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Thiolactone Chemistry-Based Combinatorial Methodology to Construct Multifunctional Polymers for Efficacious Gene Delivery Zengshi Zha, Yongyi Hu, Jean Mukerabigwi, Weijian Chen, Yuheng Wang, Chuanxin He, and Zhishen Ge Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00672 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Bioconjugate Chemistry

Thiolactone Chemistry-Based Combinatorial Methodology to Construct Multifunctional Polymers for Efficacious Gene Delivery Zengshi Zha,† Yongyi Hu,† Jean Felix Mukerabigwi,† Weijian Chen,† Yuheng Wang,† Chuanxin He,‡ and Zhishen Ge*,† †

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, Anhui, China ‡ College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, Guangdong, China. Corresponding Author: * E-mail: [email protected] ABSTRACT: Hydrophobic segments and amino moieties in polymeric nonviral gene vectors play important roles in overcoming a cascade of barriers for efficient gene delivery. However, it remains a great challenge to facilely construct well-defined multifunctional polymers through optimization of the amino and hydrophobic groups. Herein, we choose thiolactone chemistry to perform the ring opening reaction of varying hydrophobic groups-modified thiolactones by various amines to generate mercapto groups for further Michael addition reaction with poly[2-(acryloyloxy)ethyl methacrylate] (PAOEMA). Based on the combinatorial methodology, a series of multifunctional polymers were prepared and screened. The polymer (P3D) from tetraethylenepentamine and heptafluorobutyric acid-functionalized thiolactone is the most efficacious one with significantly higher gene transfection efficiency and lower cytotoxicity compared with polyethylenimine (PEI) (branched average Mw ~25,000 Da) and Lipofectamine 2000. Cellular uptake and intracellular distribution studies indicate that P3D complexes show high-efficiency endocytosis and excellent endosomal escape. Accordingly, thiolactone chemistry-based combinatorial methodology allows for facile integration of multifunctional groups to prepare simultaneous efficacious and low-cytotoxic gene delivery vectors.

Gene therapy displayed more and more potent capability to treat various intractable diseases.1-3 However, safe and effective delivery of gene materials into the precise sites has been a central challenge for the application of gene therapy.4,5 Considering the safety concerns of viral gene delivery vectors, various synthetic gene delivery carriers were developed in the recent decades.6-8 Because of facile preparation, easy modification, and a profusion of diverse chemical structures, synthetic polymers have attracted increasing attention.9-12 Notably, to achieve high-efficiency gene transfection, the gene delivery vectors must overcome a cascade of barriers in the extracellular and intracellular environment, primarily including effective complexation and condensation of gene materials, efficient cellular internalization, endosomal escape prior to trafficking into lysosomes, cytosolic transport, and disintegration of the polyplexes for nuclear transportation and transcription.13-18 These complicated barriers required the polymers with multifunctional properties aiming at all of them for efficient gene delivery. To address the delivery barriers, diverse strategies have been developed via integration of multifunctional groups and optimization of polymer structures.19,20 For example, different cationic functionalities, such as tertiary, secondary and primary amines were implemented into polymeric vectors to increase the ability of DNA condensation and gene delivery capabilities.21,22 Optimization of amines can not only condense DNA efficiently but also improve the cellular uptake and endosomal escape efficiency.23 In addition, the varying hydrophobic groups affect the properties of the complexes via reduction of charge density, increasing the complex stability and interaction with cellular membranes, and even endosomal escape capability.24,25 For example, fluorination has been proved

to dramatically improve the cellular internalization and intracellular endosomal escape of the complexes.26-32 In general, the hydrophobic moieties and amine types significantly affect the final gene transfection efficiency. Scheme 1. Thiolactone chemistry-based combinatorial synthesis of multifunctional polymers for efficient pDNA delivery via one-pot synthetic route.

Combinatorial approaches have been developed to facilitate the generation of structurally diverse libraries of gene delivery polymers through screening the hydrophobic moieties and amine types. Langer and Anderson et al.33-35 developed a li-

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brary of degradable chemically diverse poly(β-amino esters) (PAE) polymers, opening up new avenues of high-throughput synthesis of polymers for gene delivery. Recently, Wang et al.36 further developed highly branched PAE polymers. Moreover, the others reported scalable synthetic strategies to prepare multifunctional polymer libraries via copolymerization of different monomers with varying functional moieties, or postpolymerization modification of the polymers with varying functional molecules.37-39 These progresses have demonstrated that high-throughput screening and modification may accelerate the design and screening of more effective and safer polymers for gene delivery. However, more versatile and facile methodologies still need to be developed for accurate control of polymer molecular weights (MW) as well as the ratios and types of hydrophobic and amino groups, and screening more efficacious and low-toxicity polymer gene carriers. Herein, we for the first time introduce thiolactone chemistry into the synthesis of polymeric gene delivery vectors due to the combinatorial properties of multicomponent reaction so that it can be used to screen the hydrophobic and amino moieties.40,41 In this approach, we used a one-pot combination synthesis via the aminolysis of the thiolactone unit and release of a mercapto group for Michael addition reaction with poly[2(acryloyloxy)ethyl methacrylate] (PAOEMA). Briefly, thiolactone rings functioned with various hydrophobic moieties were examined to open upon aminolysis by diverse amines containing different repeating aminoethylene units, and the in situ generated thiols further reacted with the allyl double bonds of PAOEMA. Afterward, we evaluated the relationship between their structures and biological properties, including cellular uptake, endosomal escape ability, cell viability, and

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transfection efficiency. From these studies, we identified one polymer, P3D based on tetraethylenepentamine and heptafluorobutyric acid-functionalized thiolactone, showed the highest gene transfection efficiency with low associated cytotoxicity. We synthesized the polymers via thiolactone chemistry in one-pot reaction. To validate the feasibility of the proposed methodology, typical amines and alkyl moieties were selected according to the previously reported systems.23,29 Firstly, D,Lhomocysteine thiolactone hydrochloride was reacted with valeric acid, nonanoic acid, heptafluorobutyric acid via amidation reaction at room temperature to yield functionalized thiolactones containing a variety of alkyl moieties (1,2,3), which were characterized by 1H NMR and 13C NMR clearly (Figures S1, S2, and S3). Poly(2-(acryloyloxy)ethyl methacrylate) (PAOEMA) with the degree of polymerization (DP) of 84 and narrow molecular weight distribution (Mw/Mn = 1.18) was successfully prepared by the esterification between poly(2-hydroxyethyl methacrylate) (PHEMA) and acryloyl chloride (Scheme S1 and Figures S4 and S5). Notably, PHEMA with outstanding biocompatibility has been widely used in the field of biomedical materials.42-44 Subsequently, in one pot upon addition of functionalized thiolactones and amines, thiol precursors produced from aminolysis reaction of the thiolactones (1, 2, or 3) by amines (A, B, C, D, or E) were reacted with PAOEMA via Michael addition reaction (Scheme 1). The one-pot two-step reaction was very efficient and fast, the conversion of acrylate groups was more than 95% with very small amount unreacted in less than 15 minutes determined from 1H NMR analysis (Figure S6). The obtained polymers were also treated by di(tert-butyl) carbonate to protect

Figure 1. In vitro transfection efficiency of the polyplexes loading Luc-pDNA and cell viability after treatment with various concentrations of polymers. Relative gene transfection efficiency of various 15 polymers at a weight ratio of 30:1 in (A) HeLa and (C) HepG2 cell lines. Transfections by bPEI and Lipofectamine 2000 were performed according to the manufacturer’s standard protocols. The same amount of pDNA was used for all transfection experiments. The luciferase transfection results were expressed in relative light units (RLU) per mg of cellular protein. Data are expressed as the mean ± standard error (SD, n = 4). Heat map of cell viability of (B) HeLa and (D) HepG2 cell lines after incubation with a series of polymer concentrations (1, 5, 10, 20, and 50 µg/mL). Viability is given as the percentage of viable cells after treatment for two days and measured by MTT assay.


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Bioconjugate Chemistry

amino groups for gel permeation chromatography (GPC) characterization (Figure S7). The results showed monodisperse peak without shoulders indicating no crosslinked polymers obtained. To rule out the possibility of the Michael addition reaction between the amines and acrylate moieties under the same condition for the polymer preparation, we also treated the polymer PAOEMA by DET in the absence of thiolactones under the same conditions at room temperature for 15 min. 1H NMR analysis revealed that negligible amineacrylate reaction occurred likely due to the short reaction time and mild reaction conditions (Figure S8). Presumably, the reactivity of thiol group is much higher than that of amine group due to easier nucleophilic addition onto acrylate.45-47 Accordingly, thiolactones as thiol precursors followed by thiol-acrylate Michael addition reaction show the potential for preparation of well-defined multifunctional polymers. The obtained polymers were denoted according to the thiolactones and amines for the following investigation. Totally 15 kinds of polymers were synthesized, in which two parameters were varied, including hydrophobic groups and the amines with varying numbers of repeating aminoethylene units. To investigate the potential for condensation of plasmid DNA (pDNA) by these polymers, the agarose gel electrophoresis was carried out to evaluate the binding ability of these polymers. All polymers displayed excellent pDNA retarding abilities at the weight ratio of 20:1. The representative images of P3D complexes were shown in Figure S9. Moreover, polyplexes from the polymers and pDNA were characterized by measuring the size distributions and ζpotentials at pH 7.4 and 37 oC by Zetasizer. At weight ratio of 30:1, all polyplexes had a similar size of approximately 120 nm with relatively uniform size distributions, and a constant ζ-potential of nearly 13 mV (Figure S10 and S11). As compared with polyethylenimine (PEI) (branched average Mw ~25,000 Da) at N/P of 10, the polyplexes showed more compact pDNA condensation and lower charge density presumably due to incorporation of hydrophobic moieties, which were consistent with previous reports concerning polymeric gene vectors.25,48 To investigate gene transfection efficiency of the synthesized polymers, the in vitro gene transfection of luciferasecoded plasmid DNA (Luc-pDNA)-loaded polyplexes was performed in HeLa and HepG2 cells at a weight ratio of 30:1. Similar gene transfection efficiency and cytotoxicity trends can be observed in the two cell lines. The polyplexes from branched PEI and Lipofectamine 2000 were also performed as controls. As shown in Figure 1A and C, remarkably high transfection efficiencies were achieved by polyplexes from all the polymers, albeit at varying degrees of efficiency. For the polymers prepared from compounds 1 and 2, the transfection efficiencies were similar or less than those of bPEI and Lipofectamine 2000. It should be noted that proper combination of amines and compound 3-based polymers showed higher gene transfection efficiency than that of bPEI and Lipofectamine 2000, such as P3B and P3D. The results suggest that fluorinated polymers containing heptafluoropropyl in the side chains revealed significantly higher transfection efficiencies than those containing butyl or octyl moieties in the side chain, which agreed well with the previous literatures.29 Moreover, our results revealed a distinctive odd-even effect of the repeating aminoethylene units in the polymer side chain on the transfection efficiencies. Apparently, P1B, P1D, P2B, P2D, P3B, P3D polyplexes showed significantly

higher gene transfection efficiency than corresponding P1A, P1C, P2A, P2C, P3A, P3C polyplexes. This phenomenon was also observed in the gene transfection efficacies of poly(β-benzyl-L-aspartate) (PBLA) modified with ethylenediamine (EDA, A), diethylenetriamine (DET, B), triethylenetetramine (TET, C), tetraethylenepentamine (TEP, D), respectively.23 Notably, among these polymers, P3D showed the highest gene transfection efficiency, which is even 2 times higher than that of bPEI and almost 1.5 times higher than that of Lipofectamine 2000. Moreover, we also evaluated the cytotoxicity of the polymers as a function of polymer concentrations via the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromideas (MTT) assay. The synthesized polymers exhibited much better biocompatibility compared to bPEI and Lipofectamine 2000 (Figure 1B and D). These functionalized polymers showed cell viability of over 80% even at high concentrations (50 µg/mL). But for bPEI and Lipofectamine 2000, the cell viabilities dropped sharply as the concentration increased. Reasonably, the ester bonds between polymer backbones and side chains ensure that the polymers can be degraded inside cells efficiently, which significantly reduce the cytotoxicity of the polymers.

Figure 2. (A) Representative GFP images of HeLa cells after transfection with P2D, P3D, or P3C polyplexes loading GFPpDNA at a weight ratio of 30:1. Branched PEI was considered as a golden standard (N/P = 10). Scale bars represent 100 µm. (B) GFP positive cell ratios in HeLa cells after treatment with the polyplexes determined by flow cytometry. Data are expressed as mean ± SD (n = 4), **p < 0.01.

Next, to elucidate the reasons for the high transfection efficiencies of P3D complexes, we chose three representative polymers (P2D, P3C and P3D) to investigate the relationship between their structure-dependent transfection ability. First,



we further showed the green fluorescent protein (GFP) transfection efficacies of the three polymers and bPEI complexes loading GFP-coded pDNA (GFP-pDNA) in HeLa cells (Figure 2A). Note that transfection efficacy of P3D is significantly higher than that of bPEI. P2D provided a comparable GFP expression level with bPEI. We further quantified GFPpositive cell ratios as an indicator of transfected cells (Figure 2B). P3D complexes exhibited the highest ratio of GFPpositive cells, nearly two folds than bPEI. Given that cellular uptake and endosomal escape efficiencies play critical role for efficient gene transfection, we next compared cellular uptake efficiency of the four polyplexes loading Cy5-labeled pDNA (Cy5-pDNA) by flow cytometry. As showed in Figure 3, both P3C and P3D significantly increased the fluorescence of Cy5-pDNA in HeLa cells when compared to bPEI. In contrast, P2D exhibited a similar amount of intracellular pDNA to that obtained by bPEI. The above results indicated that fluorination functionalization contributes to the efficient cellular uptake. Reasonably, fluorination method has been frequently used to modify compounds for polymeric gene vectors due to both hydrophobic and lipophobic properties of fluorinated compounds which showed improved affinity to cell membrane and the endosome/lysosome membrane.2832,49,50 Therefore, fluorinated P3C and P3D showed improved cellular uptake.

plexes escaped from the endosome. Meanwhile, we calculated the colocalization ratios (overlapped green and red pixels) to evaluate the endosomal escape ability by Image J software. Consistently, the colocalization ratios of P2D and P3D polyplexes were 0.37 and 0.32, respectively, which were significantly lower than 0.76 for P3C and 0.46 for PEI, indicating superior endosomal escape ability of P2D and P3D compared with P3C and PEI. Presumably, the endosomal escape of the polyplexes may be facilitated by an increased osmotic pressure in the endosome because of a buffering effect associated with the amino groups in the constituent polycations (proton sponge hypothesis).11,51 Higher endosomal escape ability of P2D and P3D with even amino groups is attributed to their high buffering capacity with pH changing from pH 7.4 to endosomal pH 5.5 and tethered two protonated amino groups within the equivalent distance of two methylene units.23 Moreover, the strong interaction of polycations in the polyplexes with the endosomal membrane resulted in effective transport into the cytoplasm.23 High endosomal escape capability of P2D and P3D polyplexes correlated with the improved gene transfection efficiency (Figure 1). Notably, P3D also exhibited statistically lower colocalization ratio as compared with P2D (*p < 0.05) likely due to fluorination of P3D facilitating the endosomal escape. Taken together, we conclude highest gene transfection efficiency of P3D polyplexes should be attributed to efficient cellular uptake and endosomal escape into the cytoplasm.

Figure 3. Fluorescence histograms of HeLa cells obtained by flow cytometry after cell incubation at various conditions: P2D, P3C, P3D, bPEI loading Cy5-pDNA.

To evaluate the endosomal escape capability of the polyplexes, we investigated intracellular distribution of Cy5pDNA loaded in the four polyplexes by confocal laser scanning microscopy (CLSM) (Figure 4). In the CLSM images, the yellow pixels represent the colocalization of Cy5-labeled pDNA (red) with the late endosome/lysosome (green). As shown in Figure 4A, P3C and P3D polyplexes distributed more efficiently in the entire cytoplasmic region than other polyplexes. These results were consistent with the flow cytometry analysis. However, the merged images showed that a major fraction of P3C polyplexes was trapped in the lysosome indicating that P3C had low buffering capacity, which prevented its escape from the endosome.23 In contrast, P2D and P3D polyplexes showed higher red fluorescence intensity in the cytoplasm, suggesting that a major fraction of poly-

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Figure 4. (A) CLSM images of the intracellular distribution of the polyplexes loading Cy5-pDNA (red) against HeLa cells at a weight ratio of 30:1. Branched PEI was considered as a golden standard (N/P=10). The late endosomes/lysosomes (green) and nuclei (blue) were stained with LysoTracker Green and DAPI, respectively. Scale bar represents 10 µm. (B) Quantification of Cy5-pDNA (red) colocalization with LysoTracker (green). Data are expressed as mean ± SD (n = 10). *p < 0.05, **p < 0.01.


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Bioconjugate Chemistry In summary, we developed a facile combinatorial method to synthesize multifunctional gene delivery polymers by integrating different pendent functional groups based on thiolactone chemistry. A variety of typical amines and hydrophobic groups-modified thiolactones were used to perform the ring opening reaction for further Michael addition reaction with PAOEMA via one-pot synthesis to produce the final multifunctional polymers. Gene transfection and cytotoxicity evaluation demonstrated that the polymers functionalized by fluorination and the even-numbered repeating aminoethylene units in their side chains achieved highest gene transfection efficiency and exhibited low cytotoxicity. The developed novel combinatorial synthetic methodology can be extended to other amines and hydrophobic moietiescontaining thiolactones, which represents an effective method for optimization and screening high-efficiency polymeric gene delivery vectors.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Polymer synthetic routes, NMR spectra including 1H and 13C NMR, GPC characterization, DNA complexation, DLS characterization of all the polyplexes.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge financial support from National Natural Scientific Foundation of China (NNSFC) Project (21674104) and the Fundamental Research Funds for the Central Universities (WK3450000002).

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