Cationic Liposomes Modified with Polyallylamine as a Gene Carrier : Preparation , Characterization and Transfection Efficiency Evaluation

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(4), 515-520 doi: 10.15171/apb.2016.065 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Disadvantages of viral vectors are serious concerns that restricted their application.Although they are efficient systems in gene delivery, their safety is not reliable. 1These considerations favor the application of non-viral vectors over viral systems for gene delivery of genetic or acquired diseases.Regarding this fact, synthetic vectors play an important role in gene therapy.Non-viral vectors showed limited transfection efficiency in clinical applications.Most widely used synthetic DNA delivery systems generally consist of three categories: cationic polymer (polyplex), cationic lipid (lipoplex) or a mixture of these (lipopolyplex). 2,3Among these carriers, cationic polymers (polycations) are the most widely used.Polycations and DNA form compact complexes by electrostatic bonds between negatively charged DNA and positively charged polymers. 4,5The whole system carries net positive charge which facilitates the interaction with negatively charged cell surface, leading to better endocytosis. 6After endocytosis, nanoparticles undergo acidic pH of lysosomes.Endosomal escape is considered to be one of the most important steps of gene delivery. 3Polyallylamine (PAA) is one of less investigated cationic polymer which has high density of primary amino groups (as free amine or as cationic ammonium salt).High positive charge density of PAA and the other polycationic polymers is the main reason for their cytotoxicity. 6Another limitation for application of PAA in gene delivery is low buffering capacity. 3 The efficiency of gene delivery can be increased by modification of the polycation structure to reach an optimized vector.Different chemical modifications could decrease cytotoxicity and improve transfection efficiency of PAA.Boussif et al. used glycolylated derivatives of PAA. 7 Their efforts decreased the cytotoxicity of PAA-DNA complex and also increased the transfection efficiency of this complex.Nimesh et al. prepared the nanocomplexes composed of PAAdextran-DNA. 6They demonstrated transfection efficiency of these nanoparticles in HEK 293 cells increased and cytotoxicity reduced significantly compared to PAA-DNA nanoparticles.Conjugation of imidazole (as a mildly basic group) to PAA was also performed to increase the proton sponge effect and enhance endosomal escape. 8n the previous studies, we tried to modify PAA by acrylate 3 and 6-bromoalkanoic acid 9 derivatives with different chain lengths to achieve a library of compounds with more hydrophobic characteristics and we used them in polyplex structure in order to reduce toxicity and to improve the interaction with cell surface as well as maintaining the buffering capacity.
In the present study, we selected most successful derivatives of our previous studies in gene delivery and used them in lipoplex structure in order to evaluate if the transfection properties could improve or not.We expected to achieve facilitated passage through cell membrane, better endosomal release (through flip-flop effect) and decreased cytotoxicity.

Modification of PAA
The strategy of PAA modification was to convert the primary amines of PAA to the secondary amines using acrylate or 6-bromoalkanoic acid derivatives.Based on the previous studies, the reaction can take place in water free solution without the use of conjunctive reagents. 3Modification was performed with hexyl acrylate in substitution percent of 10, 30 and 50; butyl acrylate in substitution percent of 50 and also with 6bromohexanoic acid and 6-bromodecanoic acid in substitution percent of 30 and 50 of each.Chain length and substitution percent of each derivative was selected based on the results of our previous studies. 3,9he modified polymers were labeled as HAX or BAX or BHX or BDX in which X is percentage of PAA primary amines substituted with acrylate or bromoalkane derivatives, HA is hexyl acrylate, BA is butyl acrylate, BH is 6-bromohexanoic acid and BD is 6-bromodecanoic acid.Briefly, various amounts of acrylate or 6bromoalkanoic acid derivatives were dissolved in dimethylformamide (DMF).This solution was added dropwise to the stirring solution of PAA 15 kDa (0.1 g in 5 ml DMF) and the reaction was stirred for 24 overnight at room temperature.After 24 hours, the reaction mixture was dialyzed once against 0.25 M NaCl and twice against water (10,000 Da cut-off dialysis tubes) in order to remove unreacted agents.The solution of final product was freeze dried. 3,9eparation of liposomes For preparation of DOTAP:modified PAA liposomes (10 mg/ml based on DOTAP), 10:1 mole ratio of DOTAP and lipopolymer were dissolved in methanolchloroform (1:1 v/v) solvent.After complete dissolution, the organic solvent was evaporated by rotary evaporator (Heidolph, Germany) in order to form a thin lipid film.The thin film was then hydrated by deionized water at 50 °C and the container was placed in bath sonicator (40 °C) (Branson, USA) to form the liposomal vesicles.To prepare DOTAP: cholesterol liposome (10 mg/ml based on DOTAP), 1:1 molar ratio of DOTAP and cholesterol were dissolved in the same organic solvent and liposomes were prepared in the same procedure described above.In order to reduce the size, liposomal formulations were extruded through 800, 400 and 100 nm polycarbonate membranes repeatedly at 50 °C using Thermobarrel extruder (Northern Lipid, Canada).

Preparation of lipoplexes and polyplexes
Three carrier to plasmid (C/P) mass ratios (0.5, 1.5 and 3) of lipoplexes (sample and control) and corresponded polyplexes (PAA or modified PAA) were premixed and left for 20 min at room temperature to form structure.Liposome-protamine-DNA (LPD) complexes were prepared as a control.DOTAP: cholesterol liposomes were used in these structures.Particle size, polydispersity index (PDI) and zeta potential of nanocomplexes were analyzed with Zetasizer Nano ZS (Malvern Instruments, UK) after suitable dilution.

Ethidium bromide test
In order to evaluate the pDNA condensation ability of prepared vectors, ethidium bromide (EtBr) test was performed.Ethidium bromide in HBG buffer (HEPES buffer + glucose 5%) was used in this test.After adding sequential 2.5 µl of vector to the mixture of 0.5 µl pDNA solution (1 mg/ml) and 1 ml ethidium bromide (0.4 µl/ml), the spectrofluorometer (Jasco, Japan) read the light emission.The fluorescence intensity was measured at an excitation and emission wavelength of 510 and 590 nm.The lowest light emission showed the best condensation ability.penicillin at 100 U/ml.These cells were incubated at 37 °C under an atmosphere containing 5% CO 2 .Cells were seeded in 96-well plates in a density of 1 × 10 4 cells per well.Lipoplexes were prepared in C/P ratios of 0.5, 1.5 and 3 and added to the cells in 5 repetitions.After 3-4 hours in 37 °C incubator, medium was replaced and further incubation in 37 °C was done for 24 hours.Transfection and lysis buffer were added to each well.The percentage of transfected cells was determined reading Green fluorescent protein (GFP) fluorescence by fluorescent plate reader (Victor X5, Perkin-Elmer, USA).Excitation and emission wavelength was adjusted on 498 and 535 nm, respectively.For cytotoxicity evaluation, metabolic activity was measured using MTT assay.After 24 hours incubation, seeded cells were treated with the same amounts of lipoplexes used for transfection experiment.After 4 hours of incubation, the medium replacement was done.24 hours later, 10 µl of MTT solution (5 mg/ml in sterile PBS) was added to each well.After 2h hour incubation in 37 °C, the medium was removed and 100 μl of dimethyl sulfoxide added and the plates were put on shaker incubator for 30 min (5000 rpm and 37 °C).Results were read in ELISA reader apparatus (Statfax-2100, Awareness Technology, USA) at 590 nm (reference wavelength 630 nm).Cell viability was expressed as a percent relative to untreated cells.

Statistical analysis
One-way ANOVA and Tukey-Kramer test was used to analyze the obtained data.Differences were statistically significant if the P-value was less than 0.05.

Results
Mean size, polydispersity index (PDI) and zeta potential of lipoplexes were summarized in Table 1.All the complexes showed positive surface charge and a mean size between 136 to 266 nm with PDI bellow 0.5.Increasing in grafting percent did not make a significant difference in surface charges but increased mean size in some cases.All vectors condensed the pDNA at C/P ratio of 1.5 (Figure 1).Increasing in the grafting percent of polymer had no impressive effect on condensation ability of vector.Almost the same pattern was observed in all cases.Transfection efficiency was significantly decreased by using of lipoplexes containing PAA 15 KDa modified with different 6-bromoalkanoic acid compared to the same polyplexes in selected C/P ratios (P<0.05).The difference in transfection ability of all lipoplexes in all C/P ratios was not significant compared to LPD (Figure 2).Transfection efficiency of lipoplexes in C/P 3 was significantly higher than unmodified PAA (P<0.05) but they could not make a significant difference in lower C/P ratios (Figure 2).These results showed that lipoplexes have better gene transfer ability compared to unmodified PAA in higher C/P ratios.Lipoplexes containing modified PAA 15 kDa with acrylate derivatives (in different grafting degrees) significantly increased the transfection efficiency compared to analogous polyplexes in selected C/P ratios (P<0.05) and most of them showed higher transfection activity than LPD (Figure 3).All new lipoplexes in all C/P ratios made remarkable higher gene transfer in comparison with unmodified PAA.Among lipoplexes, the highest gene transfer ability belonged to lipoplex composed of PAA 15 kDa modified with 10% hexyl acrylate at C/P ratio of 3 (Figure 3).As shown in Figures 4 and 5, most vectors showed noticeable cell cytotoxicity.The majority of new nanocomplexes showed increasing cytotoxicity by increasing in C/P ratio.The differences in cytotoxicity of lipoplexes and their analogous polyplexes were not significant in C/P 0.5 and 1.5.Lipoplex composed of modified PAA with 10% hexyl acrylate (lipoplex HA10) showed significant lower cell toxicity compared to analogous polyplex in C/P 3 (P<0.05),but lipoplex containing modified PAA with 30% hexyl acrylate (lipoplex HA 30) showed higher cytotoxicity than analogous polyplex (P<0.05).Modification with acrylate derivatives could decrease cytotoxicity compared to unmodified PAA, this difference became remarkable by increasing in C/P ratio.
The difference in cytotoxicity of lipoplexes and polyplexes was not significant in C/P ratio of 0.5 and 1.5, but in C/P 3 lipoplexes showed increased cell toxicity compared to their corresponding polyplexes.Metabolic activity in C/P ratios of 1.5 and 3 in all grafting percent was higher than unmodified PAA.About the differences between these nanolipoplexes and LPD, in most cases there were no significant differences.

Discussion
Cationic polymers and lipids can cause transient destabilization of endosomal membrane, but their mechanisms of action are different.For cationic polymers some amine groups are involved in electrostatic interaction with negatively charged pDNA, and other amine groups contributed in endosomal disruption at acidic pH (proton sponge effect).Cationic lipids have fusogenic features.They help endosomal scape via bilayer-to-micelle conversion and lamellar-toinverted hexagonal transition. 1 Regarding the fact that osmotic endosomal swelling is not induced by PAA, so considering of another strategy for endosomal scape can improve the efficiency of transfection.For this purpose, using cationic lipid beside PAA (in the structure of lipoplex) can be effective. 10n the present study, we tried to improve transfection efficiency of modified PAA 15 kDa.A high density of primary amine groups is covering the structure of PAA.
We investigated the effect of substitution of primary amines with hydrophobic moieties in gene transfection ability and cell toxicity of this polymer in our previous study. 10The synthesized polymer beside DOTAP was used in liposome preparation in the present study.This modification could successfully improve gene Cationic liposomes modified with polyallylamine Advanced Pharmaceutical Bulletin, 2016, 6(4), 515-520 transfection ability of some new vectors compared to polyplexes of analogous modified PAA.PAA is a cationic polymer that has been less investigated.Cell toxicity of this polymer is not acceptable and low buffering capacity can't induce enough endosomal disruption so transfection efficiency is low. 3To overcome these limitations, some modifications have been done on this polymer.The molecular weight of 15 kDa for PAA was selected according to our previous studies results. 3,9Cationic nature of this polymer appears to be the main reason of cytotoxicity. 6We tried to increase cell viability by covering different percentages of amine groups with acrylate and 6-bromodekanoic acid derivatives.These modified polymers were used in the structure of a new nanoplex gene delivery system composing of modified PAA along with DOTAP (1:10 molar ratio) in liposomal form to evaluate transgenic characteristics and cell viability.
The ethidium bromide dye exclusion assay was done for evaluation of DNA condensation ability of new vectors.Our previous studies showed that unmodified PAA could condense pDNA in C/P ratio of 0.5. 3 These results confirmed that modification of PAA decreased surface positive charge and thereupon condensation occurred in higher C/P ratios (C/P 1.5).Polycationic polymers form nanocomplexes after interaction with negatively charged DNA.This compaction is necessary for efficient cell uptake. 11Although DNA condensation ability of vector is an advantage, DNA release from vector after cell internalization also would be important for efficient gene delivery to the nucleus.Consequently, optimal balance between condensation and separation is needed. 12Previous researches revealed that particle size is an important parameter that can determine cell internalization mechanism.Rejman et al. showed that particles less than 200 nm are entering to the cells by clathrin-mediated endocytosis and particles with a size of 200-500 nm are entering through the caveolaemediated endocytosis pathway. 13In first pathway, nanoparticles are faced with acidic pH of early and late endosome and in second one they are not faced lysosome. 14ll nanoparticles showed positive surface charge.
Positive zeta potential prevents aggregation by electrostatic repulsions and can help attachment to anionic cell surfaces. 10It expected that increasing in grafting percent decrease the positive charge of modified PAA.But the differences between surface charges of liposomal nanoparticles were not significant.This could be related to the presence of DOTAP (as a cationic lipid) along with these cationic polymers.On the other hand, the size of these structures is dependent to the level of compaction induced by their surface positivity.Consequently, the differences between their particle sizes were not significant too.Hydrophobic-hydrophilic balance, buffering capacity of modified vectors and nuclear localization are three important factors that affect their transfection efficiency.The variation was observed in transfection after modification can be related to these factors. 3,10n this study, transfection efficacy of new nano-vectors did not follow up a regular pattern.This can be related to change in hydrophobic-hydrophilic balance induced by modification which can change interaction of nanoparticles with cell membrane and cell internalization.These hydrophobic interactions are known to play a key role in the binding of the preparations to the hydrophobic surface of cell membrane and endocytosis. 10Also using modified polymer in liposomal formulation can alter endosomal escape capability as an important part of successful gene delivery.The exact effect of modification type on internalization and endosomal release is unclear.
Previously, it has shown that modification of PEI 750 KDa with 6-bromoalkanoic acids was done and 6bromohexanoic acid created the best transfection efficiency. 15Long alkyl chains have endosomolytic properties that can improve transfection activity. 16This improvement was optimum for the chain length of 6bromohexanoic acid.The transfection data indicated that the alkylation enhanced the ability of unmodified PAA to transfer a reporter gene into Neuro2A cell line.But using DOTAP along with the same modified polymers decreased gene transfer efficiency compared to their analogous polyplexes.This can be related to impaired hydrophilic-hydrophobic balance or impaired endosomal release.
In one previous study, PAMAM modification with acrylate derivatives improved gene delivery ability. 17In our study, lipoplexes showed better transfection than their analogous polyplexes in some cases.Presumably using cationic lipid beside these polymers was improved cell internalization or endosomal escape.Among all vectors of this group lipoplex containing modified PAA with 10% hexyl acrylate (lipoplex HA10) showed the best transfection efficiency.In a previous study, lipopolyplex contained this modified PAA, enhanced gene transfer ability obviously. 10Our results in both kind of modification (acrylate and 6bromoalkanoic acid derivatives) revealed better transfection activity for six-carbon chain components in lipoplex structures.The cytotoxicity of vectors was increased by increasing in C/P ratio.Cytotoxicity was dependent to the surface zeta potential and increased by increasing in positive charge.Positively charged particles can damage cells following an electrostatic interaction with cell surface. 18etabolic activity of synthesized vectors was ranged from 50%-95% of control group.The highest toxicity level was related to C/P 3 of unmodified PAA and modifications could decrease cell toxicity of vectors by decreasing positive zeta potential.

Conclusion
Among prepared vectors, lipoplex HA10 (C/P 0.5) was the best vectors for gene delivery due to their capability in transfection and their low cytotoxicity (90% cell viability).

Figure 1 .
Figure 1.Ethidium bromide test in order to evaluate the DNA condensation ability of A) vectors prepared by DOTAP:PAA modified by bromoalkane derivatives, B) vectors prepared by DOTAP:PAA modified by acrylate derivatives