymers was prepared by aqueous RAFT polymerization at 70 °C, employing 4,40 -azobis(4-cyanovaleric acid) (ACVA) as the radical initiator and 4-cyanopentanoic acid dithiobenzoate (CTP) as the RAFT chain transfer agent (CTA) with the feeds as cited in Table 1. [M]0 was defined as the initial monomer concentration. The [M]0/[CTA]0 ratio was equal to the target degree of polymerization (DP), while the [CTA]0/[ACVA]0 ratio was kept at 2/1. The typical synthesis was as follows: HPMA (7.0 mmol) was dissolved in double distilled water (2 mL) before the addition of CTP (0.12 mmol, target DP = 60) and ACVA (0.06 mmol) in 1,4-dioxane stock solution. After degassing via three freezethaw cycles, the flask was placed in a preheated oil bath for 24 h at 70 °C. The reaction was then quenched by cooling the reaction vessel in an ice bath and exposure to air. The obtained HPMA homopolymer was purified by precipitation in acetone. Then, the HPMA homopolymer was subsequently used as macro-CTA. A typical protocol described for the copolymerization of HPMA to APMA was as follows: In a 5 mL flask, APMA (1.2 mmol), HPMA homopolymer (0.02 mmol, target DP = 60), and ACVA (0.01 mmol, dissolved in dioxane) were mixed with double distilled water (2 mL). The copolymerization protocol was the same as the homopolymerization of HPMA described previously. The HPMA-b-APMA block copolymers were obtained as white powder. The content of primary amino group which 1504
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Bioconjugate Chemistry represented the ratio of APMA in the copolymer was determined by the Ninhydrin assay38 as indicated in Table 1. The composition of the copolymers was determined by 1H NMR and 13C NMR as follows. 1H NMR (300 MHz, D2O): δ (ppm) 0.991.20 (CH2CH(OH)CH3, CH2C(CH3)CO), 1.801.92 (CH2C(CH3)CO, CH2CH2CH2NH2), 3.08 (CH2CH2CH2NH2), 3.24 (CONHCH2CH(OH)CH3, CONHCH2CH2CH2NH2), 3.94 (CH2CH(OH)CH3). 13C NMR (125 MHz, D2O): δ (ppm) 19.50 (CH2CH(CH3)CH2), 22.80 (CH2CH(OH)CH3), 28.70 (NHCH2CH2CH2NH2), 39.6340.04 (CONHCH2CH2, CH2CH2CH2NH2), 47.80 (CONHCH2CH(OH)CH3), 49.72 (CH2C(CH3)CO), 56.66 (CH2C(CH3)CO), 68.21 (CH2CH(OH)CH3), 181.81 (CdO). Galactosylation of HPMA-b-APMA Block Copolymers. Galactosylated HPMA-b-APMA block copolymers (GHA) were synthesized by reductive amination reaction.26,27 Briefly, HPMAb-APMA block copolymers (127 μmol of primary amino groups) and β-lactose (12.7 μmol) were dissolved in 1 mL of sodium borate buffer (200 mM, pH 8.2), followed by the addition of sodium cyanoborohydride (5-fold molar excess of lactose). Then, the reaction mixture was stirred at room temperature for 24 h. The obtained galactosylated copolymers were purified by dialysis against distilled water (MWCO = 7000) and lyophilized. The content of galactose in the APMA block in galactosylated HPMA-b-APMA block copolymers was determined by sulfuric acid micromethod.39 Guanidinylation of Galactosylated HPMA-b-APMA Block Copolymers. The residue primary amino groups (13 mM) of galactosylated HPMA-b-APMA block copolymers were guanidinylated with 1H-pyrazole-1-carboxamidine hydrochloride (65 mM) in water which was adjusted to pH 9.0 with Na2CO3 at room temperature for 24 h.35 Then, they were extensively dialyzed against distilled water (MWCO = 7000) and lyophilized. The obtained copolymers can be named as galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG). The absolute molecular weights of the GHG copolymers were determined by a static light scattering instrument (BI-200SM, Brookhaven, USA).The chemical compositions of GHG were estimated by 1H, 13C NMR as follows. 1H NMR (300 MHz, D2O): δ (ppm) 0.961.20 (CH2CH(OH)CH3, CH2C(CH3)CO), 1.79 (CH2C(CH3)CO, CH2CH2CH2NHC(NH)NH2), 2.64 (CH2CH2CH2NHCH2CH2(OH)), 2.82 (NHCH2CH2(OH)), 3.21 (CONHCH2CH(OH)CH3, CONHCH2CH2CH2NH, CH2CH2CH2NHC(NH)NH2), 3.583.78 (CH2(OH) and CH(OH) in galactose), 3.94 (CH2CH(OH)CH3), 4.56 (CH(OCH)(CH2OH)OCH in galactose). 13 C NMR (125 MHz, D2O): δ (ppm) 19.24 (CH2CH(CH3)CH2), 22.23 (CH2CH(OH)CH3), 29.18 (NHCH2CH2CH2NH), 40.0841.65 (CONHCH2CH2, CH2CH2NHC(NH)NH2), 47.77 (CONHCH2CH(OH)CH3), 52.02 (CH2C(CH3)CO), 55.51 (CH2C(CH3)CO), 63.69 (CH2(OH) in galactose), 68.20 (CH2CH(OH)CH3), 71.1577.95 (CH(OH) in galactose), 113.57 (CH(OCH)(CH2OH)OCH in galactose), 159.50 (CH2NHC(NH)NH2), 181.93 (CdO). The guanidinylation of HPMA-b-APMA block copolymers without galactose groups were also prepared, which were named as HPMA-b-GPMA block copolymers, or abbreviated as HG. The guanidinylation was detected by 1H, 13C NMR as follows. 1H NMR (300 MHz, D2O): δ (ppm) 0.941.19 (CH2CH(OH)CH3, CH2C(CH3)CO), 1.79 (CH2C(CH3)CO, CH2CH2CH2NHC(NH)NH2), 3.21 (CONHCH2CH(OH)CH3, CONHCH2CH2
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CH2NH, CH2CH2CH2NHC(NH)NH2), 3.92(CH2CH(OH) CH3). 13C NMR (125 MHz, D2O): δ (ppm) 18.80 (CH2 CH(CH3)CH2), 22.00 (CH2CH(OH)CH3), 29.37 (NHCH2 CH2CH2NHC(NH)NH2), 39.7041.3 (CONHCH2CH2, CH2 CH2NHC(NH)NH2), 47.74 (CONHCH2CH(OH)CH3), 49.70 (CH2C(CH3)CO), 56.29 (CH2C(CH3)CO), 67.99 (CH2CH(OH)CH3), 159.21 (CH2NHC(NH)NH2), 181.62 (CdO). Agarose Gel Electrophoresis. DNA condensing ability of galactosylated HPMA-b-GPMA block copolymers (GHG) was examined by agarose gel electrophoresis, HPMA-b-GPMA block copolymers (HG) was employed as controls. In this assay, the charge ratios were presented as the molar ratio of the positively charged guanidino groups of GHG to negatively charged phosphate backbone of DNA. GHG/pDNA complexes were formed at a series of charge ratios. In each case, an appropriate amount of GHG dissolved in PBS (pH = 7.2) was mixed with 0.2 μg of pDNA to a volume of 6 μL, and incubated at room temperature for 30 min. After adding 1 μL of loading buffer, each complex solution was loaded into a well for electrophoretic separation on 1.0% agarose gel with Trisboric acid EDTA (TBE) running buffer and electrophoresced at 100 V for 10 min. The visualization of DNA was illuminated with ethidium bromide (0.2 μg/mL). Particle Size and Zeta Potential Measurements. The average sizes and zeta potential values of GHG/pDNA and HG/ pDNA complexes were examined by a dynamic light scattering instrument (90Plus/BI-MAS, Brookhaven, USA) equipped with a HeNe laser (632.2 nm, fixed scattering angle of 90°) at 25 °C. For these experiments, GHG or HG/pDNA complexes were prepared in PBS (pH = 7.2) at charge ratios (+/) of 1, 4, 8, 12, and 16. After 30 min incubation, complex solutions were diluted to a final volume of 1.5 mL containing a final pDNA concentration of 10 μg/mL. Measured sizes and zeta potential values were presented as the average values of triplicate. Cell Culture. HepG2 (human hepatocellular liver carcinoma cells) and HeLa (human cervix epithelial carcinoma cells) were cultured in DMEM medium, supplemented with 10% heat inactivated FBS, and antibiotics (10 U/mL penicillin and 10 μg/mL streptomycin). Cells were incubated at 37 °C in a 5% CO2 humidified atmosphere and split using trypsin when almost confluent. Cytotoxicity. The cytotoxicity of galactosylated HPMA-bGPMA block copolymers (GHG) was measured by MTT assay. HPMA-b-GPMA block copolymers (HG) were also evaluated in comparison with GHG, and PEI was used as a control. HepG2 cells and HeLa cells were seeded at a density of 104 cells/well in 96-well plates and grown in 100 μL DMEM medium containing 10% FBS for 24 h at 37 °C. After treating cells with GHG/pDNA complexes (0.1 μg pDNA) at different charge ratios for 6 h in 50 μL serum- and antibiotic-free DMEM medium, the medium was replaced with DMEM medium containing 10% FBS and the cells were incubated for 48 h. After this period, 20 μL of MTT solution (5 mg/mL in PBS) was added and the cells were further incubated for 4 h at 37 °C. Subsequently, medium were removed carefully and 150 μL of DMSO was added to each well to dissolve the formazan crystal formed by proliferating cells. UV absorbance was measured at 490 nm using a microplate reader (model 680, Bio-Rad, USA). The cell viability was calculated as a percentage of complex-treated cells to untreated negative control cells. All experiments were performed in sextuplicate. 1505
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Scheme 1. Synthesis of Galactosylated HPMA-b-GPMA Block Copolymer (GHG)
Transfection Experiments. The transfection efficiency of galactosylated HPMA-b-GPMA block copolymers (GHG) was measured by luciferase expression assay using plasmid PGL3 luciferase vector as a reporter gene. HPMA-b-GPMA block copolymers (HG) were also evaluated in comparison with GHG, and PEI was used as a control. HepG2 cells and HeLa cells were seeded at a density of 104 cells/well in 96-well plates and incubated in 100 μL DMEM medium containing 10% FBS for 24 h to 9095% confluence. Before transfection, the growth medium was removed from each well, and the cells
were washed twice with serum- and antibiotic-free medium. Then, cells were treated with GHG/pDNA complexes (0.1 μg pDNA) at different charge ratios for 6 h in 50 μL serum- and antibiotic-free DMEM medium. After exchange with DMEM medium containing 10% FBS, cells were further incubated for 48 h at 37 °C. Then, the medium was removed and the cells were washed twice with PBS before adding 20 μL Cell Culture Lysis reagent. The luciferase assay was carried out according to the manufacture’s instruction (Promega, USA). The luciferase activity expressed as relative light units (RLUs) was 1506
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measured with a luminometer (Chameleon V L, Hidex, Finland). The protein concentration in the cell lysates was measured by BCA protein assay kit. The final results were reported in terms of RLU/mg cellular protein. All experiments were performed in triplicate. Transfection experiments in the presence of serum were conducted in DMEM medium containing 10% FBS with the same procedure as above.
’ RESULTS AND DISCUSSION Syntheses of Galactosylated HPMA-b-GPMA Block Copolymers. Galactosylated HPMA-b-GPMA block copolymers
(GHG) were synthesized by aqueous RAFT copolymerization of HPMA and APMA, followed by galactosylation and guanidinylation as described in Scheme 1. A series of HPMA-b-APMA block copolymers HA1, HA2, HA3, and HA4 were synthesized with different target degree of polymerization (DP) as listed in Table 1. In order to control the molecular weight of HPMA-bAPMA block copolymers, 4-cyanopentanoic acid dithiobenzoate (CTP) was used as chain transfer agent. The content of primary amino groups which represented the ratio of APMA in the copolymer was determined by the Ninhydrin assay38 as indicated in Table 1. The concentration of primary amine groups in the copolymers was less than that present in the feed indicating disparate monomer reactivities. Partial primary amino groups of HPMA-b-APMA block copolymers were further modified by galactosylation for hepatocyte targeting through reductive amination reaction,26,27 and subsequently, the residue primary amino groups of galactosylated HPMA-b-APMA block copolymers were totally guanidinylated with 1H-pyrazole-1-carboxamidine hydrochloride under very gentle condition. The chemical composition of the galactose groups in galactosylated HPMA-b-GPMA block copolymers (GHG) were determined by 1H and 13C NMR measurements. The peaks at 3.583.78 and 4.56 ppm in the 1H NMR spectra of GHG were assigned to the protons from 2-carbon to 6-carbon and 1-carbon of galactose, respectively. The peaks at 63.69, 71.1577.95, and 113.57 ppm in the 13C NMR spectra of GHG were assigned to the carbon of 6-carbon, 2-carbon to 5-carbon, and 1-carbon in galactose, respectively. The quantitative composition of the galactose group to the content of GPMA block in galactosylated HPMA-b-GPMA block copolymers was determined to be 6.58.0 mol % as indicated in Table 1 by the sulfuric acid method.39 As literature reported that more than 5% galactose conjugation could show high transfection efficiency in hepatocyte-derived cell lines,26,27 the obtained galactosylated copolymers could be used as ASGP-R targeting gene carriers. The guanidinylation of residue primary amino groups was also established by 1H and 13C NMR spectroscopy. Attention was given to the region 3.03.4 ppm in the 1H NMR spectra of HA and GHG copolymers, where the proton peak of methylene next to the primary amino groups of HA copolymer was shifted from 3.08 ppm to 3.21 ppm as the guanidinylation completed.35 The disappearance of the methylene peak at 3.08 ppm indicated that the residue primary amino groups were thoroughly replaced by the guanidino ones. Furthermore, in the 13C NMR spectra of HA and GHG copolymers, the guanidinylation of primary amino group is demonstrated by the appearance of a new peak at 159.50 ppm corresponding to the C atoms of the guanidino groups. The results indicated that the residue primary amino groups of galactosylated HPMA-b-APMA block copolymers were
Figure 1. Agarose gel electrophoresis images of (A) GHG/pDNA and (B) HG/pDNA complexes at different charge ratios.
Figure 2. Average particle sizes of (A) GHG/pDNA and (B) HG/ pDNA complexes at different charge ratios examined by the dynamic light scattering instrument.
converted to guanidino groups of galactosylated HPMA-b-GPMA block copolymers after a total guanidinylation. The absolute molecular weights of galactosylated HPMA-bGPMA block copolymers GHG1, GHG2, GHG3, and GHG4 were in the range 48 60076 240 g/mol determined by static light scattering method as shown in Table 1. Agarose Gel Electrophoresis. Agarose gel electrophoresis was performed in order to examine pDNA condensing ability of galactosylated HPMA-b-GPMA block copolymers (GHG), using HPMA-b-GPMA block copolymers (HG) as controls. The results revealed that the complete complex formation occurred at charge ratios 3, 3, 2, and 0.5 for GHG1, GHG2, GHG3, and GHG4 with 16.75%, 30.23%, 41.20%, and 55.60% GPMA component in the GHG copolymers, respectively, as shown in Figure1. Meanwhile, the complete complex formation 1507
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Figure 3. Zeta potential values of (A) GHG/pDNA and (B) HG/ pDNA complexes at different charge ratios examined by the dynamic light scattering instrument.
occurred at charge ratios 3, 0.5, 0.5, and 0.5 for HG1, HG2, HG3, and HG4 copolymers, respectively. Since the guanidino group (pKa = 12.5) is fully protonated at physiological pH, all the GHG and HG copolymers can condense pDNA efficiently via electrostatic and hydrogen bond interactions between the strong positive charges of guanidino groups and negative charges of pDNA phosphates. As indicated in Figure 1, GHG1 and HG1 completely complexed with pDNA at charge ratio of 3. It is suggested that the presence of HPMA component in the GHG and HG copolymers provides the main shielding effect on the complex formation. Compared with HG2 and HG3, GHG2 and GHG3 condensed pDNA completely at relatively higher charge ratios. It is supposed that the abundant hydroxyl groups in the galactose group also enhanced the shielding effect. GHG4/pDNA and HG4/pDNA complexes both formed at charge ratio of 0.5; it might be that the component of guanidino groups took the dominant position in more than half the copolymers. Therefore, the HPMA and galactose components in galactosylated HPMA-b-GPMA block copolymers play an important role in the efficient formation of GHG/pDNA complex. Average Sizes and Zeta Potential Measurements. The average particle size of GHG/pDNA and HG/pDNA complexes prepared in PBS (pH 7.2) with different charge ratios is measured by dynamic light scattering as shown in Figure 2. The results indicated that the copolymers can condense pDNA sufficiently to form particles with the diameter less than 300 nm at charge ratio from 1 to 16. In addition, the particle size of the complexes showed the tendency to decrease with the increase in charge ratio and GPMA component in the copolymers. While the charge ratio rose to 12, the particle sizes of GHG/pDNA and HG/pDNA
Figure 4. Cytotoxicity of (A) GHG/pDNA and (B) HG/pDNA complexes at different charge ratios in HepG2 cells by MTT assay (branched PEI 25K as a control).
complexes reduced to less than 200 nm. It is revealed that GHG4 and HG4 with 55.60% GPMA component in the copolymers had the best DNA condensing ability, which formed particles with the smallest diameter of nearly 100 nm at charge ratio of 16. The particle sizes of cationic polymer/gene complexes are very important for gene expression, and the GHG/pDNA complexes with diameters of 100250 nm from the charge ratio of 4 may be to the satisfaction of this prerequisite. It is also found that the average particle sizes of GHG/pDNA complexes showed no significant difference by comparison with the corresponding HG/pDNA. The zeta potential values of GHG/pDNA and HG/pDNA complexes were shown in Figure 3. It is revealed that GHG1/ pDNA and GHG2/pDNA complexes with 16.75% and 30.23% GPMA component in the copolymers displayed positive values at the charge ratio more than 1, suggesting that the negatively charged pDNA was packed into the complex completely, while GHG3/pDNA and GHG4/pDNA complexes with 41.20% and 55.60% GPMA component in the copolymers showed positive surface values even at charge ratio of 1, demonstrating the strong pDNA condensing ability of the guanidino groups. Also, each GHG/pDNA complex had its highest zeta potential value at the charge ratio of 16, indicating complete shielding of DNA negative charges and condensed particulates with smallest size (Figure 2). 1508
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Figure 5. Cytotoxicity of (A) GHG/pDNA and (B) HG/pDNA complexes at different charge ratios in HeLa cells by MTT assay (branched PEI 25K as a control).
Figure 6. Transfection efficiencies of (A) GHG/pDNA and (B) HG/ pDNA complexes at different charge ratios in the absence of serum measured by luciferase expression assay in HepG2 cells (branched PEI 25K as a control).
Furthermore, the GHG4/pDNA complex reached the positive peak value of 18.27 at charge ratio of 16, which was consistent with the corresponding smallest particle size of 104.2 nm as shown in Figure 2. Apparently, the positive surface charge of the complexes increases gradually with the increasing GPMA component and charge ratios. Compared with HG/pDNA complexes, the zeta potential values of GHG/pDNA complexes showed no obvious decrease, indicating that the hydroxyl groups of galactose groups did not make sense in the decreasing surface potential values. The positive charge of cationic polymer/pDNA complexes is thought to be helpful for its absorption to negatively charged cellular membrane, also leading to efficient intracellular trafficking and influencing transfection efficiency.40 So, GHG/pDNA complexes with positive surface charge from the charge ratio of 4 may be advantageous for absorption onto cellular membrane. Cytotoxicity. Cytotoxicity of GHG/pDNA and HG/pDNA complexes was examined using HepG2 and HeLa cells as shown in Figures 4 and 5. PEI, the most commonly used gene carrier, was involved for comparison. The results revealed that GHG and HG copolymers exhibited less cytotoxicity than PEI at charge ratios from 4 to 16 in both cell lines, which suggested better cell viability for the application of gene delivery. All the complexes had obviously less cytotoxic effect on HepG2 cells than HeLa cells at the same charge ratios, which is supposed to be the different dependency between cell lines. In each cell line, it is found that the cytotoxicity of complexes increases with increasing charge ratio. Compared with GHG/pDNA and HG/pDNA complexes at the same charge ratio, there seems to be no
significant difference in HeLa cells. However, in HepG2 cells, GHG3/pDNA and GHG4/pDNA complexes with 41.20% and 55.60% GPMA component in the copolymers showed better cell viability than corresponding HG/pDNA complexes. It is considered to be the reduction of GHG concentration in medium by receptor-mediated endocytosis to HepG2 cells through ASGPR,26 whereas the HG copolymers without galactose groups remaining in medium bring more cytotoxic effects. Bulmus41 reported that polyHPMA could be noncytotoxic or cytotoxic depending on the choice of RAFT agent as the interplay between polymer and RAFT end-group can be a significant factor in cytotoxicity. However, the toxic effect of dithiobenzoateended polyHPMA could be eliminated at low concentrations (e200 μM). Since the concentration of the galactosylated HPMA-b-GPMA copolymers with the end-group dithiobenzoate of the RAFT agent in DMEM medium was much less than 200 μM in the cytotoxicity experiments conducted in this paper, the interplay between the copolymer and the RAFT end-group could have no significant contribution to the cytotoxicity, and the positively charged GPMA component might be the key factor in the cytotoxicity of this experiment. Transfection Experiments. The transfection of galactosylated HPMA-b-GPMA block (GHG) copolymers/pDNA complexes was performed on HepG2 expressing high levels of ASGP-R and HeLa cells lacking ASGP-R using plasmid PGL3 luciferase vector as a reporter gene. HPMA-b-GPMA block copolymers (HG) were also evaluated in comparison with GHG, and PEI was used as a control. Complexes of GHG 1509
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Figure 8. Transfection efficiencies of GHG/pDNA complexes at different charge ratios in the presence of serum (10% FBS) measured by luciferase expression assay in HepG2 cells (branched PEI 25K as a control).
Figure 7. Transfection efficiencies of (A) GHG/pDNA and (B) HG/ pDNA complexes at different charge ratios in the absence of serum measured by luciferase expression assay in HeLa cells (branched PEI 25K as a control).
and HG copolymers with pDNA were prepared at charge ratios of 4, 8, and 12, respectively. The charge ratios employed for the transfection efficiency experiments were within the range applied for the cytotoxicity evaluation as shown in the cytotoxicity section above. The transfection efficiency of GHG and HG copolymers was increased with the increase of the content of guanidino groups in the copolymers as shown in Figure 6 and Figure 7. GHG4 and HG4 with 55.60% GPMA component showed the highest transfection in both cell lines, whereas GHG1 and HG1 with 16.75% GPMA component revealed much lower transfection efficiency. The results demonstrated that the guanidino groups of the GPMA block brought a significant increase of transfection efficiency analogous to CPP mediated internalization28,30 The data shown in Figure 6 and Figure 7 also revealed that the transfection efficiency of GHG and HG copolymers was intensively influenced by the targeting groups and charge ratios. At charge ratio of 4, the transfection efficiency of GHG and HG was much higher than that of PEI control in HepG2 cells and HeLa cell. However, as charge ratio increased, most GHG copolymers in HeLa cells and HG copolymers in both cells exhibited less transfection than PEI, except HG4 with 55.6% GPMA component at charge ratio of 12 in HepG2 cells. Notably, as GHG copolymers in HepG2 cells were taken into account, GHG copolymers with guanidino groups from 30.23% revealed similar or higher transfection than that of PEI at charge ratios of 8 and 12. Furthermore, GHG4 exhibited the highest efficiency of 5 108 RLU/mg
protein at charge ratio of 12. The results can be attributed to the conjugation of galactose content in the copolymers which induced the ASGP-R mediated transfection in hepatocyte-derived cell lines. It confirmed that the galactose groups enhanced the transfection efficiency as targeting moieties. The transfection efficiency was dependent on the cell lines and cytotoxicity as well. The best transfection of GHG and HG copolymers in HeLa cells occurred at a magnitude of 107 RLU/ mg protein and charge ratio of 4 (Figure 7). As charge ratios increased, the transfection of CHG and GH copolymers was sharply reduced, which might be inhibited by the relative low cell viability of 70% at charge ratio from 4 to 12 (Figure 4) showed increasing transfection efficiency with the increase of charge ratios. In addition, the highest transfection efficiency of GHG and GH copolymers with magnitude of 108 RLU/mg protein was revealed at charge ratios of 12 (Figure 6). The GHG/pDNA and HG/pDNA complexes in HeLa cells showed relatively higher cytotoxicity than in HepG2 cells at the same charge ratios. Therefore, the different tendency of transfection efficiency in HepG2 and HeLa cells balanced the cytotoxicity contributed to the dependence of cell lines. Figure 8 showed the transfection efficiency of GHG/pDNA complexes in the presence of serum (10% FBS) in HepG2 cells at different charge ratios. The results indicated that the transfection efficiency of all the complexes decreased after addition of serum in the medium. In addition, the down regulation of transfection efficiency was reduced as the HPMA component in the copolymers increased. GHG2 and GHG3 with HPMA components of 58.80% and 69.77% had relatively high transfection efficiency. It is suggested that the hydrophilic HPMA component prevents aggregation of protein in the serum on the complexes before being taken up by cells. All the GHG/pDNA complexes showed higher transfection efficiency than PEI/pDNA complexes at charge ratios as indicated in the experiments. It is suggested that GHG copolymers have a potential for gene delivery in vivo as well as in vitro.
’ CONCLUSIONS A series of galactosylated HPMA-b-GPMA block (GHG) copolymers were synthesized by aqueous RAFT polymerization of HPMA and APMA, followed by partial galactosylation for 1510
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Bioconjugate Chemistry hepatocyte targeting and total guanidinylation for cell penetration. The GHG copolymers combined completely with pDNA to form complexes at charge ratios no more than 3. The complexes revealed positive zeta potential values with diameter 100250 nm from charge ratio of 4, which demonstrated excellent DNA condensing ability of guanidino groups in the copolymers. GHG copolymers had significantly lower cytotoxicity than PEI on HepG2 cells and HeLa cells. GHG copolymers with guanidino groups from 30.23% revealed higher transfection than PEI and GHG4 with 55.60% guanidino groups exhibited the highest efficiency at charge ratio of 12 in HepG2 cells, which confirmed that the conjugation of galactose content in the copolymers brought the ASGP-R mediated transfection. The employment of HPMA content decreases the aggregation of protein in the presence of serum transfection. Therefore, GHG copolymers combining the advantages of galactose content, guadino groups, and the HPMA component might show potential in safe hepatocyte targeting gene therapy.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (86-25) 8379-3456. Fax: (86-25) 8379-3456. E-mail:
[email protected].
’ REFERENCES (1) Liu, F., and Huang, L. (2002) Development of non-viral vectors for systemic gene delivery. J. Controlled Release 78, 259–266. (2) Thomas, M., and Klibanov, A. M. (2003) Non-viral gene therapy: polycation-mediated DNA delivery. Appl. Microbiol. Biotechnol. 62, 27–34. (3) Park, T. G., Jeong, J. H., and Kim, S. W. (2006) Current status of polymeric gene delivery systems. Adv. Drug Delivery Rev. 58, 467–486. (4) Verbaan, F. J., Bos, G. W., Snel, C., Oussoren, C., Woodle, M. C., Hennink, W. E., and Storm, G. (2004) A comparative study of different cationic transfection agents for in vivo gene delivery after intravenous administration. J. Drug Delivery Sci. Technol. 14, 1–7. (5) Gianasi, E., Wasil, M., Evagorou, E. G., Keddle, A., Wilson, G., and Duncan, R. (1999) HPMA copolymer platinates as novel antitumour agents: in vitro properties, pharmacokinetics and antitumour activity in vivo. Eur. J. Cancer 35, 994–1002. (6) Nori, A., Jensen, K. D., Tijerina, M., Kova, P. K., and Kopecek, J. (2003) Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells. Bioconjugate Chem. 14, 44–50. (7) Yuan, F., Chen, F., Xiang, Q. Y., Qin, X., Zhang, Z. R., and Huang, Y. (2008) Synthesis and characterization of HPMA copolymer-5-FU conjugates. Chin. Chem. Lett. 19, 137–140. (8) Kulkarni, S., Schilli, C., Grin, B., Muller, A. H. E., Hoffman, A. S., and Stayton, P. S. (2006) Controlling the aggregation of conjugates of streptavidin with smart block copolymers prepared via the RAFT copolymerization technique. Biomacromolecules 7, 2736–2741. (9) Yanjarappa, M. J., Gujraty, K. V., Joshi, A., Saraph, A., and Kane, R. S. (2006) Synthesis of copolymers containing an active ester of methacrylic acid by RAFT: Controlled molecular weight scaffolds for biofunctionalization. Biomacromolecules 7, 1665–1670. (10) Nicolas, J., Mantovani, G., and Haddleton, D. M. (2007) Living radical polymerization as a tool for the synthesis of polymer-protein/ peptide bioconjugates. Macromol. Rapid Commun. 28, 1083–1111. (11) Boyer, C., Bulmus, V., Liu, J., Davis, T. P., Stenzel, M. H., and Barner-Kowollik, C. (2007) Well-defined protein-polymer conjugates via in situ RAFT polymerization. J. Am. Chem. Soc. 129, 7145–7154. (12) Boyer, C., Bulmus, V., Davis, T. P., Ladmiral, V., Liu, J., and Perrier, S. (2009) Bioapplications of RAFT polymerization. Chem. Rev. 109, 5402–5436.
ARTICLE
(13) Deng, Z., Bouchekif, H., Babooram, K., Housni, A., Choytun, N., and Narain, R. (2008) Facile synthesis of controlled-structure primary amine-based methacrylamide polymers via the reversible addition-fragmentation chain transfer process. J. Polym. Sci., Polym. Chem. 46, 4984–4996. (14) York, A. W., Zhang, Y., Holley, A. C., Guo, Y., Huang, F., and McCormick, C. L. (2009) Facile synthesis of multivalent folate-block copolymer conjugates via aqueous RAFT polymerization: targeted delivery of siRNA and subsequent gene suppression. Biomacromolecules 10, 936–943. (15) Scales, C. W., Vasilieva, Y. A., Convertine, A. J., Lowe, A. B., and McCormick, C. L. (2005) Direct, controlled synthesis of the nonimmunogenic, hydrophilic polymer, poly (N-(2-hydroxypropyl) methacrylamide) via RAFT in aqueous media. Biomacromolecules 6, 1846–1850. (16) Cho, K. C., Kim, S. H., Jeong, J. H., and Park, T. G. (2005) Folate receptor-mediated gene delivery using folate-poly(ethylene glycol)-poly(L-lysine) conjugate. Macromol. Biosci. 5, 512–519. (17) Schaffer, D. V., Neve, R. L., and Lauffenburger, D. A. (1997) Use of the green fluorescent protein as a quantitative reporter of epidermal growth factor receptor-mediated gene delivery. Tissue Eng. 3, 53–63. (18) Kircheis, R., Schuller, S., Brunner, S., Ogris, M., Heider, K. H., Zauner, W., and Wagner, E. (1999) Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. J. Gene Med. 1, 111–120. (19) Kircheis, R., Kichler, A., Wallner, G., Kursa, M., Ogris, M., Felzmann, T., Buchberger, M., and Wagner, E. (1997) Coupling of cellbinding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4, 409–418. (20) Stockert, R. J. (1995) The asialoglycoprotein receptor: relationships between structure, function and expression. Physiol. Rev. 75, 591–609. (21) Bandyopadhyay, P., Ma, X., Linehan-Stieers, C., Kren, B. T., and Steer, C. J. (1999) Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethylenimine to the asialoglycoprotein receptor. J. Biol. Chem. 274, 10163–10172. (22) Choi, Y. H., Liu, F., Choi, J. S., Kim, S. W., and Park, J. S. (1999) Characterization of a targeted gene carrier, lactose-polyethylene glycolgrafted poly-l-lysine and its complex with plasmid DNA. Hum. Gene Ther. 10, 2657–2665. (23) Bettinger, T., Remy, J. S., and Erbacher, P. (1999) Size reduction of galactosylated PEI/DNA complexes improves lectin-mediated gene transfer into hepatocytes. Bioconjugate Chem. 10, 558–561. (24) Niidome, T., Urakawa, M., Sato, H., Takahara, Y., Anai, T., Hatakayama, T., Wada, A., Hirayama, T., and Aoyagi, H. (2000) Gene transfer into hepatoma cells mediated by galactose-modified a-helical peptides. Biomaterials 21, 1811–1819. (25) Hashida, M., Takemura, S., Nishikawa, M., and Takakura, Y. (1998) Targeted delivery of plasmid DNA complexed with galactosylated poly (l-lysine). J. Controlled Release 53, 301–310. (26) Cook, S. E., Park, I. K., Kim, E. M., Jeong, H. J., Park, T. G., Choi, Y. J., Akaike, T., and Cho, C. S. (2005) Galactosylated polyethylenimine-graft-poly(vinyl pyrrolidone) as a hepatocyte-targeting gene carrier. J. Controlled Release 105, 151–163. (27) Zanta, M. A., Boussif, O., Adib, A., and Behr, J. P. (1997) In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjugate Chem. 8, 839–844. (28) Funhoff, A. M., Nostrum, C. F., Lok, M. C., Fretz, M. M., Crommelin, D. J. A., and Hennink, W. E. (2004) Poly (3-guanidinepropyl methacrylate): a novel cationic polymer for gene delivery. Bioconjugate Chem. 15, 1212–1220. (29) Kim, T., Ou, M., Lee, M., and Kim, S. W. (2009) Argininegrafted bioreducible poly (disulfide amine) for gene delivery systems. Biomaterials 30, 658–664. (30) Brooks, H., Lebleu, B., and Vive’s, E. (2005) Tat peptidemediated cellular delivery: back to basics. Adv. Drug Delivery Rev. 57, 559–577. 1511
dx.doi.org/10.1021/bc100525y |Bioconjugate Chem. 2011, 22, 1503–1512
Bioconjugate Chemistry
ARTICLE
(31) Deshayes, S., Morris, M. C., Divita, G., and Heitz, F. (2006) Interactions of amphipathic carrier peptides with membrane components in relation with their ability to deliver therapeutics. J. Pept. Sci. 12, 758–765. (32) Tung, C. H., and Weissleder, R. (2003) Arginine containing peptides as delivery vectors. Adv. Drug Delivery Rev. 55, 281–294. (33) Choi, S. W., Sato, Y., Akaike, T., and Maruyama, A. (2004) Preparation of cationic comb-type copolymer having guanidino moieties and its interaction with DNAs. J. Biomater. Sci. Polym. Ed. 15, 1099–1110. (34) Gao, Y., Xu, Z., Chen, S., Gu, W., Chen, L., and Li, Y. (2008) Arginine-chitosan /DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency. Int. J. Pharm. 359, 241–246. (35) Tzivelekaa, L. A., Psarrab, A. M., Tsiourvasa, D., and Paleosa, C. M. (2007) Synthesis and characterization of guanidinylated poly(propylene imine) dendrimers as gene transfection agents. J. Controlled Release 117, 137–146. (36) Choi, J. S., Nam, K., Park, J. Y., Kim, J. B., Lee, J. K., and Park, J. S. (2004) Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J. Controlled Release 99, 445–456. (37) Koo, H., Kang, H., and Lee, Y. (2009) Analysis of the relationship between the molecular weight and transfection efficiency/cytotoxicity of Poly-L-arginine on a mammalian cell line. Bull. Korean Chem. Soc. 30, 927–930. (38) Crrotto, E., and Aros, R. (1993) Quantitative determination of chitosan and the percentage of free amino groups. Anal. Biochem. 211, 240–241. (39) Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. I., and Lee, Y. C. (2005) Carbohydrate analysis by a phenolsulfuric acid method in microplate format. Anal. Biochem. 339, 69–72. (40) Pack, D. W., Hoffman, A. S., Pun, S., and Stayton, P. S. (2005) Design and development of polymers for gene delivery. Nat. Rev. Drug Discovery 4, 581–593. (41) Pissuwan, D., Boyer, C., Gunasekaran, K., Davis, T. P., and Bulmus, V. (2010) In vitro cytotoxicity of RAFT-Polymers. Biomacromolecules 11, 412–420.
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