with High Gene Transfection Potency Across Diverse Cell Types

Dec 6, 2016 - One of the most significant challenges in the development of polymer materials for gene delivery is to understand how topological struct...
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Development of Branched Poly(5-Amino-1-pentanol-co-1,4butanediol Diacrylate) with High Gene Transfection Potency Across Diverse Cell Types Dezhong Zhou,‡,† Yongsheng Gao,‡,† Jonathan O’Keeffe Ahern,‡ Sigen A,‡ Qian Xu,‡ Xiaobei Huang,‡,# Udo Greiser,‡ and Wenxin Wang*,§,‡ §

School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China Charles Institute of Dermatology, University College Dublin, Belfield, Dublin 4, Ireland # College of Materials Science and Engineering, Sichuan University, Chengdu 610064, Sichuan, China ‡

S Supporting Information *

ABSTRACT: One of the most significant challenges in the development of polymer materials for gene delivery is to understand how topological structure influences their transfection properties. Poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32) has proven to be the top-performing gene delivery vector developed to date. Here, we report the development of branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (HC32) as a novel gene vector and elucidate how the topological structure affects gene delivery properties. We found that the branched structure has a big impact on gene transfection efficiency resulting in a superior transfection efficiency of HC32 in comparison to C32 with a linear structure. Mechanistic investigations illustrated that the branched structure enhanced DNA binding, leading to the formation of toroidal polyplexes with smaller size and higher cationic charge. Importantly, the branched structure offers HC32 a larger chemical space for terminal functionalization (e.g., guanidinylation) to further enhance the transfection. Moreover, the optimized HC32 is capable of transfecting a diverse range of cell types including cells that are known to be difficult to transfect such as stem cells and astrocytes with high efficiency. Our study provides a new insight into the rational design of poly(β-amino ester) (PAE) based polymers for gene delivery. KEYWORDS: gene transfection, poly(β-amino ester), highly branched, high efficiency, terminal functionalization, diverse cell types

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INTRODUCTION

distinguishes C32 from other cationic polymers, such as polyethylene imine (PEI), polyamidoamine (PAMAM) dendrimers, and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) etc., is the biodegradability via hydrolytically degradable ester groups, thus reducing the potential accumulative cytotoxicity post transfection.14,15 In a variety of cell types, delivery by C32 proved to result in higher gene expression than transfection mediated by the leading commercially available nonviral vectors, such as jet-PEI and Lipofectamine 2000.7,16−18 In human umbilical vein endothelial cells (HUVECs), the transfection efficiency of C32 is comparable to that of an adenovirus.7 To date, C32 has been used in a multitude of clinically relevant scenarios including gene therapy for ovarian, lung and prostate cancers,19−22 modifying stem cells for treating ischemia,15,23 and gene transfer to glioblastomas.24 While there have been numerous studies on C32, none have reported on C32 containing a

An exponential growth in the development of polymer gene delivery systems has been observed over the past decade, yet translation from the bench to a clinical setting is still hampered by a poor transfection profile.1 Rational design of polymer delivery systems with high efficiencies and reduced cytotoxicities remains a significant challenge,2 mostly as a result of our limited understanding of the structure−activity relationships for denoting fundamental polymer properties.3 Previously, Langer and Anderson et al. reported the development of linear poly(βamino ester)s (LPAEs) for gene delivery.4,5 Over 2350 structurally unique LPAEs have been developed via high throughput synthesis technology.6 Systematical biophysical and transfection screening have identified the top-performing candidate - C32, which was prepared through the conjugated addition of 5-amino-1-pentanol to 1,4-butanediol diacrylate and then end-capped with diamine (Figure 1a).6−10 C32 can effectively bind DNA to formulate 50−250 nm polyplexes showing high cellular uptake,6−8,11 and the multiple tertiary amines on the backbone can facilitate escape of polyplexes into the cytoplasm, where the condensed DNA can be released in a triggered manner.12,13 Moreover, one significant feature that © XXXX American Chemical Society

Received: September 22, 2016 Accepted: November 22, 2016

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DOI: 10.1021/acsami.6b12078 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. (a) Chemical structure of LC32. (b) Representative chemical structure of HC32. (c) Schematic structure of LC32 and HC32.

HPAEs with ease. We predict the “A2+B3+C2” Michael addition strategy, in principle, could be applied to the synthesis of HC32 and utilized for flexible modulation of its branched structures. Moreover, the multiple terminal groups would increase the chemical flexibility for post functionalization, further improving the transfection efficiency.32 We hypothesize the combination of a branched structure and terminal functionalization would achieve superior gene transfection. Herein, we report the development of HC32 (Figure 1b) and evaluate its performance in gene transfection.

branched structure and the effect of branching has on its gene transfection has yet to be explored. Displaying a three-dimensional (3D) structure and multiple terminal units, highly branched polymers are attractive alternatives to dendrimers and possess the added benefit of a more facile and cost-effective synthesis18,25 In comparison to their corresponding linear counterparts, highly branched polymers generally exhibit a high molecular weight dispersity but possess additional terminal groups for post synthesis functionalization.26 There is a growing interest in the utilization of branched polymers as gene delivery vehicles.17,25 Branched versions of PEI, 27 poly( L-lysine),28 PDMAEMA,18 and glycopolymers,29 have all been successfully developed and shown to have both superior DNA binding capabilities and higher gene transfection efficiencies than their linear counterparts. We speculate the branched structure would endow HC32 a greater level of gene transfection capability compared to C32 with linear structure (LC32). Despite of the conceivable advantages, preparation of HC32 has not been accomplished so far because the synthesis of branched poly(β-amino ester)s (HPAEs) has proven to be a long-standing challenge due to the intrinsic gelation tendency during the polymer synthesis process.30 Recently, a new strategy, so-called “A2+B3+C2” Michael addition chemistry, has been developed for the design and synthesis of HPAEs.31,32 This strategy not only circumvents the need for special monomers associated with the conventional “A2+B′BB’’” approach for HPAE synthesis,33 but also affords the ability to tune the chemical and structural properties of

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EXPERIMENTAL SECTION

Materials. Chemicals 1,4-butanediol diacrylate (C, VWR, 98%), trimethylolpropane triacrylate (T, Sigma, 99%), 5-amino-1-pentanol (32, Sigma, 99%), 1,3-diaminopropane (103, Sigma, 99%), 1,11diamino-3,6,9-trioxaundecane (122, Sigma, 98%), lithium bromide (LiBr, Sigma, 99%), 1H-pyrazole-1-carboxamidine hydrochloride (HPCH, Sigma, 99%), and N,N-diisopropylethylamine (DIPEA, Sigma, 99%) were used as received. Solvents, dimethyl sulfoxide (DMSO, Sigma, 99%), dimethylformamide (DMF, Fisher Scientific, 99%), diethyl ether (Sigma, 99%), deuterated chloroform (CDCl3, Sigma, 99.9%), phosphate buffered saline solution (pH 7.4, 0.01 M, Sigma), and Hank’s balanced salt solution (HBSS, Sigma), were used as received. Sodium acetate (Sigma, pH 5.2 ± 0.1, 3 M) was diluted to 0.025 M prior to use. For cell culture work, Dulbecco’s modified Eagle Medium (DMEM), 50% DMEM/50% F12 Ham medium, and RPMI 1640 medium were purchased from Sigma. Keratinocyte Growth Medium 2 (c-20011) was purchased from PromoCell. MesenPRO RSTM Medium was purchased from Invitrogen. Penicillin-streptomycin was purchased from Thermo Fisher Scientific. Fetal bovine serum B

DOI: 10.1021/acsami.6b12078 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces (FBS, Gibco) was filtered through 0.2 μm filters before use. Picogreen assay kit (Life Technologies), Cy3 DNA labeling kit (Mirus), trypsinEDTA solution (Sigma, 0.25%), branched polyethylenimine (PEI, Mw = 25 kDa, Sigma), SuperFect (Qiagen), BioLuxTM Gaussia Luciferase Assay Kit (New England Biolabs), Alamarblue Assay Kit (Invitrogen), 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies) were used as per manufacturers’ protocols. Cell secreted Gaussia Princeps luciferase plasmid (pCMV-GLuc) and Green Fluorescent Protein plasmid (pCMV-GFP) were obtained from New England Biolabs UK, and its expansion, isolation and purification were performed using the Giga-Prep (Qiagen) kits as per protocols. The intercalating agent, propidium iodide (PI, Life Technologies) was used to exclude dead cells from flow cytometry analysis. Synthesis of HC32 and LC32 Polymers. HC32 and LC32 base polymers were first synthesized. The LC32 base polymers were synthesized according to previous reports.7 The HC32 base polymers were synthesized using the “A2+B3+C2” Michael addition reaction.32 The detailed monomer feed ratios are outlined in Table S1. All syntheses followed similar protocols. By varying the monomer feed ratios, polymers of different composition and branched structures were synthesized. Taking the synthesis of HC32−5%-ac as an example, typically, 10 mmol (1.98 g) C, 0.5 mmol (0.148 g) T, and 9.0 mmol (0.923 g) 32 were dissolved in 3.1 mL DMSO, and then reacted at 90 °C. GPC was used to track the growth of weight molecular weight (Mw) and the reaction was stopped by diluting to 100 mg/mL with DMSO when the Mw was approaching 10 000 Da. Then, the reaction mixture was divided into three equal parts and 10 mmol of 32, 103, or 122 were dissolved in DMSO to 100 mg/mL and added to end-cap the base polymers at room temperature for another 48 h. Afterward, the polymers were purified by precipitation with diethyl ether twice to remove the excess monomers, end-capping reagents and oligomers. The final products were dried in the vacuum oven for 48 h to remove the residual solvents, and then dissolved in DMSO to obtain 100 mg/ mL stock solutions and stored at −20 °C for future studies. Guanidinylation of HC32 and LC32 Polymers. Polymer (0.1 mmol), 20.0 mmol of HPCH, and 20.0 mmol DIPEA were dissolved in 20.0 mL of DMSO and reacted at 25 °C for 72 h, and then the reaction mixtures were dialyzed in anhydrous DMSO for 3 days. These products then underwent precipitation three times in diethyl ether to remove the DMSO, unreacted HPCH, DIPEA and dried in the vacuum oven for 48 h. The final products were dissolved in DMSO to 100 mg/mL and stored at −20 °C for future studies. Polymer Characterization. Weight molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (PDI) of polymers were obtained on an Agilent 1260 Infinite gel permeation chromatography (GPC) equipped with a triple detector: (refractive index detector (RI), viscometer detector (VS DP) and dual light scattering detector (LS 15° and LS 90°)). DMF and 0.1% LiBr was used to elute the GPC column (PolarGel-M, 7.5 × 300 mm, two in series) at a flow rate of 1 mL/min at 60 °C. The linear poly(methyl methacrylate) (PMMA) standards were used to calibrate the GPC columns. To track the progress of the polymerization reaction, 20 μL reaction mixture samples were taken at different time points, followed by diluting in 1 mL of DMF and filtering through a 0.2 μm filter prior to GPC measurement. Final polymer products (LC32 and HC32) were assessed by GPC by dissolving 10 mg samples in 2 mL DMF and filtering as above. NMR was utilized to confirm chemical composition and structures of final products. Briefly, polymer samples were dissolved in CDCl3 and 1H NMR spectra was acquired on a 400 MHz Varian Inova spectrometer. All samples were reported in parts per million (ppm) relative to the solvent (7.24 ppm) or internal control (tetramethylsilane 0.00 ppm). For 13C NMR, analysis was performed at 150 MHz and reported in ppm relative to the solvent response (77.2 ppm). DNA Binding Efficiency of HC32 and LC32 Polymers. Picogreen assays were used to measure the DNA binding efficiency of polymers. One microgram of DNA was used for each sample. According to the w/w ratio, polymer solution (100 mg/mL in DMSO) and DNA solution (1 mg/mL in Tris-EDTA buffer) were diluted to a total volume of 30 μL with sodium acetate buffer, respectively, then

vortexed for 10 s to mix and allowed to incubate at room temperature for 10 min. Afterward, 60 μL of Picogreen solution (prepared by diluting 80 μL of Picogreen with 15.92 mL of sodium acetate buffer) was added and left to incubate for 5 min. DMEM cell culture media (200 μL) was added to a black 96-well plate, followed by 30 μL of polyplex/Picogreen solution. Fluorescence was measured using a SpectraMax M3 plate reader equipped with an excitation at 490 nm and an emission at 535 nm. Polyplex Size and Zeta Potential Analysis. Polyplex sizes and zeta potentials were measured with a Malvern Instruments Zetasizer (Nano-2590) at a scattering angle of 173°. For polyplex preparation, DNA solutions and polymer solutions were diluted with sodium acetate buffer, respectively to a w/w of 30:1, vortexed for 10 s to mix and then permitted to incubate for 10 min at room temperature. Following incubation 1 mL of PBS was added. Polyplex sizes and zeta potentials were measured a minimum of 4 times. Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) Morphological Assessment of Polyplexes. Polyplexes were prepared as mentioned above. Afterward, polyplexes were washed with deionized water to remove any remaining salts from centrifugation and resuspension. Next, 10 μL of polyplex solution was cast onto Formvar support films on 200 mesh copper grids and freezedried immediately. Images were captured on an FEI Tecnai 120 TEM at 120 kV in the UCD Conway Imaging Core Center. For morphological investigation of polyplexes with AFM, polyplexes were prepared as mentioned above. Polyplex suspensions (50 μL) were dropped onto a freshly cleaved mica substrate and incubated for 2 min at room temperature. Next the surface was flushed with 200 μL of distilled water three times and dried with N2 flow. An Asylum Research MFP-3D AFM was used to capture images of the polyplexes. Cell Culture. The Swiss albino mouse embryo tissue cell line 3T3 (ATCC), American green monkey kidney fibroblast-like cell line COS7 (ATCC), human-derived renal proximal tubular cell line HKC8 (ATCC), human cervical cancer cell line HeLa (ATCC), Neu7 astrocytes (Gibco) and rat adipose-derived stem cells rADSC (passage four, Gibco) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (P/S). The hepatocellular carcinoma cell line HepG2 (ATCC) was cultured in RPMI 1640 media containing 10% FBS and 1% P/S. The normal human keratinocytes NHK and the recessive dystrophic epidermolysis bullosa keratinocytes RDEBK (a gift from Dr. Fernando Larcher, Madrid, Spain) were cultured in keratinocyte growth medium 2 (c-20011 pROMOCELL) with 1% P/ S. The SHSY-5Y neuroblastoma (passage five, a gift from Dr. Ben Newland, Dresden, Germany) were cultured in 50% DMEM/50% F12 Ham media containing 10% FBS and 1% P/S. All cells were cultured at 37 °C, 5% CO2 in a humid incubator under standard cell culture techniques. Evaluation of Gene Transfection Efficiency Using Gluciferase Assays and Flow Cytometry. For gene transfection experiments, the Gluciferase coding DNA plasmid was used to quantify transfection efficiency. Briefly, cells were seeded on 96-well plates a density of 1 × 104 cells per well in 100 μL of serum containing media and cultured until 70−90% confluence. rADSC’s and SHSY-5Y cells used were below passage four and five, respectively. Prior to transfection, conditions were optimized for the commercial reagents PEI and Superfect as per manufactures protocols and previously publications.18 The commercial reagents were used in optimized formulations: w/w = 3:1 (PEI) or w/w = 9:1 (SuperFect). For astrocytes and stem cells 0.25 μg of DNA per well was used, while for immortalized cells 0.5 μg of DNA was utilized. Polyplexes were prepared as mentioned above: Briefly, equal volumes of polymer and DNA solutions were prepared by diluting with sodium acetate according to the w/w ratios. Next, polymer solutions were mixed with the DNA solution and vortexed for 10 s before incubating at room temperature for 10 min. Cell culture media was added to the polyplex solution to bring the final volume to 100 μL. The cell media was removed from the culture plates and quickly replaced with the polyplex solution and left on cells to culture for a further 48 h. Measurement of Gluciferase activity was carried out as per manufacturer’s protocol using a SpectraMax M3 plate reader with C

DOI: 10.1021/acsami.6b12078 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. Gluciferase activity of HeLa (a), RDEBK (b), and rADSC (c) cells after transfection with various LC32 and HC32 polymers at different polymer/DNA weight ratios (w/w). HC32 polymers demonstrate more favorable transfection efficiencies than LC32 polymers. Error bars indicate standard deviation, and asterisks indicate a superior Gluciferase activity in comparison with PEI (*p < 0.05). RLU = relative light units. kit was used to label DNA as per manufacturers protocol. Cells were seeded at a density of 5 × 103 cells/well on a 96 well plate; 0.1 μg of DNA per well was used for gene transfection studies at w/w of 30:1. Media was removed from cells 4 h post transfection and cells were washed three times with ice cold PBS before undergoing fixation with 4% paraformaldehyde. Cells were permeabilized with 0.1% triton-100 and stained with DAPI. Cells were visualized using a fluorescence microscope (Olympus IX81).34 Statistics. GraphPad prism (version 5 GraphPad Software, San Diego, CA, USA) was used to analyze all gene transfection data. To determine a normal distribution, D’Agostino and Pearson omnibus normality tests were applied followed by a one-way ANOVA and Tukey’s post hoc analysis once a normal distribution was evident. Statistical significance was attributed to results indicating p values of 34% transfection efficiency. Comparatively, the Neu7 astrocytes exhibited much higher gene transfection efficiency (57%), which is possibly due to the different proliferation rate in comparison with the SHSY5Y.40−42 It should be noted that fluorescence microscope images also confirmed the superior transfection capability of guanidinylated HC32−10%-122 in various cell types (Figure 5b). Importantly, the high level of gene transfection capability of HC32−10%-122 was not accompanied by an obvious reduction of cell viability, even on the fragile astrocytes and stem cells, >80% cell viability was still preserved (Figure S20). These results are very promising, as achieving high transfection efficiencies while maintaining low levels of cytotoxicity could be achieved simultaneously in diverse cell types relevant for clinical applications.

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CONCLUSIONS In conclusion, via the “A2+B3+C2” synthesis strategy, HC32 with varying degrees of branching were designed and prepared. Structure−activity relationship indicates that in comparison with the linear C32 counterpart, the branched structure can improve gene transfection efficiency by facilitating the DNA binding and formulation of smaller and toroidal polyplexes with higher surface charge. HC32 is superior to the LC32 in gene transfection. In addition, the transfection efficiency can be further enhanced via terminal functionalization. The high transfection profile, in terms of both high efficiency and safety, has been achieved on diverse cell types. It can be expected that HC32 will provide promising alternatives for many gene delivery applications. However, prior to translation to clinical applications, the transfection efficiency and safety profile of the HC32 in vivo should be further verified. The long-term stability of the HC32/DNA polyplexes should also be validated. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b12078. Polymer synthesis and characterization, cell viabilities, and polyplex uptake (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wenxin Wang: 0000-0002-5053-0611 Author Contributions †

D.Z. and Y.G. contributed equally to this work.

Notes

The authors declare no competing financial interest. H

DOI: 10.1021/acsami.6b12078 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.6b12078 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX