Efficient Gene Delivery with Osmotically Active and Hyperbranched

Bioconjugate Chem. 2009, 20, 2231–2241

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Efficient Gene Delivery with Osmotically Active and Hyperbranched Poly(ester amine)s Rohidas B. Arote,†,§ Eun-Sun Lee,‡ Hu-Lin Jiang,‡ You-Kyoung Kim,† Yun-Jaie Choi,† Myung-Haing Cho,*,‡ and Chong-Su Cho*,† Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, and College of Veterinary Medicine, Seoul National University, Seoul, 151-742, South Korea. Received April 22, 2009; Revised Manuscript Received October 22, 2009

Degradable and hyperbranched poly (ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between hydrophilic glycerol triacrylate (GTA) and low-molecular-weight polyethylenimine (LMWPEI) and evaluated as nonviral gene carriers. PEAs effectively condensed DNA with particle sizes below 200 nm and suitable surface charges (15-45 mV), suitable for intracellular delivery. PEAs degraded in a controlled fashion showing half-lives of more than 12 days and were essentially nontoxic in three different cell lines. Elevated transfection levels by luciferase assay revealed the superiority of PEAs over PEI 25K and Lipofectamine. PEAs synthesized using 1:4 mol ratio of GTA to PEI [GTA/PEI-1.2(1:4)] showed highest transfection efficiency in HepG2 cells. PEAs showed significant gene expression in Vitro as well as in ViVo through aerosol administration. Reduction in packed cell volume (PCV) of cells when treated with polyplexes supported the hyperosmotic effect of PEAs. Effect of bafilomycin A1 on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity. High transfection efficiency was attributed to the synergism from hyperosmotic glycerol backbone in the PEAs and endosomal buffering capacity of PEI amine groups. Therefore, this convergence of osmotically active biodegradable PEAs suggests their potential as a safe and efficient gene delivery vector.

INTRODUCTION The human genome project has substantially increased our knowledge about molecular mechanisms of cancer during the past decade, thus opening up new possibilities for cancer gene therapy. Gene therapy is one of the most challenging and promising therapies of inherited and acquired diseases. However, the lack of safety and efficient gene carriers limits the success of gene therapy. Viral vectors are more potent gene delivery vehicles because of their specific machinery to enter the cell and to deliver the nucleic acid into the cell and into the nucleus (1, 2). However, severe immune responses, limited insert size, and insertional mutagenesis risk limits their use as a successful gene delivery vehicle. Polymeric vectors have remarkable advantages as gene delivery vectors which make them a promising alternative to most efficient recombinant viruses (3-6). Polyethylenimine (PEI) is one of the successful and widely used gene delivery polymers which have become the gold standard of nonviral gene delivery because of its proton sponge effect (7, 8). However, several groups have reported that PEI is cytotoxic in many cell lines. Moreover, higher molecular weight reveals higher toxicity and high transfection efficiency compared with low-molecular-weight PEI (LMW-PEI) (9, 10). Several natural and synthetic biodegradable polymers such as polyesters (11, 12), linear poly(ester amine)s (13, 14), polypeptides (15), PEA networks (16), and cyclodextrins (17) are among the most preferable materials for the preparation of nonviral vectors in terms of their long-term safety and biocom* Correspondence authors. C. S. Cho, Tel: +82-2-880-4636, Fax: +82-2-875-2494, E-mail: [email protected]; M. H. Cho, Tel: +822-880-1276, Fax: +82-2-873-1268, E-mail: [email protected]. † Department of Agricultural Biotechnology. ‡ Research Institute for Agriculture and Life Sciences. § College of Veterinary Medicine.

patibility. Our group developed various biodegradable polymeric gene carriers using LMW-PEI and various cross-linkers such as poly(ethylene glycol) diacrylate (PEGDA) (18), polycaprolactone diacrylate (PCLDA) (19), poloxamer diacrylate (GDM) (20), and glycerol dimethacylate (21) to reduce cytotoxicity and increase transfection efficiency. Various strategies to enhance transfection efficiencies and reduce cytotoxicity and polyplex stability are being explored. One approach is the generation of biodegradable polymers by cross-linking LMW-PEI having low cytotoxicity with various cross-linkers so as to produce highmolecular-weight polycations with high transfection efficiency and reduced cytotoxicity. It is noteworthy that successful carriers possess sufficient DNA binding ability, effective polyplex uptake by cells, subsequent release into cytoplasm, trafficking of polyplex to nucleus, and immediate dissociation of the polyplex. On the basis of the reports by Zauner et al., polyplex uptake and its subsequent release from endosomal environment to the cytoplasm have been considered as one of the rate-limiting steps for successful delivery of gene (22). In correlation with the above assumptions, our approaches to enhance transfection efficiency, reduce cytotoxicity, and increase cellular uptake are designed on the synthesis of biodegradable poly(ester amine) (PEA) containing LMW-PEI and hydrophilic glycerol triacrylate (GTA) as a cross-linker. PEAs based on GTA and LMW-PEI will facilitate cellular uptake by exerting a hyperosmotic effect owing to the presence of a glycerol backbone while PEI residues will enhance the endosomal release by the “proton sponge effect”.

MATERIALS AND METHODS Materials. Branched PEI (1.2 and 25 kDa), glycerol triacrylate (GTA) (MW: 254 Da), and anhydrous methanol were purchased from Sigma (St. Louis, MO, USA) and were used as received. Cell Titer 96 AQueous One Solution Reagent for cell viability, luciferase reporter 1000 assay system for in Vitro

10.1021/bc900184k  2009 American Chemical Society Published on Web 11/24/2009

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Table 1. Characteristics of Synthesized PEAs mol. wt. of reactants code

PEI

GTA

feed ratio GTA/PEI

PEI mola %

yield of copolymer %

molecular weightb (Da)

GTA/PEI-1.2 (1:1) GTA/PEI-1.2 (1:2) GTA/PEI-1.2 (1:4)

1200 1200 1200

254 254 254

1:1 1:2 1:4

52.18 65.35 74.24

25.06 29.45 40.42

4190 5880 5480

a

Determined by 1H NMR. b Determined by GPC-MALLS.

transfection and pGL3-control vector with SV-40 promoter, and enhancer encoding firefly (Photonus pyralis) luciferase were obtained from Promega (Medison, WI, USA). Plasmid pEGFPN2, which has the early promoter of CMV and enhanced green fluorescence protein (EGFP) gene, was obtained from Clontech (Palo Alto, CA, USA). The plasmid was amplified with a competent E. coli strain JM109. Synthesis of PEAs. PEAs were successfully synthesized following modified Michael addition reaction as reported previously (19). Briefly, GTA and LMW-PEI (Mn: 1.2 kDa) were separately dissolved in anhydrous methanol, and GTA solution was slowly added to PEI solution at three different GTA/PEI stoichiometric ratios (Table 1). The reaction mixtures were heated at 60 °C with constant stirring for 24 h. Subsequently, the reaction mixtures were dialyzed at 4 °C for 24 h using Spectra/Por membrane (molecular mass cutoff 3.5 kDa; Spectrum Medical Industries, Inc., California) against distilled water. Finally, the polymers were lyophilized and stored at 0 °C. Characterizations of PEAs. 1H NMR samples were prepared by dissolving polymers in CDCl3 at a concentration of 10 mg/ mL. NMR spectra were recorded using Avance 500 spectrometer (Bruker, Germany). Absolute molecular weights of the polymers were measured by gel permeation chromatography coupled with multiangle laser light scattering (GPC-MALLS) using Sodex OHpack SB-803 HQ (Phenomenox, USA) column (column temperature at 25 °C; flow rate 0.5 mL/min). Ammonium acetate (0.5M, pH 5.5) was used as a mobile phase (19). Degradation of PEAs. The extent of polymer degradation was estimated by measuring the reduction in molecular weight of PEAs. Polymers dissolved in PBS (0.1 g/mL) were incubated in a shaking incubator (37 °C; 100 rpm) and sampled at various time intervals. Subsequently, the lyophilized samples were subjected to GPC for molecular weight determination. Complexation Study. Polymer/pDNA complexation was carried out in sterilized distilled water by gently mixing DNA (pGL3 control; 0.1 µg) with polymer solutions (10 µL) at various N/P ratios to mitigate the possibilities of mechanical damage of DNA. The complexes were incubated at room temperature for 30 min, and a 12 µL volume including loading dye mixture was loaded on 0.8% agarose gel containing ethidium bromide (0.1 µg/mL). The gel was run with tris-acetate buffer at 100 V for 40 min. The gel image was captured under UV illumination. Protection and Release Assay of DNA. Protection and release of DNA in complexes were measured using electrophoresis, according to the method in our previous reports (19). Particle Size and Zeta Potential Measurement. Particle sizes and zeta potentials of polymer/DNA complexes (prepared in water at various N/P ratios) were measured using dynamic light scattering spectrophotometer (DLS) (ELS8000, Otsuka Electronics, Osaka, Japan), with 90 and 20° scattering angles, respectively. Polymer/DNA complexes were prepared in water from N/P ratios 1 to 30. Transmission Electron Microscopy (TEM). The morphology of PEA/DNA complexes was observed using energy filtered TEM (EF-TEM) (JEM 1010, JEOL, Japan).TEM specimens were prepared by placing a drop of PEA/DNA complexes on a copper grid and stained with 1% uranyl acetate solution for 5 s.

The specimens were allowed to dry further for 10 min and were then examined under the microscope. Cell Lines and Cell Culture. Human cervix epithelial carcinoma cells (HeLa), human hepatoblastoma cells (HepG2), and human kidney transformed cells (293T) were maintained in a DMEM culture medium supplemented with 10% heatinactivated FBS, penicillin/streptomycin. Cells were grown under standard conditions, in a 37 °C humidified incubator containing 5% CO2. Cell Viability Assay in Vitro. Cell viability of PEAs was investigated by Cell Titer 96 Aqueous One Solution Cell Proliferation Kit (Promega). Cells seeded in 96 well plate at an initial density of 1 × 104 (HeLa and 293T) or 2 × 104 (HepG2) cells/well in 0.2 mL growth medium were incubated for 18-20 h to reach 80% confluency prior to the addition of PEAs. Growth medium was replaced by fresh serum-free media containing PEA solutions at various concentrations (5, 10, 20, and 40 µg/mL), and the cell viability study was carried out by the method reported in our previous reports (19). In Vitro Cell Transfection. All assays were conducted in 24 well plates. One milliliter of HeLa, 293T and HepG2 cell cultures (for HeLa and 293T, 1 × 105 cells/mL; and for HepG2, 2 × 105) was grown to ∼70-80% confluency under standard incubation conditions. The media in the wells were replaced with either serum-free media or 10% serum-containing media supplemented with 1 µg of polymer/pGL3 complexes of various N/P ratios, and incubated at 37 °C for 6 h. Subsequently, serumfree media were replaced with fresh media containing serum and incubated for additional 24 h under standard incubation conditions. The luciferase assay was carried out per manufacturer’s protocol (Promega). Relative light units (RLU) were measured with chemiluminometer (Autolumat, LB953, EG&G Berthold, Germany). The obtained RLUs were normalized with respect to protein concentration in the cell extract determined using BCA protein assay kit. Flow Cytometry and Confocal Microscopy Study. The transfection efficiency was also verified by flow cytometric measurement using EGFP reporter gene. 293T cells after being transfected with PEA/pEGFP-N2 were washed with PBS and trypsinized. Transfection efficiency was evaluated by scoring the percentage of cells expressing green fluorescence protein using a FACS Calibur System from Becton-Dickinson (San Joes, CA). The experiments were performed in triplicate and 20 000 cells were counted in each experiment (21). 293T cells were plated on collagen-coated glass coverslips in 24 well plates (5 × 104 cells/well) and were grown to ∼60-70% confluency. The media were then replaced with serum-free media containing PEA/pEGFP-N2 (1 µg) complexes (N/P ratio 30). After 6 h of incubation, serum-free media were changed with fresh media containing serum and incubated for additional 18 h. Cells were washed twice with PBS and immobilized by adding 200 µL of 2% glutaraldehyde solution. The coverslips were washed with PBS and enclosed in glycerol, and finally, the cells were visualized by confocal laser scanning microscopy (CLSM) (Micro Systems LSM 410, Carl Zeiss, Germany) equipped with a 488 nm argon/krypton laser. Fluorescence emission was collected using a 515 nm/30 nm

PEAs as Efficient Gene Carriers

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identical conditions using different concentrations of glycerol instead of PEA/DNA complexes as control. Effect of Bafilomycin A1. 293 T cells seeded in 12 well plates (2 × 105 cells/well) were incubated for 18 to 20 h to reach 80% confluency. The cells were incubated for 10 min before transfection, with endosome proton pump inhibitor, bafilomycin A1 (a specific inhibitor of vacuolar type H+-ATPase). The cells were then subjected to in Vitro luciferase assay as described above. The cells without treatment with bafilomycin A1 were taken as control. In ViWo Transfection. Aerosol administration in mice was carried out to determine the in ViVo transfection ability of PEAs. Thirteen-week-old female BALB/c mice, maintained at 23 ( 2 °C and relative humidity of 50 ( 20%, were placed in noseonly exposure chamber (NOEC; Dusturbo, Seoul, Korea) and exposed to aerosol administration. Aerosol was prepared using 1 mg of pEGFP-N2 plasmid DNA, and transfection ability of PEAs was evaluated according to a method previously reported (19).

RESULTS AND DISCUSSION Figure 1. Synthesis scheme of PEAs. GTA linker reacts with primary and secondary amines of PEI resulting in formation of ester bonds.

bandpass filter. Cells treated with only PBS or polymers were taken as controls. Packed Cell Volume Measurement. Trypsinized 293T cells were centrifuged and resuspended in DMEM supplemented with 10% FBS. To monitor the effect of glycerol content (wt %) in PEAs on packed cell volume (PCV), different concentrations of PEAs and/or PEA/DNA complexes were then added to the cell suspensions. For each set, 1 mL of cell suspension was transferred to a mini-PCV tube (TPP, Trasadingen, Switzerland) and centrifuged for 1 min at 5000 rpm. The volume of the packed cells in the graduated capillary was reported as the % PCV and compared with the experiments done under the

Synthesis and Characterization of PEAs. In general, PEAs are formed from nucleophilic addition of amines to acrylates under normal conditions. The main objective of this study is to rationally design, characterize, and evaluate a novel class of potentially safe, biodegradable, and hyperbranched polymers with poly(ester amine) backbone based on hydrophilic glycerol triacrylate and LMW-PEI. Hyperbranched PEAs were obtained using LMW-PEI (Mn: 1200 Da) as amine nucleophile and glycerol triacrylate (GTA) as a cross-linker at different stoichiometric ratios in anhydrous methanol through Michael addition reaction (Figure 1). The absence of water during synthesis should prevent hydrolysis of sensitive ester linkages. Triacrylate linker reacts with PEI by Michael addition reaction to either primary or secondary amines, generating ester-based

Figure 2. Representative 1H NMR spectrum of PEA [GTA/PEI-1.2(1:4)] in CDCl3.


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Figure 3. Degradation pattern of PEAs.

polymers. An excess of cross-linker resulted in gelation and originated highly cross-linked polymers insoluble in aqueous media. Unlike the reaction condition mentioned in our earlier previous work, the reaction was carried out at slightly elevated temperature (60 °C) to increase the cross-linking and thereby the molecular weight of final polymer (19, 21). The characteristics of synthesized PEAs at different GTA/PEI stoichiometric ratios are summarized in Table 1. Unlike the reports by Langer group, no significant difference in the MW of PEAs was observed through variation in stoichiometric ratio of GTA to LMW PEI (13). The compositions of synthesized PEAs were confirmed through 1H NMR spectroscopy (Figure 2). The signals at δ 1.3 ppm are related to GTA protons, which are highlighted by arrows. The signals of PEI protons were found in the range δ 2.2-3.0 ppm. The ratios of integrals of GTA protons to PEI protons and molecular weight are shown in Table 1. Unlike our previous reports, the composition of PEAs was found to match those of the stoichiometric feed ratios of the reactants (21). Cross-linking of these small polycations using potentially degradable linkages was conceived as a way of increasing molecular weight and thus enhanced DNA binding capacity and polyplex stability while maintaining their nontoxic nature and high transfection efficiency. One feature that has to be fulfilled by the compacting domains is high DNA binding affinity and, moreover, the ability to form stable DNA complexes. As DNA binding activity of polyplexes is strongly influenced by charge density and molecular weight (23), the biophysical characterization provided preliminary information about the original structures. It has been shown from the reports of Lynn et al. that DNA condensation, transfection efficiency, and cytotoxicity are significantly affected by the molecular weight of the carrier. In addition, monomer structure, choice of solvent, and reaction temperature affects the average molecular weight of polymer which lies in the range 2000-50 000 Da (8, 24). GPC-MALLS measurements were performed to characterize PEAs, and it was found that maximum molecular weights were observed at equivalent stoichiometric ratios as reported by Langer group and our previous reports (14, 19). The average molecular weights of synthesized PEAs were found not to vary widely (4190 to 5480 Da), with polydispersity indices close to 2 indicating the difficulty in controlling crosslinking by simple change in reactant feed ratio (Table 1). To determine the degradability, PEAs were subjected to aqueous degradation environment incubated at 37 °C and aliquots were removed at appropriate time points. The degradation was calculated by measuring the reduction in the molecular weight. Figure 3 shows the degradation profile of PEAs as a function of time. The results showed that PEAs degraded slowly and

Figure 4. Agarose gel electrophoresis of PEA/DNA (pGL3-control) complexes at various N/P ratios: (a) GTA/PEI-1.2 (1:1), (b) GTA/PEI1.2 (1:2), and (c) GTA/PEI-1.2 (1:4). 0.1 µg pDNA was used to prepare complexes with PEAs.

the half-life of degradation was approximately more than 12 days. Absence of cytotoxicity is one of the significant features for in ViVo application of any polymeric gene carriers. Generally, cytotoxicity arises from accumulation of nondegraded and nondischarged polymers in the cell (16). The ester bonds in PEAs are susceptible to hydrolysis at physiological conditions to form the respective diol linker and amino acid, thereby generating low-molecular-weight nontoxic byproduct. PEA/DNA Polyplex Characteristics. One feature that has to be fulfilled by the compacting domains is high DNA binding affinity and, moreover, the ability to form stable DNA complexes. The ability of polymers to complex with plasmid DNA (pGL3) was demonstrated using agarose gel electrophoresis. The band of pGL3-control was retarded as the amount of PEAs increased. As shown in Figure 4, all PEAs have a great capacity to condense DNA effectively where pGL3-control was completely retarded at N/P ratio 5. In contrast, the pDNA band was completely moved. N/P ratio 5 at which all PEAs showed complete DNA binding was considered in future characterization. Effective DNA condensation by polycation and polyplex stability are important requirements for DNA stability against degradation by nucleases (25). PEA efficacy to protect DNA from attack of DNase-I was confirmed by agarose gel electrophoresis. PEAs were able to guard DNA as compared with naked DNA, which was completely degraded by DNase-I (data not shown). Successful transfection efficiency of gene carrier depends on its ability to condense negatively charged DNA into nanosized particles with positive charges so as to enter into the cell. The size and surface charge of PEA/DNA complexes were measured using dynamic light scattering as shown in Figure 5. It was found that all three PEAs effectively condensed DNA into nanosized, positively charged polymer/DNA complexes. Lower amounts of polymer tended to give larger nanoparticles owing to the aggregation. An increase in PEA/DNA charge ratio produced smaller-sized nanoparticles. All three PEAs showed



Figure 5. Biophysical properties of PEA/DNA complexes at various N/P ratios: (a) particle size measurement of PEA/DNA complexes, (b) zeta potential measurement of PEA/DNA complexes, and (c) EF-TEM images of GTA/PEI-1.2(1:4)/DNA complexes at N/P ratio 30 (n ) 3). (*p < 0.1; **p < 0.05; ***p < 0.01, two-tailed Student’s t-test).

polyplex sizes below 200 nm which are suitable for intracellular delivery. In all three PEA cases, the polyplexes had positive surface charges in the range 15-45 mV. An increase in the charge ratio from 5 to 30 increased the surface charge of the polyplexes from 15 to 45 mV as shown in Figure 5b. In correlation with our previous findings, the particle size and zeta potential results of PEA/DNA polyplexes typically match the DNA condensation pattern where DNA was initially condensed into larger particles upon addition of cationic PEAs through a charge neutralization mechanism (19, 21). The particle sizes and zeta potential measurements indicated that polymer/DNA complexes were cationic with effective diameter below 200 nm and surface charges cationic enough for attachment to anionic cell surfaces. These features suggest that PEA/DNA particles are cationic enough for association with negatively charged cell membranes and subsequent endocytosis. For efficient endocytosis and transfection, the polyplexes must be small, compact, and spherical. The morphology of PEA/DNA polyplexes was observed by EF-TEM. Figure 5c shows morphology of GTA/PEI-1.2(1:4) at N/P ratio 30. The GTA/PEI1.2(1:4)/pDNA polyplexes reveal the formation of spherical nanoparticles with an average diameter smaller than that obtained in particle size measurement. Cell Cytotoxicity. Cell compatibility studies were performed by incubating PEA solutions in different concentrations on cells and assessing the metabolic activity by MTS-based cell viability method after 24 h. PEAs prepared from hydrophilic glycerol triacrylate and LMW-PEI have remarkably low cytotoxicity in all three cell lines compared with highly cytotoxic PEI 25K. As depicted in Figure 6, all PEAs showed reduced cytotoxicity with concentrations as high as 60 µg/mL. In contrast, PEI 25K showed significant cytotoxicity reflected by the fact that less than 40% of the cells were viable when the concentration of PEI 25K was higher than 20 µg/mL. Among the many attempts

to reduce the cytotoxicity of the gene carrier, the introduction of biodegradable linkage, i.e., ester linkage, has been the prominent method until today (11-14, 18-21). Reduced cytotoxicity of PEAs is undoubtedly a significant advantage of the degradable ester linkage over nondegradable PEI 25K. It also appears that the reduced cytotoxicity may be correlated with PEA degradation. The increased cell viability is undoubtedly due to the nontoxic building blocks and biodegradation products appearing from the ester backbone of PEAs. It is noteworthy that cytotoxicity of PEAs is cell-line dependent and free polymers are usually more toxic to cells and tissues, while the cytotoxicity is reduced when polymers are complexed with DNA (10, 26). As shown in Figure 6, interestingly all PEAs revealed at least 70% cell viability in all three cell lines at concentration as high as 60 µg/mL. In contrast, in case of nondegradable PEI, it aggregates on cell surface, thereby impairing important membrane functions (10), and reduces cell viability. MTS assay results clearly indicated the significant cell viability of PEAs, and thereby, it is potential as a safe gene carrier. In Vitro Transfection by Luciferase Assay. The ability of PEAs based on GTA and LMW-PEI to deliver DNA into cells was determined by performing luciferase reporter gene transfections. Figure 7 shows the transfection efficiency of PEAs in HeLa, HepG2, and 293T cells. In Vitro luciferase activity of PEAs was assessed in triplicate using 1 µg of pGL3 complexed with PEAs at various N/P ratios from 5 to 30. After 48 h gene expression time, lysates of transfected cells were assessed for luciferase activity by measuring the relative light units (RLUs) normalized by total protein content. All PEAs showed increase in reporter gene expression owing to effective DNA binding ability thereby forming nanosized polyplexes. Interestingly, all PEAs were able to mediate higher levels of gene expression despite lower levels of molecular weight. Consistent with our


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Figure 6. Cytotoxicity of PEAs at various concentrations in different cell lines: (a) HeLa, (b) HepG2, and (c) 293T cells (*p < 0.1; ** p < 0.05; ***p < 0.001, two-tailed Student’s t-test) (n ) 3, error bar represents standard deviation).

previous work (21), PEAs composed of GTA and LMW-PEI also revealed higher gene expression levels owing to the presence of biodegradable ester linkage, thereby reducing the cytotoxicity. It was noteworthy that GTA/PEI-1.2(1:4) showed 30-35-fold higher transfection levels compared with “golden standard” PEI 25K at N/P ratio of 30 in all three cell lines. Reporter gene expressions in HeLa and 293T cells remained at rather similar levels in the case of all three PEAs probably due to a smaller difference in the molecular weights, as transfection efficiency of PEAs was shown to be dependent on molecular weight, in correlation with the reports by our group and Mikos group (9, 19, 21). As expected from our previous results, transfection results of GTA/PEI-1.2 copolymers followed a somewhat similar pattern where the introduction of an ester bond with hydrophobic/hydrophilic balance boosted the gene-carrying activity on one hand and reduced cytotoxicity on other hand, which is also in agreement with reports by Anderson et al. (19, 27). Unlike our previous reports, the extent of hydrophobicity in GTA/PEI-1.2 copolymers is higher than GDM/PEI-1.2 owing to the absence of a free hydroxyl group (21). Even though PEI 25K is a highly transfection efficient polymer used in gene delivery, its transfection was decreased with increasing charge ratio owing to its cytotoxicity. As depicted in Figure 7, it was found that the transfection efficiency of PEAs in HepG2 cells was in the order GTA/PEI-1.2(1:1) < GTA/PEI-1.2(1:2) < GTA/PEI-1.2(1:4).

Figure 7d shows the transfection efficiency of PEA/pDNA complexes with and without serum in HeLa cells at N/P ratio of 30. A normal difference was found between the transfection abilities of PEAs in the absence and presence of serum. Polyplexes prepared from PEAs and pDNA were stable and showed a quite similar transfection profile in the presence and absence of 10% serum owing to the hydrophilic property of glycerol moieties in PEA backbone, which correlates with our previous reports (21). This also matches the hypothesis reported by Kabanov et al. where complexes formed by P123-g-PEI (2K) and DNA in the presence of free polaxamer reveals stability in a variety of conditions especially in the presence of serum (28). All three PEAs showed a little decrease in the gene expression level when tested in the presence of serum compared with serum-free transfection. On the other hand, PEI 25K showed a significant reduction in the transfection level when tested in the presence of serum. Confocal Microscopy and Flow Cytometry. The transfection ability of PEAs was also checked using GFP gene by CLSM and flow cytometry. Figure 8a shows the GFP reporter gene expression shown by PEAs using confocal microscopy in A549 cells. Optimized charge ratios (N/P ratio 30) of PEA/pDNA complexes (checked by Luciferase assay) were used for confocal experiments. Polyplexes containing PEA/pEGFP-N2 transfection complexes transfected into A549 cells and revealed interesting



Figure 7. Luciferase activity of PEA/pGL3-control at various N/P ratios in three different cell lines: (a) HeLa, (b) HepG2, (c) 293T, and (d) with and without serum on HeLa cells (*p < 0.1; **p < 0.05; ***p < 0.01, two-tailed Student’s t-test) (n ) 3, error bar represents standard deviation).

fluorescence compared with PEI 25K in correlation with luciferase assay results. The transfection efficiency of PEAs using pEGFP-N2 was also monitored by flow cytometry. Flow cytometry studies in 293T cells revealed a significantly high level of transfection as shown in Figure 8b. With increasing N/P ratio, the maximum in transfection efficiency shifted to polyplexes made with GTA/ PEI-1.2(1:4) at N/P ratio of 30. The mean fluorescence intensity of pEGFP-N2 positive cells can serve as the average expression of GFP per cell. In contrast, PEI 25K showed optimum transfection at N/P ratio of 10 and further decreased up to 30 owing to its cytotoxicity. This significant increase in transfection efficiency of PEAs is undoubtedly the advantage of ester linkage which on one hand increases the momentum of transfection efficiency and on the other hand reduces the cell cytotoxicity. The above results of transfection level as a measure of luciferase and GFP expression clearly indicate the excellence of PEAs over PEI 25K as a potential gene carrier. Effect of Bafilomycin A1. To elucidate the mechanism of transfection efficiency of PEAs, we analyzed the effect of bafilomycin A1, an endosome proton pump inhibitor (29) on the transfection efficiency of PEA in 293T cells. Reporter gene expression shown by PEAs was reduced by more than 20-25fold when transfected in the presence of 200 nM bafilomycin A1, compared to the experiments without inhibition of endosomal acidification as shown in Figure 9a. The decrease in reporter gene expression levels shown by PEAs in the presence

of bafilomycin A1 is undoubtedly the advantage of the presence of PEI moieties in the PEA backbone which acidifies these endosomal vesicles, thereby allowing the influx of water and swelling and bursting the endosome. Thus, PEAs with significant bursting capacity in the lysosomal pH range show a much higher transfection capability. The evidence of the reduction in transfection efficiency of PEAs in the presence of bafilomycin A1 strongly suggests the involvement of the proton sponge effect in PEI-mediated transfection. Estimation of Hyperosmolarity of PEAs. Nevertheless, it has not been shown yet whether the presence of glycerol moiety in the PEA backbone has any effect on increased cell cellular uptake and thereby increased transfection efficiency. To elucidate the mechanism of increased transfection efficiency of PEA through increased cellular uptake, we analyzed the reduction in packed cell volume in 293T cells when treated with PEA/ pDNA polyplexes. To determine the impact of the osmotic effect of polyplexes containing PEAs, 293T cells were suspended in DMEM with 10% FBS and GTA/PEI-1.2(1:4)/pGL3 complexes with varying glycerol content (3, 4, and 5 wt %) in the polymer backbone and the same protocol followed as reported by us previously (21). As shown in Figure 9b, GTA/PEI-1.2(1:4)/ pGL3 complexes with increasing glycerol content (viz. 3, 4, and 5 wt %) reduced PCV by 18%, 17%, and 27%, respectively. The differences in the osmolarity of different glycerol solutions and polymer/DNA polyplexes are also shown in Table 2. Pure glycerol was used as a control in the same amounts of


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Figure 8. (a) GFP expressed in the A549 cell line transfected with PEA/pEGFP-N2 complexes at N/P ratio of 30. A1, B1, C1, and D1 represent the fluorescent images of the pEGFP-N2 reporter gene complexed with GTA/PEI-1.2(1:1), GTA/PEI-1.2(1:2), GTA/PEI-1.2(1:4), and PEI 25K, respectively. Phase contrast images are also shown along with fluorescent images, respectively (A2 to D2). (b) pEGFP-N2 expressed in 293T cells transfected with PEA/DNA (pEGFP-N2) complexes at various N/P ratios and analyzed by flow cytometry (*p < 0.1; **p < 0.05; ***p < 0.01, two-tailed Student’s t-test) (n ) 3, error bar represents standard deviation).

concentration (3, 4, and 5 wt %) and revealed 20%, 20%, and 38% reduction in PCV, respectively, whereas almost a 50%

decrease in cell volume was observed as a result of exposure to 10 wt % glycerol in DMEM.

Figure 9. (a) Effect of bafilomycin A1, an inhibitor of vacuolar type H+-ATPase, on pGL3 expression with PEAs at N/P ratio of 30 in serum-free transfection medium determined by luciferase assay. (b) Effect of high osmolarity of GTA/PEI-1.2(1:4)/pGL3 complexes and glycerol concentration on cell volume. The approximate volume of 293T cells was determined using mini-PCV tubes following exposure to the conditions indicated for 5 min. As a control, the cells were suspended for 5 min in DMEM prior to the PCV determination. Each bar represents the average of three independent experiments (*p < 0.1; **p < 0.05; ***p < 0.01, two-tailed Student’s t-test) (n ) 3, error bar represents standard deviation).



Table 2. Osmolarity Measurement of Various Solutions and Complexes osmolarity [mOsm] DMEM + 10% FBS Glycerol GTA/PEI-1.2(1:4)/DNA complexes

3% 4% 5% 3% glycerol (wt %) 4% glycerol (wt %) 5% glycerol (wt %)

354 ( 3 751 ( 4 914 ( 3 1102 ( 3 601 ( 2 649 ( 2 759 ( 7

Grosjean et al. reported the significant decrease in cellular volume and thereby an increase in transient reporter gene expression in nonsynchronized CHO cells after post-transfection shocks (30). In addition, the synergistic effect of glycerol and poly(L-lysine) (PLL) was able to lyse human erythrocytes as well as internal vesicles (microsomes) of H225 cells more efficiently than either glycerol and PLL alone as per the reports of Zauner et al. It was found that vesicular membranes became susceptible to PLL-mediated disruption owing to glycerol labilization, thereby releasing the vesicular content into the cytoplasm. However, unlike calcium phosphate mediated transfection, post-transfection osmotic shocks with glycerol barely increased gene transfer activity in transferrin-PLL mediated gene delivery stating the need for synergism between glycerol and polyplexes during transfection (22). Hyperosmotic glycerol

generally causes shrinkage of cells and thus increases cellular uptake; however, a reduction in transfection efficiency was observed owing to increased cytotoxicity by using glycerol concentration higher than 15% according to the reports of Wilson et al. (31). In the case of polyplexes formed from PEA, the reduction in PCV can be explained by the evidence that the glycerol backbone in PEAs exerted osmotic pressure on the cell membrane, which in turn labilizes the vesicular membrane and facilitates cellular uptake by improving membrane permeability. pEGFP-N2 Expression after Aerosol Administration. Encouraging in Vitro results of PEA-mediated gene delivery, especially reduced cytotoxicity, increased cellular uptake, and significant transfection efficiency, prompted us to investigate its in ViVo gene delivery potential by aerosol administration. One would expect that the presence of glycerol backbone and PEI moieties in PEAs boosts the delivery of nucleic acid in ViVo after aerosol administration. PEA [GTA/PEI-1.2(1:4)] showing optimum transfection efficiency in Vitro was selected for aerosol administration in mice. pEGFP-N2 expression in the lungs after aerosol administration is shown in Figure 10. Although some groups (32, 33) reported the superior efficacy of simple positively charged polyplexes with linear PEI 22 K by aerosol administration, the application of such polyplexes remains restricted because of severe toxicity in the pulmonary region (34). In addition, according to the reports of Koshkina

Figure 10. In ViVo pEGFP-N2 expression analysis after aerosol administration to lungs. A1, B1, and C1 are the fluorescent images of control, PEI 25K, and GTA/PEI-1.2(1:4), respectively. A2, B2, and C2 are the merged images of control, PEI 25K, and GTA/PEI-1.2(1:4), respectively. A3, B3, and C3 are phase contrast images of control, PEI 25K, and GTA/PEI-1.2(1:4), respectively (n ) 3, error bar represents standard deviation).


et al., even though both aerosol and intravenous (i.v.) routes deliver gene to lung, aerosol administration is more promising and advantageous, because it results in substantial deposition of DNA and its extended retention in the pulmonary region (35). GTA/PEI-1.2(1:4)/pEGFP-N2 showed promising GFP fluorescence compared to PEI 25K after aerosol administration as shown in Figure 10. This increased GFP expression level shown by GTA/PEI undoubtedly evidences its ability as a potential gene carrier in Vitro and in ViVo. Because of the large surface area of the pulmonary region, alveolar cells are much more eligible to internalize PEA/pEGFP-N2 polyplexes, resulting in elevated levels of transfection efficiency. On the contrary, smaller levels of gene expression shown by PEI 25K may be supported by its low uptake into the lung and its nonbiodegradability, which leads to cytotoxicity. Despite development of various successful cationic polymers by our group which showed remarkable transfection efficiency both in Vitro and in ViVo, the superior efficacy of the present polymeric system and an earlier one (GDM/PEI-1.2 copolymers) over others is the hyperosmotic effect that it exerts because of the presence of glycerol moieties in the PEA backbone. This present polymer system exhibits a higher gene expression level presumably due to the synergistic effect of endosomal buffering capacity (resulting from PEI amine groups) and the hyperosmotic effect (resulting from glycerol backbone).

CONCLUSIONS The approach described in this paper represents an easy and efficient method to obtain a fairly stable vector system with hyperbranched, degradable, and osmotically active PEAs. These PEAs were successfully synthesized by the Michael addition reaction between GTA and LMW-PEI and exhibited remarkable biophysical properties such as polyplex size below 200 nm and surface charge in the range 15-45 mV suitable for intracellular gene delivery carrier. The presence of ester linkage indicated the controlled hydrolytic degradation of PEAs with a half-life of more than 12 days and was essentially nontoxic in all three cell lines (HeLa, HepG2, and 293T cells) at higher doses as compared with PEI 25K. Furthermore, PEAs exhibited higher levels of transfection efficiencies in Vitro and in ViVo compared with the “gold standard” PEI 25 K and Lipofectamine as a commercial control. The higher transfection levels of PEAs over PEI could be attributed to the synergism arising from the hyperosmotic effect from glycerol backbone and the “proton sponge effect” from PEI residues. Currently, we are conducting more comprehensive studies such as structure-activity relationships of this vector system with regard to the cellular uptake, reduction in cytotoxicity, and increased transfection efficiency.

ACKNOWLEDGMENT This research work was supported by National Research Laboratory of Korean Science and Engineering Foundation by the Korean Government (MEST) (ROA-2008-000-20024-0). National Instrumentation Center for Environmental Management (NICEM) is acknowledged for providing analytical facilities. R. A. was supported by Korea Research Foundation.

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BC900184K

Efficient Gene Delivery with Osmotically Active and Hyperbranched

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