ARTICLE pubs.acs.org/bc
Linear Cationic Click Polymers/DNA Nanoparticles: In Vitro StructureActivity Relationship and In Vivo Evaluation for Gene Delivery Yu Gao, Qi Yin, Lingli Chen, Zhiwen Zhang, and Yaping Li* Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
bS Supporting Information ABSTRACT: The aim of this work was to explore the structure activity relationships (SAR) of a series of novel linear cationic click polymers with various structures for in vitro gene delivery and in vivo gene transfer. The experimental results revealed that the minimal structure variation could result in a crucial effect on DNA-binding ability, buffering capacity, and the cellular delivery capacity of polymer, all of which brought about the obvious effects on their transfection efficiencies. The polymer synthesized from diazide monomer containing bis-ethylenediamine unit and dialykene monomer containing bis-ethylene glycol unit (B2) could effectively condense DNA into complex nanoparticles (B2Ns), which showed the highest in vitro transfection efficiency. The biodistribution and transfection efficiency of B2Ns in nude mice bearing tumor demonstrated the ability of effectively delivering DNA into tumor tissue. These results implied that this gene vector based on linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.
’ INTRODUCTION One of the hurdles to successful application of gene therapy is the development of a safe and efficient gene vector.1,2 Nonviral vectors, in particular, cationic polymers, have received increasing attention due to easy manufacturing and low immunogenicity, which are commonly associated with the viral vectors.3,4 However, nonviral vectors show less efficacy than viral vectors because of a lack of adequate function to overcome the intracellular barriers for efficient gene delivery. Recently, many strategies have been undertaken in polymer science to realize the full potential of gene therapy. The modification of the existing polymers with functional groups such as imidazoles or targeting ligands to overcome the entrybased barriers is an effective tool to improve transfection efficiency.57 Synthesis of biodegradable polycations containing a hydrolyzable ester bond or enzyme-degradable amide bond is one of the potential means to reduce the intrinsic toxicities of polymers.810 The use of low molecular weight polymers, which can be eliminated via the kidney, is also a logical choice to increase safety.11 However, there are still many problems in the existing cationic polymers, and the ideal polymers for gene transfer have not been found yet. Thus, the rational design of the biocompatible polymers that are well-defined and chemically tailored for clinical application of gene therapy remains a significant challenge. Cationic polymers mediate gene transfer via condensing genes into nanometer-scale structure small enough to enter cells via r 2011 American Chemical Society
endocytosis and intracellular trafficking of the genes.12 Several recent reports have revealed that the related cationic polymers with small structural changes could result in a significant difference in transfection efficiency.1315 Polyplexes from poly(aspartamide) bearing two different side chains with one methylene variation showed completely different transfection and cytotoxicity profiles.13 Altering the hydroxyl number and stereochemistry by the carbohydrate moieties in poly(glycoamidoamine)s resulted in dissimilar gene expression profiles.14 Therefore, parallel synthesis and study on the structure activity relationships (SAR) could be a powerful approach for the rapid identification of novel polymers for efficient gene delivery.16,17 The copper-catalyzed azidealkyne cycloaddition has emerged as a unique ligation method combining selective reactivity and easy manufacturing,18,19 and is used in a variety of polymer syntheses including the linear polymer, in which the azide- or alkyne-functionalized monomers could be easily tailored to form the different copolymers with different chemical and biological properties.20,21 The amidetriazole moiety, which was formed during polymerization, could be an H-bond acceptor and increase the hydrophobic interactions of the polymer with DNA, thus improving the polymerDNA binding stability.22 Therefore, we Received: January 5, 2011 Revised: March 17, 2011 Published: May 12, 2011 1153
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Bioconjugate Chemistry could take advantage of the characteristics of click polymerization to develop a gene delivery system based on linear cationic click polymers (CPs). The amidetriazole moiety in the CP backbone could promote the strong polymerDNA complexation and increase the serum stability of complexes. With the azide- or alkyne-functionalized monomers varied, the differences in efficacy between two polymers could be easily investigated to find out how the structural units affected the characteristics of the polymers or which structural unit played a key role during gene transfer. Consequently, the polymers that were designed to be nontoxic and effective could be found through the SAR study. In our previous work, a series of novel linear CPs were designed and synthesized with various structural changes including the type of charge groups, the number of methylene or ethylene glycol groups, and the distance between charge groups, and the biocompatibility of CPs were evaluated.23 From study based on the structure-biocompatibility relationships, we chose two CPs with good biocompatibility to preliminarily evaluate their in vitro transfection efficiency. The two CPs showed in vitro effective gene transfection, which indicated that CPs could be promising gene carriers and inspired us to investigate the behaviors of these CPs during gene transfer further. In this work, we aimed to explore the in vitro SAR of these click polymers and in vivo gene transfer. We examined the DNA-binding efficiency, buffering capacity, cellular uptake of DNA, and in vitro transfection efficiency of these polymers and tried to find out how the structural units affected the characteristics of the polymers, and which structural unit played a key role during gene transfer. Finally, a candidate polymer from the SAR was selected for the investigation of in vivo gene transfer.
’ EXPERIMENTAL PROCEDURES Materials. Trypsin-EDTA, phosphate buffered saline (PBS), and agarose were obtained from Gibco-BRL (Burlington, ON, Canada). The Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium, antibiotics, DNA loading buffer, and fetal bovine serum (FBS) were purchased from Invitrogen GmbH (Karlsruhe, Germany). YOYO-1 was purchased from Molecular Probes (Eugene, OR, USA). Ethidium bromide, trypan blue, and branched polyethylenimine (PEI, 25 kDa) were purchased from Sigma (St. Louis, MO, USA). Sixteen click polymers were synthesized using “click chemistry” and named A1A4, B1B4, C1C4, and D1D4 as described in our previous work (Scheme 1).23 All other chemicals and solvents if not mentioned were of analytical grade and used as received without additional purification. Plasmid EGFP-N1 (4.7 kb) encoding enhanced green fluorescent protein driven by immediate early promoter of CMV was purchased from Clontech (Palo Alto, CA, USA), and plasmid DNA containing random sequences was used as negative control (neg-DNA). pGL3-control vector was purchased from Promega (Madison, WI, USA). The plasmid DNA (pDNA) grown in DH5R strain of E. coli was isolated with the EndFree Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). The purity was confirmed by spectrophotometry (A260/A280), and DNA concentration was measured by UV absorption at 260 nm. Cell Cultures. The cell lines HEK 293 (human embryonic kidney cell) and MDA-MB-468 (human breast cancer cell) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HEK 293 cells were grown in DMEM containing 10% fetal bovine serum (FBS), 100 U/mL
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Scheme 1. Cationic Click Polymers Used in This Study
penicillin G sodium, and 100 μg/mL streptomycin sulfate. MDAMB-468 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin G and 0.1 mg/mL streptomycin). Cells were maintained at 37 °C in a humidified and 5% CO2 incubator. Animals. BALB/c nude mice (1820 g, 6 weeks old) were purchased from Shanghai Experimental Animal Center (Shanghai). All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Preparation and Characteristics of CP/DNA Complex Nanoparticles (CPNs). CPNs were prepared by the following procedures: briefly, pDNA (100 μg/mL) dissolved in sterile water was added to the CP solution diluted in sterile water with equal volume to obtain the desired polymer-amine to DNA-phosphate ratio (N/P ratio). Subsequently, solution was immediately vortexed for 30 s (XW-80A Votex mixer, Shanghai). The complex nanoparticles were allowed to sit at room temperature for 30 min. As positive control, PEI 25K/DNA complex nanoparticles (PEINs) were prepared with the same procedure as CPNs. The particle size and zeta potential of CPNs were determined by the laser light scattering measurement using a Nicomp 380/ ZLS zeta potential analyzer (Particle Sizing System, USA). The CPNs were prepared as described above, and the size measurement was performed at 25 °C at a 90° scattering angle and recorded for 300 s for each measurement. The formation of CPNs was evaluated by agarose gel electrophoresis. After the DNA loading buffer was added to CPNs, CPNs containing 0.1 μg DNA at various N/P ratios were applied to a 1.0% agarose gel in Tris-acetate-EDTA (TAE) buffer containing 0.5 μg/mL ethidium bromide. Electrophoresis was carried out at 80 V for 45 min in TAE buffer. The resulting DNA migration pattern was revealed under UV irradiation. AcidBase Titration. The buffer capacity of CP was determined by acidbase titration assay over the pH values ranging from 10 to 2 as described previously.24 Each CP was dissolved in 150 mM NaCl solution at an amino group concentration of 5 mM. The pH of the polymer solution was raised to 10 using 1 M NaOH. The resulting solution was then incrementally titrated with 0.1 M HCl. The pH of the solution was monitored with a Sartorius PB-10 pH meter. The experiment was performed at room temperature, each data point was collected at about 5 min after addition of the acid, and the time interval was the same 1154
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Bioconjugate Chemistry for all the samples. The pH profile was obtained for each polymer, and graphs of the data were generated accordingly. Cellular Uptake Experiment. MDA-MB-468 cells were seeded in a 24-well plate with 0.5 mL growth medium and allowed to attach for 24 h. Nanoparticles were prepared at N/P ratio 32 for CPs with YOYO-1-labeled DNA concentration of 50 μg/mL. The DNA/YOYO-1 complex was prepared with 1 nM dye molecule/μg DNA and incubated 1 h at room temperature in the dark. The cells were then incubated with CPNs (DNA concentration 2.5 μg/well) in complete growth medium for 2 h at 37 °C followed by washing three times with PBS (pH 7.4), then trypsinized and resuspended in the medium. The residual fluorescence out of the cell membrane was quenched with 0.4% trypan blue for 2 min.25 Then, cells were centrifuged and washed three times with PBS. The cell resuspension was finally subjected to flow cytometry (FACSCalibur, Becton Dickinson, USA) and analyzed with CellQuest software through fluorescence channel 1 (FL1). Observation of Intracellular Distribution of CPNs. MDAMB-468 cells were seeded on 35 mm glass-based dishes with 0.5 mL growth medium and allowed to attach for 24 h. After incubation with CPNs (N/P ratio 32, DNA concentration 2.5 μg/well) for 2 h at 37 °C, the medium was removed, and the cells were washed three times with PBS. The intracellular distribution of CPNs was observed by confocal laser scanning microscopy (CLSM) after staining lysosomes with LysoTracker Red (Molecular Probes, Eugene, OR, USA) and nuclei with Hoechst 33342 (Sigma, USA). The CLSM observation was performed using Leica TCS SP2 confocal spectral microscope (Leica Microsystems, Germany) with a 63 objective (Leica, Wetzlar, Germany) at excitation wavelengths of 633 nm (HeNe laser), 488 nm (Ar laser), and 351 nm (UV laser) for LysoTracker Red (red), YOYO-1 (green), and Hoechst 33342 (blue), respectively. In Vitro Transfection Experiment. Cells (HEK 293 and MDA-MB-468) were seeded in 24-well plates at a density of 1 105 cells per well in 500 μL of complete medium and incubated for 24 h prior to transfection. Then, the media were replaced with fresh complete growth medium containing CPNs with DNA concentration 2.5 μg/well at various N/P ratios. Media were replaced by fresh culture medium after 24 h, and cells were incubated for an additional 24 h. The expressed green protein could be observed under a fluorescence microscope. Finally, cells were collected and resuspended in PBS (pH 7.4). The transfection results were measured using a FACSCalibur through FL1 and the percentage of cells which expressed EGFP was counted (Supporting Information Figure S1). As control, naked DNA and click polymers/neg-DNA complex nanoparticles were used on cell cultures and examined as described above. As positive control, PEINs with mass ratio of 3 were prepared and transfected with media without FBS for 2 h before the medium was replaced with fresh complete growth medium. In Vivo Biodistribution and Transfection Efficiency. For biodistribution study, female nude mice were inoculated with 100 μL of phosphate buffered saline (pH 7.4) containing 1 106 MDA-MB-468 cells by subcutaneous injection in the left flank. Tumors were allowed to grow to a volume of 200300 mm3. For qualitative evaluation, B2/DNA nanoparticles (B2Ns) with pDNA labeled by the cell-impermeable fluoresecnt dye YOYO-1 at N/P ratio 32 was injected into mice bearing breast tumor via the tail vein at a dose of 2 mg/kg. Mice were sacrificed at different time intervals after injection (n = 4 at each time point), and the principal organs were isolated. The extracted tissues were
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homogenized with lysis buffer (Promega, Madison, WI, USA), and the samples were centrifuged at 10 000 rpm for 5 min. The supernatant was measured using a microplate reader (Infinite F200, TECAN, Austria) with an excitation at 485 nm and exmission at 520 nm. The data were normalized to the tissue weight. For in vivo transfection efficiency, female nude mice were inoculated with 100 μL of phosphate buffered saline (pH 7.4) containing 1 106 MDA-MB-468 cells by subcutaneous injection in the left flank. Experiments were performed at day 10 after inoculation when the tumor volume reached approximately 100 mm3. For quantitative evaluation, B2Ns formed with pGL3-contral vector at N/P ratio 32 were injected into the mice bearing breast tumor via the tail vein at a dose of 2 mg/kg in a 200 μL injection volume. For comparison, PEINs at mass ratio 1 were intravenously injected at the same DNA dosage. Animals were killed 48 h after i.v. injection, and the tissues were isolated. The organs were washed with cold saline and then homogenized with 1 mL lysis buffer using a tissue homogenizer. The homogenate was centrifuged at 15 000 g for 15 min, and the luciferase activity was quantified by Luciferase Assay System using a chemiluminometer (NOVOSTAR, German). Relative light units (RLU) were measured for 5 s at room temperature, and the transfection efficiency was expressed as RLU/g tissue. Statistical Analysis. Statistical analysis was performed using a Student’s t-test. The differences were considered significant for p value < 0.05 and p < 0.01 indicative of a very significant difference.
’ RESULTS AND DISCUSSION Buffering Capacity of CPs. It was assumed that the buffering capacity of cationic polymers like PEI could facilitate the endosomal escape of polyplexes by so-called “proton sponge effect”. This capacity was a relevant physicochemical parameter in the transfection efficiency of the cationic polymer.24,26 In order to understand the relationship between polymer buffering capacity and polymer structure, and how the buffering capacity affected the transfection efficiency of polymer, the acidbase titration experiment was performed to estimate the buffering capacity of CPs (Figure 1). The titration was monitored over the range of pH 102.0 in 150 mM NaCl solution to mimic the physiological condition. It was expected that the presence of different amino groups with different pKa would increase the buffer capacity of polymer. However, the titration curves did not show such trends. The flat slope in pH 57 which was relevant to the overall gene transfection process indicated the higher buffer capacity of CPs containing only one type of amine than that of CPs with two types of amine. C1, C2, D1, or D2 showed some buffering capacity in pH 57 (Figure 1C,D). When tertiary amine or secondary amine was added to CP, the buffering capacity of C3, C4, D3, or D4 decreased significantly, which was also found in polymers A4 and B4. C4 and D4 showed higher buffering capacity than C3 and D3, respectively, which demonstrated that CPs containing primary amine and tertiary amine had higher buffering capacity than polymers with primary amine and secondary amine (Figure 1C,D). A4 and B4 showed obviously low buffering capacity, which indicated that polymers containing both secondary amine and tertiary amine had weak ability in accepting hydrogen ions. From the above-mentioned, it was found that the different types 1155
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Figure 1. Titration curves of CPs at an amino group concentration of 5 mM.
of amines in CPs contributed differently to the buffering capacity of CPs. The different polymers with the same secondary amine showed different buffering capacities. The buffering capacity of polymers in pH 57 decreased as the number of amines increased in the repeat unit (A1 > B1, A2 > B2), and also decreased as the number of amines increased in different monomers (A1 > A3, B1 > B3) (Figure 1A,B), which could be due to the protonation of an amine group electrostatically suppressed the protonation of the neighboring amines.21 As a result, the correlation between the buffering capacity and polymer structure was not as direct as the correlation between the amount of the buffering groups and the structural variations. Finally, the effects of methylene moiety and the ethylene glycol unit on the buffering capacity of CPs were evaluated. No obvious difference was found between polymers containing monomer C and D (Figure 1C,D), which indicated that the influence of the methylene moiety on the buffering capacity was negligible. The buffering capacities of polymers synthesized by monomers A, B, and C, but not monomer D, increased with ethylene glycol group in the polymer backbone increasing. DNA Binding Ability. For efficient gene transfer and expression, the polymer should have the ability to condense DNA into nanoparticles and consequently mediated cell entry via endocytosis. CPs were examined for their ability to bind negative plasmid EGFP-N1 using a gel electrophoretic shift assay (Figure 2). Each polymer was combined with pDNA to form CPNs at various N/P ratios between 1 and 64. N/P ratios were calculated by the number of amine groups in the polymer repeat unit but not the amide nitrogens. All four CPs synthesized by monomer A or B could form complexes with DNA efficiently, it could be observed that the free DNA band completely disappeared at N/P ratio
2 for A1-4 or B1-4. A3 and B3 showed the strongest DNA binding ability with complete binding DNA at N/P ratio 1. However, CPs synthesized from monomer C or D could not bind DNA efficiently. D1 could not bind DNA completely even at N/P ratio 64. The differences in number and type of amines in the polymer backbone strongly affected its DNA binding ability. In the case of one type of amine in the polymer backbone, it was obvious that the secondary amine showed a stronger ability in DNA binding than primary amine in CP backbone by comparing A1-2 with C1-2. In the case of two types of amines in the polymer backbone, polymers with different combinations of amine types showed different DNA binding abilities. C4 showed greatly improved DNA binding ability compared with C1-2 when tertiary amine was introduced into the backbone of the original polymer which only has a primary amine. However, no obvious difference was observed between C3 and C1-2. By comparing A4 with A3 or B4 with B3, we could find that the DNA binding ability decreased a little by replacing the secondary amine with the tertiary amine. Comparing C series with D series, it was found that one more methylene group in the polymer backbone could greatly reduce the DNA binding ability no matter the types of amines on the polymer backbone. No obvious difference was observed between polymers containing 1 and 2, which indicated that the influence of the ethylene glycol moiety on the DNA binding ability was neglectable. These results demonstrated that, besides the electronic interaction between polymer and DNA, other interactions such as hydrophobic interactions or ππ interactions might also be very important. Particle size is one of the important factors influencing the internalization of complexes. The size of particles less than 200 nm could be preferably internalized into most types of cells,27 and a positive surface charge could lead to great interaction with the 1156
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Figure 2. Agarose gel electrophoresis of CPNs at various N/P ratios. Lane 1: naked DNA control. Lanes 2 to 7: complexes prepared at N/P ratios of 1, 2, 4, 8, 16, 32, and 64 in water, respectively.
Figure 3. Average particle size of CPNs at different N/P ratios.
negative cell membrane.28 In this study, all CPs could form nanoparticles with DNA in water. The average particle size of CPs as a function of N/P ratio was evaluated. Figure 3 showed some clear correlations between CP structure and the particle size. Polymer A series and B series formed smaller particles than polymer C series or D series, and showed constant particle sizes as N/P ratios increasing from 4 to 64. These results were in good agreement with the agarose gel electrophoresis assay that polymer A or B series showed stronger DNA binding ability with condensing DNA into smaller particles than polymer C or D series. Replacement of the azide-functionalized monomers
containing ethylene glycol groups with monomers containing secondary amines or tertiary amines could significantly decrease the particle size of complex nanoparticles. The particle size of complex nanoparticles of A, B, or C series obviously increased with the ethylene glycol group in polymer backbone increasing. These results demonstrated that the polymers with higher amine density could yield stronger DNA condensation ability. Except C4, the average sizes of polymer C series or D series increased with N/P ratio increasing, especially the polymers synthesized by azide-functionalized monomers containing the ethylene glycol groups. It was reported that the different polyplexes showed 1157
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Figure 4. Cellular uptake (A) and intracellular distribution (B) of YOYO-1 labeled pDNA complexed with CPNs at N/P ratio 32 in MDA-MB-468 cells. Cells were incubated with CPNs with DNA concentration 5 μg/mL for 2 h. The nucleus and lysosome were stained by Hoechst 33342 (blue) and LysoTracker (red), respectively.
different structural property such as rods, toroids, aggregates, flower-like structures, and nanoparticles, and the molecular morphologies were strongly dependent on the charge ratios and polymer properties.29 Polymer-DNA showed different condensate formation at different charge ratios. We assumed that complex nanoparticles formed by C1-2 or D1-2 could have more incompact structures at higher charge ratios. Cellular Delivery of CPNs. The ability of different polymers to mediate the cellular uptake of DNA was measured in MDA-MB468 cells by introducing a fluorescence based flow cytometry protocol. The pDNA was labeled with YOYO-1, an intercalating dye with a high affinity to DNA. The average fluorescence intensity per cell could be quantitatively assessed via flow cytometry. By this technique, the pDNA delivery efficiency could be directly determined as a function of polymer structure. Cells were incubated with complexes formed at the same N/P ratio of 32 for 2 h. There seems to be a positive correlation between DNA binding ability of CPs and cellular uptake of the corresponding CPNs. As shown in Figure 4A, the polymers synthesized by monomer A or B showed superior cellular uptake to monomer C or D. Due to the inefficiency in complexing DNA, nanoparticles formed by polymer D series showed negligible cellular uptake. In polymer C series, the cellular uptake of CPNs could increase by introducing a secondary amine or tertiary amine into the polymer backbone. The stronger DNA binding ability of C4 could be the main contribution to the cellular uptake. In polymers synthesized by monomers A and B, the replacement of the ethylene glycol unit with a tertiary amine in the polymer backbone could increase cellular uptake of CPNs, but not the secondary amine, which demonstrated that the polymer with a different amine would facilitate cellular DNA uptake. More ethylene glycol units in the polymer backbone synthesized using monomer B could increase cellular uptake, while the opposite result was acquired in polymers synthesized using monomer A. The intracellular distribution of CPNs was further investigated with pDNA labeled by YOYO-1 (green), and nucleus and lysosome stained by Hoechst 33342 (blue) and LysoTracker (red), respectively. The intracellular distribution of different CPNs in MDAMB-468 cells after 2 h incubation was significantly different
(Figure 4B). The polymers synthesized using monomer A or B could facilitate the YOYO-1 labeled DNA into the whole cytoplasm. However, the green fluorescence was mostly around the cell membrane in cells treated with CPNs formed by polymers synthesized using monomer C or D, which were accordant with the result from flow cytometry. With the cellular uptake improved by introducing tertiary amine into the polymer backbone, the green fluorescence could be seen inside cells when incubated with complexes formed by C4. However, the colocalization of green and red fluorescence could not be easily observed, which demonstrated that the complexes formed by C4 endocytosed into cells were not sequestrated in lysosomes. In confocal images of cells treated with CPNs formed by A3 or B3, obvious yellow regions where YOYO-1 labeled DNA was localized in lysosomes could be observed, which suggested the segregation of YOYO-1 labeled DNA in lysosomal compartments without diffusing into cytoplasm. The confocal images of cells treated with CPNs formed by A1, A2, B1, or B2 displayed much more green regions in cytoplasm, which indicated that A1, A2, B1, or B2 complexes showed good endosomal escaping behavior which was consistent with the results of the buffering capacity. However, to our surprise, the green fluorescence was mostly in neclei when cells were incubated with complexes formed by A4 or B4, which had poor buffering capacity. Membrane destabilization of cationic polymers under different pH conditions was reported recently.20 Membrane destabilization happened at either the acidic pH in the late endosomal compartment or the physiological pH at the cytoplasm, and would perturb either the membranes of cytoplasmic vesicles or organelles at physiological pH. In our previous study about the structurebiocompatibility relationship of these click polymers, polymers A4 and B4 demonstrated severe cytotoxicity and could disturb the erythrocyte membrane with high hemolysis ratio.23 So, we inferred that these two polymers could have strong membrane destabilization effects, and the injury of the nuclear membrane would facilitate the nuclear delivery of DNA. Transfection Efficiency of CPNs. In our pre-experiment, we found that most of the 16 CPNs showed the best transfection 1158
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Figure 5. Transfection efficiency of CPNs at N/P ratio 32 with DNA concentration 5 μg/mL in HEK 293 cells (A) and MDA-MB-468 cells (B).
efficiency at N/P ratio 32, and considering that most CPs had good DNA binding ability and DNA condensing efficiency at N/P ratio 32, as a result, the transfection efficiency of 16 CPNs at N/P ratio 32 was compared to investigate the relationships of structure/transfection efficiency. Figure 5A,B showed the different transfection efficiency of 16 CPNs on the HEK293 and MDA-MB-468 cells. From the histograms, we could find that CPNs demonstrated the similar relationship of structure/transfection efficiency in the two cell lines. Complexes of D series could not transfect both of the cell lines. Complexes formed by C3 or C4 demonstrated the improved transfection efficiency compared with C1 and C2, which indicated that cellular uptake was a limiting factor with polymers of the C series. Among 16 CPNs, the complexes formed by B2 (B2Ns) mediated the highest transgene expression in both cell lines, which was comparable to the PEINs group. Cells transfected with complexes formed with A1 or B1 also showed high gene expression. Complexes formed by polymers of B series displayed higher transfection efficency than that of A series, which indicated that the number of secondary amines in the polymer backbone could affect the gene expression. Complexes formed by polymer A3 or B3 demonstrated lower gene expression than B2Ns, which indicated that endosomal escape of these polymers could be a limiting factor in promoting high gene expression. Cytotoxicity of polymers also is a very important parameter affecting transfeciton efficiency. From the results of cellular uptake, it could be found that the complexes formed by polymer A4 or B4 delivered a relatively high amount of DNA into each cell (Figure 4A), but they revealed a relatively low gene expression compared with B2Ns (Figure 5). Among all 16 polymers, A4 and B4 showed the strongest cytotoxicity and induced the highest hemolysis rate.23 The strong membrane destabilization effects of these two polymers could facilitate the nuclear delivery of DNA, but at the same time, destroy the normal function of cells, thus affected gene transfection and protein expression. The above results demonstrated that DNA-binding ability, buffering capacity, cytotoxicity, or the cellular delivery capacity of polymers could affect their complex transfection efficiencies. In Vivo Biodistribution and Transfection Efficiency. According to the above structureactivity relationship, B2Ns demonstrated high transfection efficiency with low toxicity. As a result, polymer B2 was chosen for further in vivo studies as follows. The lethal toxicity was first investigated in MDA-MB-468 breast tumor-bearing mice. No lethal toxicity was observed for B2Ns at N/P ratio 32 at 3 mg DNA/kg. Because the i.v. injection of B2Ns in mice had a maximum volume, and the complex nanoparticles
were apt to assemble under high concentration, the toxicity of B2Ns at over 3 mg DNA/kg was difficult to determine, but it was easy to find that B2Ns did not induce the obvious toxicity in mice under the experimental dose level (2 mg DNA/kg). The biodistribution characteristics of nonviral vectors were among the most important factors affecting gene transfection efficiency. The difference in polymer structures and surface properties of nanoparticles leads to the difference in vivo biodistribution. It was reported that over half of the administered PEI/ siRNA nanoparticles distributed to the liver in mice,30 but the polyphosphazene/DNA nanoparticles mainly distributed into the lung in a short period of time.31 As a result, it is necessary to investigate the biodistribution characteristics of newly synthesized click polymer/DNA complex nanoparticles. To investigate the biodistribution of B2Ns in tumor bearing mice, fluorescent dye YOYO-1 was chosen to label DNA. It is a cell-impermeant stain which shows over a thousandfold increase in its green fluorescence when bound to dsDNA. Tumor was allowed to grow to a volume of 200300 mm3 to facilitate qualitative observation of the fluorescence in each tissue under a real-time in vivo fluorescence imaging system. Then, the fluorescence was quantitatively evaluated. At the DNA concentration range 05 μg/mL, there is a perfect linear relationship between DNA concentration and fluorescence intensity. The biodistribution profiles of B2Ns in MDA-MB-468 solid tumor-bearing mice were shown in Figure 6A. B2Ns could rapidly distribute into several principle tissues at 15 min after intravenous administration. A large amount of B2Ns accumulated in the liver which could be due to the Kupffer cells scavenging charged particles.30 At 15 min, some B2Ns could be detected in tumor, and maximal accumulation of B2Ns in tumor tissue was observed 1 h after administration. The distribution in tumor tissue was much higher than in heart, spleen, lung, and kidney, which could be attributed to the EPR effect.32,33 The accumulation in each tissue decreased over time, and after 24 h, almost all B2Ns was eliminated. These results demonstrated that the complex nanoparticles fromed by click polymers could not accumulate in the body. The in vivo transfection efficiency of B2Ns was compared with PEINs in MDA-MB-468 solid tumor-bearing mice. Due to the severe toxicity of PEI, the mass ratio of the administered complexes could not be over 1 under the experimental dose level (2 mg DNA/kg). The luciferase expression in heart, liver, spleen, lung, kidney, and tumor at 48 h after i.v. was shown in Figure 6B. The highest levels of luciferase expression were found in tumor, and much higher luciferase activity was detected in liver 1159
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polymer selected from SAR study were evaluated in this work. Comparative analysis of 16 polymers revealed that the minimal structure variation had a crucial effect on buffering capacity, DNA-binding ability, or the cellular delivery capacity of polymer, all of which produced striking effects on their complex transfection efficiencies. The parallel screening led to the identification of a candidate polymer B2, which could effectively deliver DNA into tumor tissue. These results implied that gene vector based on this linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.
’ ASSOCIATED CONTENT
bS
Supporting Information. Representative FACS histograms of HEK 293 control cells, cells after transfected with naked DNA, B2/neg-DNA complex nanoparticles, or B2Ns. This material is available free of charge via the Internet at http://pubs. acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Address: 501 Haike Road, Shanghai 201203, China. Tel.: þ8621-2023-1979. Fax: þ86-21-2023-1979. E-mail address: ypli@ mail.shcnc.ac.cn.
Figure 6. (A) Biodistribution of B2Ns in nude mice bearing MDA-MB468 tumor. Data were given as mean ( SD (n = 3). (B) Quantitative gene expression in vivo. Luciferase expression 48 h after intravenous administration of B2Ns formed by polymer B2 into nude mice bearing MDA-MB-468 tumor at a dose of 2 mg DNA/kg. **p < 0.01 compared with PEINs. Luciferase expression is plotted as RLU/g tissue. Data were given as mean ( SD (n = 3).
and kidney than other organs via injection of B2Ns. The high tumor gene expression could be due to the targeting of nanoparticles to tumor via the EPR effect. In tumor, the gene expression of B2Ns was a little higher than PEINs. In heart, spleen, and lung, the luciferase activity of PEINs was higher than that of B2Ns, expecially in the lung. It was reported that PEI could induce severe aggregation of erythrocytes after intravenous injection, and the aggregation was apt to block pulmonary capillary, which provided enough time for gene expression in the lung.34 However, in our previous work, B2 did not induce the obvious erythrocyte aggregation,23 and the strong binding ability of triazole-amide to DNA could effectively overcome aggregation of nanoparticles in blood circulation. Therefore, the gene expression of B2Ns in lung was significantly lower than that of PEINs (p < 0.01). The reduced distribution to lung tissue gave B2Ns more opportunity to distribute to tumor and acquire more gene expression.
’ CONCLUSIONS In vitro structureactivity relationship of 16 linear cationic click polymers and in vivo gene transfection of a candidate
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