Brushlike Cationic Polymers with Low Charge Density for Gene

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Brush-like cationic polymers with low charge density for gene delivery Jonathan O'Keeffe Ahern, Sigen A, Dezhong Zhou, Yongsheng Gao, Jing Lyu, Zhao Meng, Lara Cutlar, Luca Pierucci, and Wenxin Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01267 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 11, 2017

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Brush-like cationic polymers with low charge density for gene delivery a a *,a a a Jonathan O’Keeffe Ahern‡, Sigen A‡, Dezhong Zhou Yongsheng Gao, Jing Lyu, Zhao Meng, a

a

a a *,a Lara Cutlar, Luca Pierucci, and Wenxin Wang

Charles Institute of Dermatology, University College Dublin, Dublin 4, Ireland.

Keywords: Cationic Polymers, Gene Delivery, Brush-like Polymers, Hydrophobicity, Charge Density

Abstract

Using a combined synthesis approach comprising reversible addition fragmentation transfer (RAFT) polymerization and ring opening reaction (ROR), a series of poly glycidyl methacrylate (polyGMA) polymers were designed and synthesized for gene delivery. These polymers characterised by low cationic charge respective to established gene delivery vectors such as PEI were studied to further elucidate the key structure activity parameters which mediate efficient and biocompatible gene delivery. Compared to PEI these brush-like polymers facilitated markedly improved safety and gene delivery efficiency.

Introduction

ACS Paragon Plus Environment

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Gene therapy has become a promising therapeutic approach for the treatment of various inherited or acquired human diseases1. Despite significant advances in this field, the most challenging aspect remains the design of safe and efficient biocompatible delivery vectors, in a cost-effective manner2, 3. Non-viral gene delivery vectors, particularly in the form of poly-cation based systems have garnered significant interest in recent years owing to their facile synthesis, tunable conformation and enhanced nucleic acid packaging capability3-8 High cationic charge density materials including: polyethylenimine (PEI), polyamidoamine (PAMAM) and poly(2dimethylaminoethyl methacrylate) (PDMAEMA) have all been extensively studied for gene delivery over the last number of decades9, 10. Despite the vast number of cationic materials that have been studied as gene delivery vectors, the key parameters which mediate successful gene delivery has yet to be comprehensively understood11. Charge density plays a decisive role in DNA condensation, shielding from enzymatic degradation, polyplex cellular uptake, endosomal escape, and cytotoxicity2-8,

10, 12

. Given that polymer-plasmid DNA complexes are formed

through electrostatic interactions between the cationic polymer and the anionic DNA, the charge density of polycations is a key determinant of whether a loose or compact polyplex formation is achieved2-4,

6-8, 13

. Possession of a high charge density aids plasmid DNA complexation and

condensation and moreover, polymers containing high nitrogen content enhance the proton buffering capacity of polycations to facilitate escape from early endosomes through rupturing of the endosomal membrane2, 3, 14-17. Since the positive charge of polycations is derived from the protonable nitrogen within the amine groups, high nitrogen content has been identified as an indispensable component of polycation gene delivery vector design rationale. However, while polymers require sufficient charge density to bind to plasmid DNA for successful gene delivery, excess positive charge on the surface of polymers leads to high levels of cytotoxicity due to


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unspecific interactions with negatively charged serum molecules or membranes of cellular components followed by rapid clearance via the reticuloendothelial system4,

12, 14, 16, 17

. A

multitude of different approaches have been utilized to lower the charge densities of polymers to date4, 16-18. Of note, coating polymers with hydrophilic polymers such as poly (ethylene glycol) (PEG) or acetylation of functional groups are readily employed methods for “shielding” and reducing the excess positive charge density on polymers17-20. Hydrophobicity is an oftenunderappreciated factor for conferring successful gene delivery despite its role in promoting polyplex charge inversion, increasing DNA binding through cooperative binding and enhancing endocytosis via strengthening interactions with phospholipid membranes14,

16, 21, 22

. Previous

reports of low charge density polymers, such as poly(N-methyldiethyleneamine sebacate) (PMSC) highlighted the importance of hydrophobicity for polymers with low charge densities. Here, adjusting hydrophobicity was found to influence gene transfection efficiency and was pivotal to compensating for weak ionic interactions between polymers and DNA through strong interactions between hydrophobic units which formed domains that non-covalently crosslinked polyplexes and conferred enhanced particle stability for gene delivery14,

16

. Owing to the

undesirable effects associated with polymers with large charge densities such as PEI (with a nitrogen content around 33%), the exploration into the development of efficient non-viral gene delivery vectors with low charge density and low cytotoxicity is important. Reversible addition-fragmentation chain transfer (RAFT) polymerization is one the most widely used reaction systems owing to its robustness, versatility and broad-spectrum applicability9,

10, 12, 23

. Using RAFT polymerization, libraries of cationic polymers can be

synthesized and tailored to yield gene delivery vectors of desired molecular weights, defined architectures, and post synthesis functionalizable end groups12, 23-25. Given the role that polymer


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characteristics play in facilitating gene delivery, the controllability and versatility of RAFT mediated polymerizations have led to their rise to the forefront of usage as gene delivery vectors 15, 23, 25

. The monomer glycidyl methacrylate (GMA) is widely used in the biomedical field owing

to its low production cost, and the presence of both vinyl and epoxy functional groups12, 26. Its polymer, PolyGMA is a well- studied parent polymer and has been used for the production of a broad spectrum of multifunctional daughter polymers for gene delivery

16, 26

. Herein we report

the synthesis of a series of cationic polyGMA brush-like polymers via RAFT polymerization and subsequent post-synthesis amination through ring-opening reactions (ROR) to yield polymers with low nitrogen content and cationic charge moieties that can facilitate efficient gene delivery in a cytocompatible manner compared to PEI. Whilst these delivery vectors are characterized by significantly lower charge densities in comparison to established gene delivery vectors such as PEI, PAMAM and PDMAEMA we demonstrate that they are capable of complexing with plasmid DNA and facilitating gene delivery. Moreover, given the modular design of our delivery vectors, further trends regarding hydrophobicity and alkyl chain length are examined for their influence on overall transfection efficiency and biocompatibility11.

Materials and Methods Glycidyl methacrylate (GMA, 97%), 2,2’-azobis (2-methylpropionitrile) (AIBN, 98%,) were purchased from Sigma Aldrich and used as received. The chain transfer agent (CTA), 2cyanoprop-2-yl dithiobenzoate was purchased from Sigma Aldrich. Diethyl ether (99.5%, Sigma-Aldrich), chloroform-d (99.8 atom % D, Sigma-Aldrich), methyl ethyl ketone (MEK, 99%, Fisher), ethanol (99.5%, Sigma-Aldrich), acetone (99.97%, Fisher) and dimethyl formamide (DMF, 99.99%, Fisher) were used as received. The amines: undecylamine


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(UDA,98%), dodecylamine (DDA, 99%), tridecylamine (TRDA,98%) and tetradecylamine 1

(TEDA, 95%) were purchased from Sigma Aldrich and used without any purification. H-NMR spectra used for chemical structure and composition confirmation was collected on a Varian NMR system 400 MHz spectrometer. Chloroform-d (7.26 ppm) was used as the deuterated solvent and tetramethylsilane (TMS, 0 ppm) was used as the internal standard at the concentration of 0.03% (v/v). Molecular weight (Mn, Mw) and polydispersity index (PDI) of the polymers were tracked with a gel permeation chromatography (GPC) equipped with a refractive index detector (RI), a viscometer detector (VS DP) and a dual angle light scattering detector (LS o

15° and LS 90°). GPC measurements were performed at 60 C, with columns being eluted with Dimethyl Formamide (DMF) containing 0.1% LiBr at a flow rate of 1mL/min. Preparation of PolyGMA via RAFT polymerization: Initially the homopolymerization of GMA via RAFT was synthesized as follows: GMA (1.42 g, 10 mmol), AIBN (32.8 mg, 0.2 mmol), CTA (88.5 mg, 0.4 mmol) and MEK (30 mL) were added into a round bottomed flask and sealed. The solution was purged with argon for 30 min to remove the oxygen, followed by immersion into an oil bath at 60 °C and monitoring of reaction progress by GPC. Once the desired Mn/Mw had been achieved the polymerization was stopped by exposing the reaction to air. The monomer was removed by precipitating the solution into a large excess of diethyl ether. Ring opening reactions of PolyGMA with amines of different chain length: Aminated PolyGMA polymers were prepared by ring opening reactions. Briefly, PGMA (48 mg, 0.01 mmol) and UDA/DDA/TRDA/TEDA (54.8 mg, 59.3 mg, 63.8 mg, 68.3 mg, 0.32 mmol) o

were dissolved in 400µL of ethanol, placed in a glass scintillation vial and reacted at 90 C for 48 h at a ratio of 1:2 such that no unreacted amines would remain.


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Nuclear Magnetic Resonance (NMR) Analysis: 1H-NMR was performed on a 400MHz Varian NMR system spectrometer. The spectra were analyzed using MestReNova processing software. Chloroform-d and Methanol-d4 were used as the solvents. The chemical shifts were referenced to the lock chloroform-d (7.26 ppm) and methanol-d4 (3.31 ppm), respectively. Zeta Potential Measurements: Aminated PolyGMAs and DNA were prepared as per transfection studies. Firstly polymers and DNA were diluted in 25 mM sodium acetate. Following this, polymers and DNA were mixed together at a polymer: DNA w/w ratio of 30:1 using 1 µg DNA and a 1:1 v/v ratio. After incubating for 15min at room temperature to allow complexation, polyplex solutions were added into a disposable cuvette. Zeta potential was analyzed using a Malvern Zetasizer Nano ZS. All measurements were taken in triplicate at 25 O

C.

Plasmid DNA Binding Assay: DNA encapsulation was measured using the nucleic acid stain, ®

picogreen . Aminated PolyGMAs were firstly dissolved in 25 mM sodium acetate and then mixed with DNA (3 µg) at a 1:1 v/v ratio for polymer: DNA w/w’s of 10:1, 20:1, 30:1. PolymerDNA solutions were incubated for 15 min to allow polyplex formation. Picogreen working solution was prepared as per manufacturer’s protocol (diluting 10 µL of stock into 1.9 mL sodium acetate) and added into polyplex solution at a 1:1 volume ratio. After 5-min incubation, 30µL of polymer-DNA-Picogreen solution was mixed with 200µL DMEM medium (without serum) in a black 96-well plate. Fluorescence was measured on a plate reader (SpectraMax® M3) at excitation of 490 nm and emission of 530 nm. The relative DNA binding efficiency (relative fluorescence (RF)), was calculation by the following relationship: RF = ( FDNA - FSample)/ (FDNA – FBlank) × 100%


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where FDNA is the fluorescence value of a sample with DNA-Picogreen without polymer as a control group, Fsample is the fluorescence value of the polymer-DNA-Picogreen sample and Fblank is the fluorescence value of a sample with DNA-Picogreen without polymer or DNA as a blank group (Picogreen only). Cell Culture: The human derived embryonic kidney cell line (HEK293) were cultured in Dulbecco’s modified Eagle Medium (DMEM) with 10% FBS and 1% Penicillin/Streptomycin o

(P/S). Cells were cultured at 37 C, 5 % CO2 in a humid incubator under standard cell culture techniques. Evaluation of Gene transfection efficiency of aminated PolyGMAs: 24 h hours prior to 4

transfection, cells were seeded in 96-well plate format at a density of 2x10 cells per well in 100 µL cell culture media. 0.25 µg DNA (GFP/Gluciferase) was used per well, and polymer to DNA weight ratios (w/w) 10:1, 20:1, 30:1 utilized, with PEI acting as a positive control at a w/w of 1:1. To prepare polyplex solutions, polymers and DNA were separately dissolved in 25 mM sodium acetate and then DNA solution was added to the polymer solution and vortexed for 10 seconds. Polymer-DNA solutions were then incubated for 15min at room temperature to allow for polyplex formation. After incubation, polyplex solutions were added onto cells in serum free DMEM media. 4 h post transfection serum free media was replaced with full serum media. Transfected cells were analyzed for GLuciferase activity and GFP expression using the Gaussia Luciferase Assay Kit (BioLux®) and a fluorescence microscope (Olympus) respectively 48 h post transfection. Determination of Cell Viability: The biocompatibility of aminated PolyGMA-DNA complexes were evaluated by alamarBlueTM assay (Thermo scientific), which assessed metabolic activity of


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transfected cells. HEK293 Cells were seeded and transfected as described above, with different PolyGMA polymers complexed with 0.25 µg DNA at weight ratios (w/w) 10:1, 20:1, 30:1. 4 hafter transfection under serum free conditions, media was replaced with full serum media. 48 h post-transfection, cell viability was assessed by following the alamarBlueTM assay manufacturers’ protocol. After 2 h incubation with 100 µL alamarBlue reagent, absorbance at ®

570 and 600 nm was recorded on a multi-plate reader (SpectraMax M3) and alamarBlue reduction percentage was calculated.

Statistical Analysis: All data expressed as average ± standard deviations (SD), with SD represented by error bars. Statistical comparisons between the control and treated groups were performed using Student t-tests. Average value and SD’s were calculated from at least three independent experiments. The levels of statistical significance were set at P < 0.05 (*).

Results and Discussion A series of cationic brush-like polymers were synthesized through two steps, as outlined in Scheme 1. First the controlled homopolymerization of PolyGMA was successfully carried out using the RAFT polymerization approach, with AIBN as the initiator. 2-cyanoprop-2-yl dithiobenzoate was utilized as the chain transfer agent (CTA) as it has previously shown to efficiently control the polymerization and yield a homopolymer with a narrow polydispersity index (PDI) and defined molecular weight

26, 27

. Gel permeation chromatography (GPC) was

used to monitor the polymerization reaction (Fig. S1) and upon reaching an Mn of 4.8 kDa, the reaction was terminated by exposure to air and cooling to room temperature. Confirmation of chemical structure and composition of the homopolymer was acquired using 1H-NMR (Fig. S2).


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The second step in synthesizing the brush-like polymers was the post synthesis amination of the PolyGMA homopolymer via ring opening reactions (ROR) of the epoxy groups with primary amines of different chain lengths (Table S1). To ensure the complete functionalization of the epoxy groups, polyGMA was reacted with different amines at a ratio of 1:2, in terms of epoxy to NH, for 48 h at 90 oC as confirmed by 1H-NMR spectra (Fig. S3). Given that successful gene delivery is a multifactorial process, optimal gene delivery agents must satisfy certain physicochemical parameters that modulate gene delivery. It is widely accepted that a prerequisite for gene delivery with polycations is the condensing of DNA into nanoparticle complexes that protect and shield the plasmid DNA from degradation3,

4, 16

. As such plasmid DNA binding

efficiency was assessed using the picogreen assay for each aminated polymer at increasing w/w ratios. Given the low charge density of our aminated PolyGMA polymers, there is a limited capacity for binding of polycations and plasmid DNA and would as such lead us to believe that it would be quite difficult for our polymers to effectively condense and shield the plasmid DNA 15, 16

. However, Figure 1 demonstrates that each polymer was able to successfully bind with at least

50% efficiency and a direct correlation was observed between binding efficiency and increasing w/w. PolyGMA-DDA at 30:1 w/w exhibited the strongest binding of above 90% whereas polyGMA-TEDA at each w/w displayed the lowest binding of DNA. In contrast to polymers with a high charge density where strong ionic interactions dictate the facile condensing of plasmid DNA into nanoparticles, for polymers with low charge density ionic interactions alone might not explain the ability to form stable polyplexes [3a, 6b, 7b]. The ability of our polymers to successfully bind and shield the plasmid DNA despite their limited charge density could be due to modifying hydrophobicity in our polymers which has been identified to promote enhanced


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DNA condensation through cooperative binding16, 21. Our polymer design rationale sought to utilize hydrophobic modifications of the PolyGMA homopolymer with long chain amines of differing lengths of hydrophobic repeat units to take advantage of the compensatory role that hydrophobicity has for low charge density polymers in conferring successful gene delivery. Surface charge is another important factor in the gene delivery process as it modulates cellular uptake. The neutralization of negatively charged plasmid DNA and conferring of moderate positive surface charge onto polymers is favourable to mediate interactions of polyplexes with cells and promoting of polyplex uptake21, 24, 28-38. Given the low charge density of our aminated PolyGMA polymers, under neutral media conditions their Zeta Potential is almost zero. However, upon complexation in sodium acetate, as Fig. S4 demonstrates all polymers successfully negated the charge of the plasmid DNA and contain positively charged surfaces. Taken together with the picogreen DNA binding results, these brush-like polymers can effectively condense DNA and have the desired positive surface charge to mediate traversing of the cell membrane for gene delivery. Having established the ability of these polymers to successfully complex plasmid DNA and have the desired positive surface charge which is known to be desirable in mediating gene delivery, the question remained of whether these low charge density materials could in fact deliver plasmid DNA into cells in an efficient and biocompatible manner. Building on the initial biophysical analyses of the aminated PolyGMA deliver vectors, in-vitro gene transfection capabilities were assessed in human derived embryonic kidney cells (HEK293). Transfection efficiency was firstly evaluated using the gene reporter GFP (Fig. 2). PolyGMA-DDA exhibited the highest GFP expression of all the cationic brush-like polymers, with PolyGMA-TEDA demonstrating the lowest GFP expression. Both PolyGMA-UDA and PolyGMA-TRDA mediated similar transfection capabilities. Furthermore, GLuciferase gene


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transfection was quantified in HEK 293 cells for brush-like polymers at various w/w’s and compared to that of the widely used “gold standard” non-viral gene delivery vector PEI (Fig. 2). For PolyGMA-UDA/DDA/TRDA there was an observed tendency of increased luciferase gene expression with increasing w/w ratios. The reverse of this was evident for PolyGMA-TEDA whereby a significant decrease in GLuciferase expression was seen with increasing w/w ratios. Overall, PolyGMA-UDA/DDA/TRDA all demonstrated comparable to superior GLuciferase expression at each w/w (10:1, 20:1, 30:1) compared to PEI (1:1), with the lowest w/w (10:1) of PolyGMA-TEDA also exhibiting superior transfection efficiency. Successful gene delivery must also be coupled with high levels of cytocompatibility, with safety one of the most important parameters for assessing gene deliver vectors

5, 15, 28-32

. Polymers with high charge densities and

nitrogen content such as PEI bring about cell cytotoxicity through disruption of cellular compartment membranes resulting in cell necrosis and apoptosis4, 15, 16. In contrast, our polymers would be expected to maintain high levels of cell viability. As envisaged our polymers at various w/w ratios maintained superior cell viability in HEK293 cells compared to PEI (Fig. 3). At w/w ratios of 10:1, 20:1 and 30:1 over 80% cell viability was maintained for PolyGMAUDA/DDA/TRDA, conversely PEI complexes induced significant cytotoxicity with cell viability below 60% post transfection. In particular, all aminated PolyGMA delivery vectors bar PolyGMA-TEDA exhibited substantially lower cytotoxicity than PEI at each w/w. For PolyGMA-TEDA a dose-dependent cytotoxicity was observed with cell viability decreasing sharply with increasing w/w ratios.

Conclusion


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In summary, a series of well-defined brush-like, aminated PolyGMA polymers with low charge density were designed and synthesized via RAFT polymerisation and post-polymerization ROR’s as gene delivery vectors. Utilizing the robust RAFT polymerisation system and postpolymerization functionalisation facilitated the structural tailoring of PolyGMA polymers and provides a versatile platform for constructing novel gene delivery vectors. Compared to PEI, whose nitrogen content makes up one third of its elemental composition, the nitrogen content of our aminated PolyGMAs is extremely low. Despite this low charge density, aminated PolyGMAs were able to successfully condense plasmid DNA and demonstrated in-vitro transfection efficiency superior to that of PEI in addition to maintaining higher levels of cell viability. As demonstrated by the results, it is evident that a high charge density in materials is not a necessity to successfully complex DNA and deliver it into cells. All of the low charge density brush-like polymers evaluated here were not only able to complex with DNA to form nanocomplexes of a positive surface charge, but in addition were able to confer efficient gene delivery in a cytocompatible manner. Results from in-vitro transfection efficiency and cell viability demonstrate that overall, PolyGMA-DDA was the optimal polymer formulation as it showed the highest transfection efficiency and cell viability, with PolyGMA-TEDA displaying the lowest levels of transfection efficiency and cell viability. Taking the results into account, the gene delivery and cytotoxicity profile of PolyGMA-TEDA could be explained by its lower DNA binding capability at the highest w/w ratios compared to its shorter alkyl chain length analogues. Moreover, the marked increase in DNA binding in PolyGMA-DDA at increasing w/w ratio’s to over 95 % compared to other PolyGMA delivery vectors and resulting higher gene transfection efficiency and biocompatibility profile highlights the importance that minute changes in the chain length of the flanking amine unit has on modulating transfection efficiency and


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cytotoxicity. The RAFT synthesized PolyGMA vectors designed here provide further insight into overcoming the widely established problem of excess charge for polycations and opens a new avenue for the rational design of efficient, tailorable non-viral gene delivery agents with low nitrogen content.

Figure 1. Assessment of DNA binding capability of aminated PolyGMA delivery vectors at various w/w ratios using picogreen assay.


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Figure 2. GFP and GLuciferase expression of HEK 293 cells 48 h post transfection with the low nitrogen content brush-like polymers. Cells were initially transfected under serum free conditions with PolyGMA-DNA complexes, after 4 h media was replaced with full serum media (containing 10% FBS).


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Figure 3. HEK293 cell viability with PolyGMA-DNA polyplexes at increasing w/w ratios (10:1, 20:1, 30:1) compared to PEI, using 0.25 µg DNA. Cells were transfected under serum free conditions with PolyGMA-DNA complexes, 4 h post transfection media was replaced with full serum media. 48 h post transfection cell viability was assessed using the alamarBlueTM assay.


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Scheme1. Synthesis of brush-like polymers via RAFT polymerization and amination by ROR strategies.


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Author Information Corresponding Author *Corresponding authors: [email protected], [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was funded by Science Foundation Ireland (SFI) Principal Investigator Award (13/IA/1962), Investigator Award (12/IP/1688), Health Research Board (HRA-POR-2013-412) and Irish Research Council CAROLINE Fellowship (CLNE/2017/358). Notes The authors declare no competing financial interests.

Supporting Information Supporting Information Representative GPC and NMR analysis of PolyGMA and Aminated PolyGMA polymers. Zeta potential of PolyGMA-DNA polyplexes and molar concentrations of monomers required for polymer synthesis r. The Supporting Information is available free of charge on the ACS Publications website.


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Table of Contents


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Brushlike Cationic Polymers with Low Charge Density for Gene

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