Manipulation of Transgene Expression in Fibroblast Cells by a

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Manipulation of Transgene Expression in Fibroblast Cells by a Multifunctional Linear-Branched Hybrid Poly(#-Amino Ester) Synthesized through an Oligomer Combination Approach Ming Zeng, Dezhong Zhou, Fatma Alshehri, Irene Lara-Sáez, Yuanning Lyu, Jack Creagh-Flynn, Qian Xu, Sigen A, Jing Zhang, and Wenxin Wang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04098 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Manipulation of Transgene Expression in Fibroblast Cells by a Multifunctional Linear-Branched Hybrid Poly(β-Amino Ester) Synthesized through an Oligomer Combination Approach Ming Zeng,†,‡ Dezhong Zhou,*,†,# Fatma Alshehri,† Irene Lara-Sáez,† Yuanning Lyu,† Jack Creagh-Flynn,† Qian Xu,† Sigen A,† Jing Zhang,† and Wenxin Wang*,† †Charles

Institute of Dermatology, School of Medicine, University College Dublin, Dublin 4, Ireland

‡Department of Dermatology, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China #School of Chemical Engineering and Technology (SCET), Xi'an Jiaotong University, Xi'an, Shaanxi, China

ABSTRACT: Delivery of functional genetic materials into fibroblast cells to manipulate the transgene expression is of great significance in skin gene therapy. Despite numerous polymeric gene delivery systems having been developed, highly safe and efficient fibroblast gene transfection has not yet been achieved. Here, through a new linear oligomer combination strategy, linear poly(β-amino ester) oligomers are connected by the branching units, forming a new type of poly(β-amino ester). This new multifunctional linear-branched hybrid poly(β-amino ester) (LBPAE) shows highperformance fibroblast gene transfection. In human primary dermal fibroblasts (HPDFs) and mouse embryo fibroblasts (3T3s), ultra-high transgene expression is achieved by LBPAE: up to 3292-fold enhancement in Gluciferase expression and nearly 100% of green fluorescence protein expression are detected. Concurrently, LBPAE is of high in-vitro biocompatibility. In-depth mechanistic studies reveal that versatile LBPAE can navigate multiple extra- and intra-cellular barriers involved in the fibroblast gene transfection. More importantly, LBPAE can effectively deliver minicircle DNA encoding COL7A1 gene (a large and functional gene construct) to substantially upregulate the expression of type VII collagen (C7) in HPDFs, demonstrating its great potential in the treatment of C7-deficiency related genodermatoses such as recessive dystrophic epidermolysis bullosa. KEYWORDS: gene therapy, fibroblast transfection, linear-branched hybrid poly(β-amino ester), linear oligomer

combination, type VII collagen

INTRODUCTION Following almost three decades of development, gene therapy has become a predominant part of the rapidly increasing armamentarium of nanomedicine for improving health conditions and correcting genetic disorders.1 Although multiple clinical trials using viral gene delivery vectors have been undergone, the risks of triggering immunogenic responses and transgene insertional mutagenesis, limitations associated with largescale production and low cargo capacity, along with the unpredictability of vector mobility, remain unaddressed.2,3 From this perspective, non-viral gene delivery vectors (e.g., liposomes4,5 and cationic polymers6,7) would be more promising because of their minimal immunogenicity, non-tumorigenicity, cost-effective manufacturing, high payload of nucleic acid, and localized gene expression. From 2010 onwards, the number of clinical trials of gene therapy using non-viral gene vectors has remarkably increased, plasmid DNA and small interfering RNA (siRNA) have been formulated in at least 40 nanoparticle-based gene therapies for gene correction, therapeutic protein expression and antigen vaccination, with 12 major liposome systems investigated

in 27 clinical trials and 7 polymer-based systems in 13 clinical trials.3 Among the polymer based clinical trials, the off-the-shelf cationic polymer polyethylenimine (PEI) has showed some promise. However, PEI is nondegradable and severely hampered by its safety concerns.8 Therefore, tremendous efforts have been made to improve the gene transfection efficiency and safety of the polymeric gene vectors so that polymer-based gene therapy can be brought closer to clinical applications. Among polymeric gene delivery vectors, poly(β-amino ester)s (PAEs) are one type of the most promising candidates. PAEs were first designed and synthesized by Langer and co-workers by the copolymerization of amines with diacrylates through a one-step Michael addition process.9 The tertiary amines on the backbone and primary amines at the terminals serve as the cationic units to condense DNA into nanometric particles through electrostatic interactions and facilitate polyplex escape from endo/lysosomes via the “proton sponge effect”, the ester bonds on the backbone can be hydrolytically degraded under aquatic conditions to dissociate the polyplexes and release DNA as well as reduce the

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cytotoxicity after gene transfection.10,11 After intensive structure and property optimizations,12–14 several PAEs have been identified for DNA transfection with favorable safety profile and high transfection efficiency, both in vitro and in vivo.10,15,16 However, until 2015, almost all the studies with PAEs had been focused on their linear structure. Considering that branched polymers may have greater potential for gene transfection because their three-dimensional (3D) structure and multiple terminal functional groups would bestow additional advantages to the polymeric gene vectors, we have successfully developed highly branched poly(β-amino ester)s (HPAEs) via a facile one-pot “A2+B3+C2” Michael addition strategy.17–21 Over a wide range of cell types, HPAEs exhibited much higher gene transfection ability in comparison with their corresponding linear counterparts, demonstrating their greater potential in gene delivery. The high gene delivery ability of HPAEs was further demonstrated in vivo using the recessive dystrophic epidermolysis bullosa (RDEB) skin disease model.18,19,22 RDEB is a rare, devastating, hereditary mechanobullous disorder caused by the mutation of COL7A1 gene that encodes type VII collagen (C7), which is a key component of anchoring fibrils (AFs) that serve to secure the epidermal-dermal adherence.23 The deficiency of C7 leads to skin fragility, widespread bullae, and erosions that characteristically heal with exuberant scarring and milia formation.24 In both the RDEB knockout mouse model18 and human RDEB skin graft mouse model,19,22 HPAEs mediated high level and up to 10-week restoration of C7 expression, highlighting their huge potential for clinical skin gene delivery. Fibroblasts play a pivotal role in maintaining the skin tissue integrity and skin biological function, regulating cellular microenvironment and are associated with multiple skin diseases. The ability to manipulate gene expression within fibroblasts is fundamental for functional genomics, pathway analysis and biomedical applications. For example, primary human dermal fibroblasts (HPDFs) are an accessible source of phenotypically and karyotypically normal human skin cells, biologically more relevant to in vivo applications in comparison with the immortalized cell lines.25 To date, allogeneic HPDF-based cell therapy has been an appealing strategy for C7 restoration in RDEB patients.26,27 The intradermal injection of HPDFs did show the formation of AFs in RDEB patients. Thus, it can be envisaged that after genetic engineering by transfection, fibroblasts can be diversely adapted and made more suitable for clinical gene therapy. The enhancement expression of C7 in HPDFs would have profound effects on improving the re-constructed AFs, optimizing the dosing schedule and reducing the administration frequency in RDEB. However, non-viral gene transfection of fibroblasts has always been challenging. The most common methods include the expensive electroporation,

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magnetofection and relatively inefficient and toxic chemical formulations.25,28,29 For instance, only 27% and 44% of enhanced green fluorescence protein (EGFP) delivery efficiency in human fibroblasts was detected by different electroporation systems.30,31 The maximum transfection efficiency with the leading cationic lipid reagents TransFectin, Lipofectamine LTX and electroporation in the mouse embryonic fibroblast was 15.7%, 11.8% and 48.1%, respectively.29 Therefore, the development of a reliable non-viral gene delivery system to transfect fibroblasts with high efficiency and safety is imperative and of great significance. In this work, we report the successful development of linear-branched hybrid poly(β-amino ester) (LBPAE). A novel linear oligomer combination strategy is proposed to prepare LBPAE for high performance fibroblast transfection. Linear poly(β-amino ester) oligomers are first prepared from the A2 type amines with C2 type diacrylates, and then the B3 type triacrylates are incorporated to combine the as-synthesized linear oligomers to yield LBPAE. The sequential linear oligomer growth and branching impart the resulting LBPAE more uniform distribution of the linear segments and branching units, which is substantially different from our previous one-pot “A2+B3+C2” Michael addition strategy. In terms of the tailored polymer design and structurefunction relationship, using Gluciferase (Gluc) and green fluorescence protein (GFP) plasmid DNA, we hypothesize that the newly-developed LBPAE would achieve robust transgene expression in reporter gene transfections and possess high levels of biocompatibility in the difficult-totransfect HPDFs and the commonly used mouse embryo fibroblasts (3T3s). Besides, to decipher the possible mechanisms behind the performance of LBPAE in fibroblast transfection, physiochemical and biological properties associated with multiple extra- and intracellular barriers in the gene transfection steps were investigated. Finally, the gene cargo capacity and potency of LBPAE were further confirmed by delivering a large and functional gene - minicircle DNA encoding COL7A1 gene (MCC7) with 12 kb in length, the optimized and miniaturized DNA construct devoid of bacterial sequence. After transfection, LBPAE showed significant enhancement of C7 expression, holding great promise for C7-deficiency disorders such as the devastating and debilitating RDEB. RESULTS AND DISCUSSIONS LBPAE prepared by linear oligomer combination. Previously, the synthesis of dendritic poly(β-amino ester) was challenging because of the intrinsic gelation proneness of multifunctional monomers during the


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Scheme 1. Schematic illustration of the synthesis of LBPAE through the linear oligomer combination strategy. In step 1, A2 type amine reacts with C2 type diacrylate to form the linear A2-C2 base oligomer, which is further end-capped by a second amine to generate the linear A2-C2 oligomer. In Step 2, the linear A2-C2 oligomer is combined by the B3 type triacrylate by branching to yield LBPAE. The box shows the monomers and end-capping agent used for the synthesis of LBPAE. polymerization process.32 In 2016, we developed a one-pot “A2+B3+C2” Michael addition platform for the design and synthesis of HPAEs with different composition, structure and functionalities.19 However, during the one-pot “A2+B3+C2” Michael addition process, A2 type amine is copolymerized with B3 type triacrylate and C2 type diacrylate simultaneously, leading to a random distribution of the branching units and linear segments, which thus potentially compromises the gene transfection performance of PAEs. Here we propose a linear oligomer combination strategy to synthesize LBPAE to circumvent this limitation. As illustrated in Scheme 1, this strategy involves two sequential steps: linear oligomer formation and branching. In the first step, A2 type amine reacts with C2 type diacrylate to generate acrylate terminated base oligomer which is further end-capped with a second amine. After purification to remove the unreacted monomers and excess end-capping agent, the linear A2C2 oligomer is formed. In the second step, B3 type triacrylate is introduced to combine the linear A2-C2 oligomer and yield the LBPAE. In comparison with the one-pot “A2+B3+C2” method for HPAE synthesis, advantages of the linear oligomer combination strategy

for LBPAE synthesis are two-fold: 1) The length of the linear segments in the obtained LBPAEs would be predetermined and thus can be tailored easily; 2) The branching units in LBPAEs would be more evenly distributed between the linear segments. To validate our hypothesis, 5-amino-1-pentanol (AP), trimethylolpropane triacrylate (TMPTA), 1,4-butanediol diacrylate (BDA) and 1,11-diamino-3,6,9-trioxaundecane (DATOU), which have been demonstrated to be effective monomers in the synthesis of PAEs for gene transfection, were used as A2, B3, C2 types monomers and end-capping agent for LBPAE synthesis, respectively. BDA and AP, with a stoichiometric ratio of 1.2 : 1, were reacted in dimethyl sulfoxide (DMSO) at 90°C and the weight average molecular weight (Mw) was monitored with gel permeation chromatography (GPC). After 24 hours, when Mw of the reaction mixture was approaching 3000 Da, the reaction was stopped by cooling down to room temperature and diluted with DMSO. Excess DATOU was then added to end-cap the acrylate terminated base oligomers for 48 hours at 25°C. After removing the unreacted monomers of a Mw < 3000 Da by dialysis in acetone, the linear A2-C2 oligomer with a Mw


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Figure 1. Transfection efficiency and cell viability assessment. a) Gluc activity and cell viability of HPDFs 48 h post transfection by the LBPAE/DNA, PEI/DNA and SuperFect/DNA polyplexes at a series of w/w ratios. b) Gluc activity and cell viability of 3T3s. Significant difference from the *PEI and #SuperFect group in Gluc activity (p ＜ 0.05, Student’s t-test). around 3500 Da and a polydispersity index (PDI) 1.69 was obtained (Figure S1). To generate LBPAE, the linear A2-C2 oligomer and TMPTA were dissolved in DMSO (the molar ratio of A2-C2 : TMPTA was set as 3 : 1) and reacted at room temperature. When Mw was around 10 kDa, the reaction was stopped and excess DATOU was incorporated to consume all the unreacted vinyl groups. The polymer was then precipitated in diethyl ether and dried in vacuum oven to give the final LBPAE product. GPC measurement shows that LBPAE has a Mw of 9.4 kDa with a PDI 2.5 (Figure S1). The Mark-Houwink (MH) plot alpha value of 0.36 validates its highly branched structure (Figure S2). The chemical composition of LBPAE (Figure S3) was confirmed by proton nuclear magnetic resonance (1H NMR). LBPAE achieves robust Gluc transfection efficiency and excellent cell viability. For cationic polymer based gene delivery vectors, the polymer/DNA weight ratio (w/w) is one of the most critical parameters determining both the transfection efficiency and cytotoxicity.9,15 Therefore, we first optimized the w/w ratio systematically. Considering that primary cells (e.g., HPDFs) are usually fragile to cationic polymers, the LBPAE/DNA w/w ratio used for HPDF transfection was increased gradually from 10 : 1 to 50 : 1. In order to set up a strong benchmark for comparison, the w/w ratios used for the two dendritic commercial gene transfection reagents, PEI10,11,33 and SuperFect4,19,34, were also optimized according to manufacturers’ protocols and previous publications. Figure 1a outlined the Gluc activity and cell viability of HPDFs after transfection. It is clearly shown that the optimal w/w ratios for PEI and SuperFect gene transfection are 1 : 1 and 3 : 1, respectively. Further increase in the w/w ratio not only obviously lowers the Gluc activity, but also substantially increases the cytotoxicity. For example, in comparison with that at the w/w ratio of 3 : 1, Gluc activity of HPDFs after transfection with the SuperFect/DNA polyplexes at the w/w ratio of 9 : 1 was 3.4-fold lower and the cell viability was decreased from over 89% to under 44%. In sharp contrast, over the

range of the w/w ratios tested, even at the lowest w/w ratio 10 : 1, Gluc expression of HPDFs after transfection by the LBPAE/DNA polyplexes was still stronger than that mediated by the PEI/DNA polyplexes and SuperFect/DNA polyplexes at their optimal w/w ratios. Gluc activity of HPDFs was especially high when transfected by the LBPAE/DNA polyplexes at the w/w ratio of 40 : 1, where it was up to 103-fold higher than that mediated by the PEI/DNA polyplexes. Importantly, LBPAE did not induce obvious cytotoxicity. Even at the highest w/w ratio of 50 : 1, more than 95% cell viability was still preserved. In 3T3s, the PEI/DNA polyplexes exhibit the similar trend of gene transfection efficiency and cytotoxicity with that observed in HPDFs. Although at the w/w ratio of 6 : 1 and 9 : 1, SuperFect/DNA polyplexes showed a higher gene transfection efficiency, but preserved only 62% and 49% cell viability, respectively (Figure 1b). Again, at all the tested w/w ratios, LBPAE/DNA polyplexes exhibit both strong gene transfection ability and high cell viability. Gluc activity of 3T3s after transfection with the LBPAE/DNA polyplexes was orders-of-magnitude higher than that mediated by the PEI/DNA and SuperFect/DNA polyplexes at their optimal w/w ratios. Amazingly, at the w/w ratio of 70 : 1, LBPAE/DNA polyplexes mediated up to 3292-fold higher Gluc activity in comparison with the PEI/DNA polyplexes, while more than 90% cell viability was still maintained. This further demonstrates that LBPAE performs as a viable vector which can not only achieve high gene transfection efficiency, but also induce minimal cytotoxicity in fibroblast transfections. LBPAE demonstrates high lethal concentration 50 (LC50) values. Although > 2500 candidates have been developed and screened for gene transfection,35 so far, toxicological study data with the PAE polymer in fibroblasts is limited. To this end, LC50 values of polyplexes were determined to further validate the biocompatibility of LBPAE in fibroblast transfection. LBPAE/DNA polyplexes were used to transfect HPDFs


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Figure 2. LC50 assessment of LBPAE/DNA polyplexes and SuperFect/DNA polyplexes in HPDFs and 3T3s. a) Representative live/dead images of the untreated cells or cells transfected with the LBPAE/DNA polyplexes at the concentration of 555 μg mL−1 or SuperFect/DNA polyplexes at the concentration of 35 μg mL−1. The scale bars are 50 μm. b) LBPAE/DNA polyplex concentration-dependent cell viability measured by Alamarblue assay. c) SuperFect/DNA polyplex concentration-dependent cell viability measured by Alamarblue assay. and 3T3s with polyplex concentration increased from 355 μg mL−1 to 755 μg mL−1. Gluc activity results showed that the overall gene transfection efficiency mediated by SuperFect was better than PEI, therefore, SuperFect was employed for comparison here. SuperFect/DNA polyplexes were used and the concentration was increased from 15 μg mL−1 to 55 μg mL−1. 24 hours post transfection, cells were simultaneously stained with the green-fluorescent Calcein-AM (C-AM, for live cells) and red-fluorescent ethidium homodimer-1 (EthD-1, for dead cells). The representative fluorescence images of untreated cells and those treated with the LBPAE/DNA polyplexes at the concentration of 555 μg mL−1 and SuperFect/DNA polyplexes at the concentration of 35 μg mL−1 are shown in Figure 2a. It can be seen that, although treated with one order-of-magnitude higher concentration of the LBPAE/DNA polyplexes, HPDFs and 3T3s showed similar cell viability with that treated by the SuperFect/DNA polyplexes. Polyplex concentration– dependent cell viability was determined with Alamarblue assay and results are shown in Figure 2b and Figure 2c, from which it is calculated that LC50 values for the

SuperFect/DNA polyplexes in HPDFs and 3T3s are 35.2 μg mL−1 and 39.5 μg mL−1, respectively. In contrast, LC50 values of the LBPAE/DNA polyplexes are 538.4 μg mL−1 and 552.3 μg mL−1 respectively, corresponding to 15 and 14-fold the value of the SuperFect/DNA counterparts. SuperFect is a partially degraded polyamidoamine (PAMAM) dendrimer which possesses a spheroidal architecture with branches radiating from a central core and terminal amino groups.4 It has been widely used for gene transfection due to its improved biocompatibility.36,37 The toxicity of PAMAM dendrimers has been demonstrated to depend on the structure and surface charge density. More terminal cationic groups from a higher PAMAM generation are believed to interact and disrupt with negatively charged cell membranes.38 LBPAE is showing a much lower cell-killing effect and demonstrating its extremely high biocompatibility, which is of great significance in gene transfection. This is especially beneficial for the hard-to-transfect cell types because considerably high polyplex doses or multiple repeat transfections can be used to enhance the transfection efficiency.


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Figure 3. Comparison of GFP expression and MFI mediated by different gene delivery systems. a) GFP images of HPDF cells after the treatment with LBPAE/DNA, PEI/DNA and SuperFect/DNA polyplexes. Untreated (UT) cells were used as the negative control. Scale bar, 200 μm. b) Histogram distribution of HPDF populations after transfection with different polyplexes. c) Percentage of GFP-positive HPDFs and the MFI of cells after transfection. d) GFP images of 3T3s. Scale bar, 200 μm. e) Histogram distribution of 3T3 populations after transfection with different polyplexes. f) Percentage of GFPpositive 3T3s and the MFI of cells after transfection. Significant difference from commercial reagent groups in the *percentage of GFP-positive cells and #MFI (p ＜ 0.05, Student’s t-test). LBPAE exhibits ultra-high GFP expression. The Gluc reporter assay was used to quantify the overall transgene expression level mediated by LBPAE. GFP DNA was further used to quantify the percentage of successfully transfected cells. HPDFs and 3T3s were transfected with the LBPAE/DNA polyplexes at the same w/w ratios as above. As evidenced by the fluorescence images shown in Figure 3a, at all the w/w ratios, many more HPDFs were transfected by the LBPAE/DNA polyplexes in comparison with that by PEI/DNA and SuperFect/DNA counterparts at their optimal w/w ratios. Flow cytometry measurements show that the percentage of GFP-positive HPDFs achieved by the PEI/DNA and SuperFect/DNA polyplexes is only 50% and 44%, respectively. In contrast, LBPAE/DNA polyplexes achieve much higher level of GFP-positive population. This is reflected by the far shift of the cell populations responding to the GFP spectrum channel in the histogram distributions (Figure 3b). The lowest GFP-positive population mediated by the LBPAE/DNA polyplexes is 68% at the w/w ratio of 10 : 1. When the w/w ratio is above 40 : 1, 94% of the HPDFs are GFP-positive. Furthermore, median fluorescence intensity (MFI) of

HPDFs transfected by the LBPAE/DNA polyplexes is up to 140-fold higher than that by the PEI/DNA and SuperFect/DNA counterparts (Figure 3c). In 3T3s, the percentage of GFP-positive cells achieved by the LBPAE/DNA polyplexes increased from 35% at the w/w ratio of 30 : 1 to 91% at the w/w ratio of 70 : 1, in contrast to 5% and 11% achieved by the PEI/DNA and SuperFect/DNA polyplexes, respectively (Figure 3d, 3e and 3f). Our previous work showed that the highest efficiency in 3T3s achieved by the top-performing HPAE is < 60%,20 much lower than that achieved by LBPAE (> 90%) developed here. In addition, at the w/w ratio of 70 : 1, MFI of 3T3s mediated by the LBPAE/DNA polyplexes was 272 and 230-fold higher compared with that of the PEI/DNA and SuperFect/DNA counterparts (Figure 3f). These results indicate that LBPAE not only transfects more cell numbers, but also significantly promotes the level of protein expression in the individually transfected cells. Primary fibroblasts are difficult-to-transfect cell types, the fact that LBPAE can mediate more than 90% gene transfection efficiency in the primary HPDFs, demonstrating its robust gene transfection ability. Given that LBPAE has proven to be highly biocompatible


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Figure 4. Physicochemical characteristics of the LBPAE/DNA polyplexes. a) DNA condensation ability determination with agarose gel electrophoresis. b) DNA binding affinity measurement with PicoGreen assay. c) Polyplex size and zeta potential measurements. d) Polyplex morphology observation with TEM. Scale bar, 200 nm. over a wide range of w/w ratios with superior gene transfection ability, it can be envisaged that LBPAE will have broad applicability in fibroblast gene transfection. DNA condensation and binding affinity of LBPAE. Effective DNA condensation, which can not only protect DNA from degradation by endonucleases but also facilitate polyplex cellular uptake, is the prerequisite for a successful gene transfection.37 For cationic polymers, DNA condensation is mainly driven by electrostatic interactions. For the LBPAE studied here, there are two types of amines which can partially protonate to generate positive charges: one is the multiple terminal primary amines derived from the end-capping agent DATOU, the other is the multitude backbone tertiary amines derived from both the AP and DATOU. The DNA condensation ability of LBPAE was determined with agarose gel electrophoresis. As shown in Figure 4a, at all the w/w ratios, no DNA shifting bands were observed, indicating that the negatively charged DNA is shielded by the positively charged LBPAE effectively and thus retained in the agarose wells without migration. Both of the commercial gene transfection reagents PEI and SuperFect show high DNA condensation ability. This is especially true for the SuperFect, which condenses the DNA so tight

that it is difficult for the DNA staining dye to gain access to the DNA, and thus the DNA band is of a lighter shade. The binding affinity between the DNA and LBPAE was further quantified with PicoGreen assay. As shown in Figure 4b, LBPAE exhibits strong DNA binding affinity at all w/w ratios. In general, the DNA binding affinity increases with the w/w ratio, e.g., from 86% at the w/w ratio of 10 : 1 to 96% at the w/w ratio of 70 : 1, demonstrating that more LBPAE leads to stronger electrostatic interaction between the LBPAE and DNA. Comparatively, both PEI and SuperFect show even stronger DNA binding affinity of nearly 100%. DNA binding affinity of the LBPAE, PEI and SuperFect correlates very well with their DNA condensation ability. However, it should be noted that a moderate DNA binding affinity is more favorable for gene transfection since an excessive interaction would compromise DNA release from polyplexes.39,40 LBPAE/DNA polyplex size, zeta potential and morphology. Nanometric size and positive surface charge can facilitate particle cellular uptake through the endocytosis pathway.41,42 As shown in Figure 4c, in the physiological solution, over all tested w/w ratios, the average sizes of the LBPAE/DNA polyplexes, which were


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Figure 5. Cellular uptake of diverse polyplexes. a) Fluorescent images of cells 4 hours post transfection with different polyplexes. The nucleus was stained with DAPI (blue), DNA was labeled with Cy3 (red). Scale bar, 20 μm. b) Polyplex uptake efficiency in HPDFs quantified with flow cytometry. c) Polyplex uptake efficiency in 3T3s quantified with flow cytometry. d) Percentage of Cy3 positive HPDFs and the normalized MFI of cells. e) Percentage of Cy3 positive 3T3s and the normalized MFI of cells. Significant difference from PEI and SuperFect in the *MFI quantification (p ＜ 0.05, Student’s t-test). measured with dynamic light scattering (DLS), were all less than 250 nm. In the w/w ratio range from 10 : 1 to 60 : 1, the particle size of the polyplexes ranged from 188 nm to 228 nm. However, when the w/w ratio was further increased to 70 : 1, the polyplex size substantially decreased to 97 nm. Correspondingly, all the polyplexes exhibited a positive zeta potential. At the lowest w/w ratio 10 : 1, the LBPAE/DNA polyplexes had a very low zeta potential of 6 mV. When the w/w ratio is higher than 10 : 1, the zeta potential was significantly increased to over 30 mV with the highest 34 mV achieved at the w/w ratio of 40 : 1. At the same testing conditions, the SuperFect/DNA polyplexes had a very small size of around 92 nm and a high zeta potential around 37 mV. These observations correlate with the DNA condensation ability and binding affinity of the polymers very well. In contrast, despite of high DNA condensation and binding capacity, the PEI/DNA polyplexes had a substantially large size of over 500 nm. Transmission electron microscopy (TEM) was used to further observe the polyplex size and morphology. As shown in Figure 4d, all the LBPAE/DNA and SuperFect/DNA polyplexes manifested uniform spherical morphologies with the size between 60 nm and 250 nm, which was similar to that measured by the DLS. Importantly, there is no obvious polyplex aggregation, indicating the high stability of these polyplexes. In contrast, the PEI/DNA polyplexes exhibit

an ellipsoid morphology and the size is much bigger than that of other polyplexes. It is widely accepted that polyplexes with the size below 250 nm and moderately positive surface charge are more favorable for cellular uptake while avoiding to induce potential cytotoxicity caused by excessive positive charge.11,12 The abovementioned gene transfection studies have shown that the best w/w ratios for HPDF and 3T3 transfection are 40 : 1 and 70 : 1 respectively, when using LBPAE/DNA polyplexes. This indicates that the most favorable polyplex size and surface charge for effective fibroblast gene transfection may vary substantially according to the fibroblast subtype. Here, within a broad range of w/w ratios, the LBPAE/DNA polyplexes always demonstrate an average size below 250 nm and a moderate zeta potential of slightly above 30 mV which provides the evidence of self-assembly cationic particle synthesis for efficient fibroblast gene transfer. Cellular uptake of LBPAE/DNA polyplexes. The cellular uptake of LBPAE/DNA polyplexes was further investigated. As shown in Figure 5a, with the same cell density, 4 hours post transfection, all the polyplexes show high cellular uptake efficiency. Comparatively, much more LBPAE/DNA polyplexes were taken up by the HPDFs and 3T3s in comparison with the PEI/DNA and SuperFect/DNA polyplexes. This was evidenced by the


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Figure 6. Proton buffering capacity, degradation and DNA release assessment of the LBPAE. a) Proton buffering capacity determined by acid-base titration. b) Degradation profile determined using GPC. c) Evaluation of DNA release from polyplexes assessed with PicoGreen assay. much stronger red fluorescence observed from the Cy3 labelled DNA. Flow cytometry quantification reveals that the PEI/DNA and SuperFect/DNA polyplexes respectively achieve 96.5% and 98.4% cellular uptake efficiency in the HPDFs (Figure 5b). Even so, the uptake efficiency of the LBPAE/DNA polyplexes is still slightly higher and is almost 100% (99.3%). In 3T3s, a similar trend was also observed (Figure 5c). Furthermore, in HPDFs, the normalized MFI of the LBPAE/DNA polyplexes is 3.05 and 1.39-fold higher than that respectively achieved by the PEI/DNA and SuperFect/DNA counterparts (Figure 5d). In 3T3s, the LBPAE/DNA polyplexes out-perform PEI/DNA and SuperFect/DNA polyplexes by 1.98 and 1.68-fold, respectively (Figure 5e). All these results indicate that the PEI/DNA polyplexes have the lowest cellular uptake efficiency while the LBPAE/DNA polyplexes out-perform both the PEI/DNA and SuperFect/DNA counterparts significantly in this aspect. Collectively, LBPAE promotes the initial cellular uptake efficiency and the ultimate transfection efficiency in fibroblast transfection. Proton buffering capacity measurement. Endo/lysosomal escape is another major bottleneck to overcome for efficient non-viral gene delivery. Given the mechanism of the “proton sponge effect”, cationic polymers with high content of protonatable secondary and tertiary amines with a pKa close to the endosomal/lysosomal pH are more favorable for polyplex escape from the endo/lysosomes.36 PEI and SuperFect are the most typical representatives for endo/lysosomal escape. To verify the proton buffering capacity of LBPAE, acid-base titration was conducted. As shown in Figure 6a, for the given amount of polymers dissolved in the NaCl solution, it is not surprising that PEI shows the strongest proton buffering capacity with a relatively more gradual slope in the acid-base titration curve between the pH 7.4 and 5.1. Since PEI has very high content of primary, secondary and tertiary amines with every third atom a nitrogen in the backbone. After normalization, it is found that the proton buffering capacity of PEI, SuperFect and LBPAE is 5.1 mmol H+ g−1, 4.6 mmol H+ g−1 and 1.6 mmol H+ g−1, respectively (Table S1). Indeed, LBPAE exhibits

lower proton buffering capacity than PEI and SuperFect. However, due to its weaker cytotoxicity, to effectively promote endo/lysosomal escape of the LBPAE/DNA polyplexes, the w/w ratio can be significantly increased in practical applications. For instance, for the HPDF and 3T3 gene transfection, the LBPAE/DNA polyplexes were used at the w/w ratio of 40 : 1 and 70 : 1, respectively. Under these conditions, LBPAE achieved 13 and 22-fold the proton buffering capacity of PEI at the w/w ratio of 1 : 1, and 5 and 8-fold the value of SuperFect at the w/w ratio of 3 : 1. The high proton buffering capacity of LBPAE would cause the increase of the osmotic pressure, leading to swelling and rupture of the endo/lysosomes, and thus the successful endo/lysosome escape of polyplexes to prevent the DNA digestion in these acidic compartments. Degradation and DNA release assessment of LBPAE. A versatile gene delivery vector can not only effectively condense DNA and protect it from degradation by enzymes, but also potentially release the condensed DNA from the polyplexes before the transcription. For cationic polymers, a series of strategies have been proposed to promote polymer degradation and thus facilitate DNA release and reduce accumulative cytotoxicity after gene transfection. However, for efficient gene transfection, a moderately long half-life is required for the gene vectors because too short a half-life will lead to insufficient DNA protection and premature DNA release while too long a half-life would result in difficulty for polyplex/DNA disassociation and subsequent DNA release. There are multiple ester bonds on the backbone of PAEs. Under physiological conditions, the ester bonds can be degraded by hydrolysis to yield biocompatible small molecular β-amino acids and diols. It is reported that, depending on the chemical composition, PAEs have a half-life spanning from 1.5 hours to over 6 hours in aqueous environments.11 For our LBPAE, 43% degradation was observed after 2 hours of incubation at 37 °C. This degradation further progressed to 81% after 6-hours incubation, and then to 85% after 8-hours incubation (Figure 6b). The corresponding DNA release from the polyplexes was determined by PicoGreen assay. As shown


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Figure 7. Immunofluorescence staining of C7 expression in HPDFs. a) Fluorescent images of HPDFs four days post transfection with the LBPAE/MCC7 and SuperFect/MCC7 polyplexes. The nucleus was stained with DAPI (blue) and the C7 with antibodies (red). Scale bar, 20 μm. b) Flow cytometry quantification of C7 expression of HPDFs. c) Degree of C7 expression upregulation and the MFI of HPDFs after transfection with LBPAE/MCC7 and SuperFect/MCC7 polyplexes. d) Schematic illustration of C7 in maintaining skin integrity: C7 exerts its activity to secure the epidermal-dermal adherence. Significant difference from SuperFect in the *percentage of C7 upregulation and #MFI (p ＜ 0.05, Student’s t-test). in Figure 6c, at the lowest w/w ratio of 10 : 1, LBPAE/DNA polyplexes had the fastest DNA release rate, after 2 hours of incubation. More than 60% of the DNA had been released. Comparatively, at the moderate and high w/w ratios of 40 : 1 and 70 : 1, the condensed DNA was released from the polyplexes at a slower but similar rate. However, after 8 hours, more than 60% of the DNA was released. The DNA release profile matches the LBPAE degradation profile, demonstrating that LBPAE can release the condensed DNA via hydrolysis spontaneously in physiological conditions without necessitating any additional external triggers in a timely and efficient manner. Collectively, polymer DNA condensation and binding, proton buffering capacity, degradation, polyplex size, zeta potential, morphology, cellular uptake and DNA release are the key parameters that dictate gene transfection efficiency.3,19,36 We think that the synergistic effects of strong DNA condensation and binding ability, uniform and nanoscaled polyplex size, moderately positive charge, high polyplex cellular uptake efficiency, strong proton buffering capacity, facilitating DNA release and reduced cytotoxicity impart the LBPAE ultra-high gene transfection efficiency.

LBPAE delivers functional COL7A1 to manipulate C7 expression in HPDFs. Many gene delivery vectors show high level of reporter gene expression, however, the translation of such success in delivering a large and functional gene construct is far more challenging.43 Hence, the effectiveness of multifunctional LBPAE was further assessed by delivering a functional COL7A1 gene to promote the expression of C7 in HPDFs. Currently, there is no effective cure available beyond palliative care for RDEB. Although both keratinocytes and dermal fibroblasts are capable of producing and secreting C7,44 the latter are more robust than the former as the target cell types in gene therapy of genodermatosis diseases.26,45 Minicircle (MC) DNA has shown a 10-1000 fold higher and more stable non-integrative transgene expression than normal plasmids without the risk of immunogenic responses from the bacterial backbone in regular plasmids.46,47 Considering that the COL7A1 gene is quite large with about 9 kb cDNA/mRNA transcript, MCC7 encoding the ∼9 kb full-length COL7A1 cDNA with the cytomegalovirus promotor was used to transfect HPDFs. As shown in Figure S4, MCC7 contains ∼9 kb COL7A1 cDNA and 3 kb backbone in length, 2 kb shorter than the pcDNA3.1COL7A1 normal plasmid. It should be noted


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that the maximum cargo size of retrovirus and adenoassociated virus (AAV) vectors is usually less than 8 kb,48 but both the size of the pcDNA3.1COL7A1 and MCC7 exceeds the gene packaging capacity of the majority viral vectors. Therefore, efficient COL7A1 gene transfection by LBPAE may have great significance to the gene therapy of RDEB. By combining LBPAE and MC DNA, herein, we delivered MCC7 to manipulate the C7 expression in HPDFs with the expectation to enhance its utility in gene therapy of RDEB. Figure 7a outlined the cytoimmunofluorescence staining images of HPDFs four days post transfection with the LBPAE/MCC7 polyplexes. As expected, no obvious C7 expression (red fluorescence) was observed in the untreated group without antibody (Ab) incubation and the group only incubated with the anti-C7 secondary Ab. The wild-type HPDFs and the group treated with SuperFect exhibit moderate fluorescence, indicating the production of C7. In comparison, HPDFs transfected with the LBPAE/MCC7 polyplexes showed the strongest fluorescence, demonstrating more recombinant C7 expression obtained by LBPAE. Flow cytometry was used to further quantify the C7 expression efficiency. In consistence with previous reports,49 the wild-type of HPDF showed about 41% percent of C7 expression. After transfection with the LBPAE/MCC7 polyplexes, the C7 expression efficiency was significantly increased to 74.4%, in comparison with 44.9% achieved by the SuperFect/MCC7 polyplexes (Figure 7b). Moreover, 40% enhancement in MFI was also realized by the LBPAE/MCC7 polyplexes, in contrast to 10% achieved by the SuperFect/MCC7 counterparts (Figure 7c). All these results demonstrate that LBPAE can efficiently deliver MCC7 with 12 kb in full length into HPDFs. In addition, our preliminary study showed that in the C7 null RDEB fibroblasts (RDEBFs), after transfection with the LBPAE/MCC7, the C7 expression efficiency was restored to around 40%. Further optimization of the transfection and quantification of the C7 expression in RDEBFs are still undergoing. Our findings point out that LBPAE has strong payload capacity to deliver the large cDNA into skin primary cells. The primary dermal fibroblasts can be further engineered by this polymeric vector to secret potent cellular C7, which is pivotal for strengthening the dermal-epidermal junction (Figure 7d). Although the non-viral gene therapy requires repeated applications for the correction of dysfunctional C7 genetic skin diseases, given the apparent wound sites and the good access to medication, multiple topical administrations are much safer than systemic gene deliveries. Therefore, LBPAE holds great promise for fibroblast-based gene therapies to restore or enhance the C7 expression and thus reverse the disease phenotype of RDEB. CONCLUSIONS Fibroblast gene delivery has yet to show the required efficiency for therapeutic applications. To overcome this

limitation, a novel LBPAE gene delivery vector prepared via a new linear oligomer combination strategy has been developed. Apart from the advantages of the nonintegrating polymeric vector with a large gene payload capacity, the bottom-up designs of LBPAE composition and structure bestow it multifunctional properties in gene delivery: 1) Evenly distributed linear segments and branching units; 2) Adequate amounts of primary and tertiary amines that can be used as the DNA condensing units and buffering moieties; 3) Biodegradability due to hydrolysis of the ester bonds. First, LBPAE achieves ultrahigh reporter gene transfection efficiency with minimal cytotoxicity in the difficult-to-transfect fibroblasts, HPDFs and 3T3s. It significantly outperforms the commercially available reagents branched PEI and SuperFect, with up to 3292-fold enhancement in Gluc expression and nearly 100% of GFP expression. Secondly, high LC50 values of LBPAE polyplexes demonstrate their favorable biocompatibility in fibroblast transfections, allowing for flexible adjustment of the polymer/DNA formulations and future systemic gene delivery applications. Thirdly, the mechanism studies reveal that LBPAE offers strong DNA condensation ability and binding affinity, and LBPAE/DNA polyplexes are facilely synthesized by self-assembly to cationic nanosized particles ranging from 60-250 nm with uniform spherical morphology, achieving nearly 100% of cellular uptake efficiency. Afterwards, strong buffering capacity of LBPAE is observed, indicating the possibility of efficient polyplex endo/lysosomal escape after the internalization. Besides, biodegradable LBPAE facilitates DNA release in a timely and efficient manner, which is considered very important for promoting the transcription levels after the nucleus import. Finally, LBPAE can efficiently deliver a large plasmid DNA (12 kb) encoding for COL7A1 with an optimized and miniaturized gene construct into HPDFs, improving C7 expression which is critical in maintaining skin integrity. Our work demonstrates that LBPAE is a versatile, efficient and biocompatible vector in fibroblastbased gene delivery, holding considerable potential and capacity in genodermatosis treatments and regenerative therapies.

ASSOCIATED CONTENT Supporting Information This material is available free of charge on the ACS Publications website at DOI:

Experimental section, GPC results of linear oligomer and LBPAE, MH Alpha curve and value of LBPAE, chemical composition analysis of LBPAE by 1H NMR, agarose gel results of MCC7 and pcDNA3.1COL7A1 and buffering capacity of LBPAE and commercial reagents.

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


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ORCID

Ming Zeng: 0000-0001-5919-6237 Dezhong Zhou：0000-0002-5783-8707 Jing Zhang：0000-0002-5726-3974 Wenxin Wang：0000-0002-5053-0611 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the technical support of Dr. A. Blanco of the Conway Flow Cytometry Core and D. Scholz of the Conway Electron Microscopy Core, University College Dublin. 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-2013412), Irish Research Council CAROLINE Fellowship (CLNE/2017/358), University College Dublin and DEBRA Ireland and National Natural Science Foundation of Jiangsu Province (BK20171013).

REFERENCES (1) Naldini, L. Nature 2015, 526, 351–360. (2) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Nat. Rev. Genet. 2014, 15, 541–555. (3) Foldvari, M.; Chen, D. W.; Nafissi, N.; Calderon, D.; Narsineni, L.; Rafiee, A. J. Control. Release 2016, 240, 165– 190. (4) Yamano, S.; Dai, J.; Moursi, A. M. Mol. Biotechnol. 2010, 46, 287–300. (5) Allen, T. M.; Cullis, P. R. Adv. Drug Deliv. Rev. 2013, 65, 36–48. (6) Aied, A.; Greiser, U.; Pandit, A.; Wang, W. Drug Discov. Today 2013, 18, 1090–1098. (7) Lai, W.-F.; Wong, W.-T. Trends Biotechnol. 2018, 36, 713–728. (8) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. J. Control. Release 2006, 114, 100–109. (9) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 10761–10768. (10) Green, J. J.; Zugates, G. T.; Tedford, N. C.; Huang, Y.H.; Griffith, L. G.; Lauffenburger, D. A.; Sawicki, J. A.; Langer, R.; Anderson, D. G. Adv. Mater. 2007, 19, 2836– 2842. (11) Sunshine, J. C.; Peng, D. Y.; Green, J. J. Mol. Pharm. 2012, 9, 3375–3383. (12) Akinc, A.; Lynn, D. M.; Anderson, D. G.; Langer, R. J. Am. Chem. Soc. 2003, 125, 5316–5323. (13) Anderson, D. G.; Lynn, D. M.; Langer, R. Angew. Chemie Int. Ed. 2003, 42, 3153–3158. (14) Anderson, D. G.; Akinc, A.; Hossain, N.; Langer, R. Mol. Ther. 2005, 11, 426–434. (15) Sunshine, J. C.; Sunshine, S. B.; Bhutto, I.; Handa, J. T.; Green, J. J. PLoS One 2012, 7, e37543. (16) Mangraviti, A.; Tzeng, S. Y.; Kozielski, K. L.; Wang, Y.; Jin, Y.; Gullotti, D.; Pedone, M.; Buaron, N.; Liu, A.; Wilson, D. R.; Hansen, S. K.; Rodriguez, F. J.; Gao, G.-D.;

Page 12 of 14

DiMeco, F.; Brem, H.; Olivi, A.; Tyler, B.; Green, J. J. ACS Nano 2015, 9, 1236–1249. (17) Huang, J.-Y.; Gao, Y.; Cutlar, L.; O’Keeffe-Ahern, J.; Zhao, T.; Lin, F.-H.; Zhou, D.; McMahon, S.; Greiser, U.; Wang, W.; Wang, W. Chem. Commun. (Camb). 2015, 51, 8473–8476. (18) Zhou, D.; Gao, Y.; Aied, A.; Cutlar, L.; Igoucheva, O.; Newland, B.; Alexeeve, V.; Greiser, U.; Uitto, J.; Wang, W. J. Control. Release 2016, 244, 336–346. (19) Zhou, D.; Cutlar, L.; Gao, Y.; Wang, W.; OKeeffeAhern, J.; McMahon, S.; Duarte, B.; Larcher, F.; Rodriguez, B. J.; Greiser, U.; Wang, W. Sci. Adv. 2016, 2, e1600102. (20) Zhou, D.; Gao, Y.; O’Keeffe Ahern, J.; A, S.; Xu, Q.; Huang, X.; Greiser, U.; Wang, W. ACS Appl. Mater. Interfaces 2016, 8, 34218–34226. (21) Zeng, M.; Zhou, D.; Ng, S.; Ahern, J. O.; Alshehri, F.; Gao, Y.; Pierucci, L.; Greiser, U.; Wang, W. Polymers (Basel). 2017, 9, 161. (22) Cutlar, L.; Zhou, D.; Hu, X.; Duarte, B.; Greiser, U.; Larcher, F.; Wang, W. Exp. Dermatol. 2016, 25, 818–820. (23) Woodley, D. T.; Chen, M. J. Invest. Dermatol. 2015, 135, 1705–1707. (24) Fine, J.-D.; Bruckner-Tuderman, L.; Eady, R. A. J.; Bauer, E. A.; Bauer, J. W.; Has, C.; Heagerty, A.; Hintner, H.; Hovnanian, A.; Jonkman, M. F.; Leigh, I.; Marinkovich, M. P.; Martinez, A. E.; McGrath, J. A.; Mellerio, J. E.; Moss, C.; Murrell, D. F.; Shimizu, H.; Uitto, J.; Woodley, D.; Zambruno, G. J. Am. Acad. Dermatol. 2014, 70, 1103–1126. (25) Fountain, J. W.; Lockwood, W. K.; Collins, F. S. Gene 1988, 68, 167–172. (26) Wong, T.; Gammon, L.; Liu, L.; Mellerio, J. E.; Dopping-Hepenstal, P. J. C.; Pacy, J.; Elia, G.; Jeffery, R.; Leigh, I. M.; Navsaria, H.; McGrath, J. A. J. Invest. Dermatol. 2008, 128, 2179–2189. (27) Petrof, G.; Martinez-Queipo, M.; Mellerio, J. E.; Kemp, P.; McGrath, J. A. Br. J. Dermatol. 2013, 169, 1025– 1033. (28) Nakayama, A.; Sato, M.; Shinohara, M.; Matsubara, S.; Yokomine, T.; Akasaka, E.; Yoshida, M.; Takao, S. Cloning Stem Cells 2007, 9, 523–534. (29) Lee, M.; Chea, K.; Pyda, R.; Chua, M.; Dominguez, I. J. Biomol. Tech. 2017, 28, 67–74. (30) Sharifi Tabar, M.; Hesaraki, M.; Esfandiari, F.; Sahraneshin Samani, F.; Vakilian, H.; Baharvand, H. Cell J. 2015, 17, 438–450. (31) Jordan, E. T.; Collins, M.; Terefe, J.; Ugozzoli, L.; Rubio, T. J. Biomol. Tech. 2008, 19, 328–334. (32) Liu, Y.; Wu, D.; Ma, Y.; Tang, G.; Wang, S.; He, C.; Chung, T.; Goh, S. Chem. Commun. (Camb). 2003, 21, 2630-2631. (33) Bhise, N. S.; Shmueli, R. B.; Gonzalez, J.; Green, J. J. Small 2012, 8, 367–373. (34) Theoharis, S.; Krueger, U.; Tan, P. H.; Haskard, D. O.; Weber, M.; George, A. J. T. J. Immunol. Methods 2009, 343, 79–90. (35) Green, J. J.; Langer, R.; Anderson, D. G. Acc. Chem. Res. 2008, 41, 749–759.


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(36) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug Discov. 2005, 4, 581–593. (37) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259–302. (38) Otto, D. P.; de Villiers, M. M. J. Pharm. Sci. 2018, 107, 75–83. (39) Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A. Biotechnol. Bioeng. 2000, 67, 598– 606. (40) Zelphati, O.; Szoka, F. C. Proc. Natl. Acad. Sci. 1996, 93, 11493–11498. (41) Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Biochem. J. 2004, 377, 159–169. (42) Verma, A.; Uzun, O.; Hu, Y.; Hu, Y.; Han, H.-S.; Watson, N.; Chen, S.; Irvine, D. J.; Stellacci, F. Nat. Mater. 2008, 7, 588–595. (43) Oliveira, P. H.; Mairhofer, J. Trends Biotechnol. 2013, 31, 539–547. (44) McGrath, J. A.; Ishida-Yamamoto, A.; O’Grady, A.; Leigh, I. M.; Eady, R. A. J. J. Invest. Dermatol. 1993, 100, 366–372. (45) Goto, M.; Sawamura, D.; Ito, K.; Abe, M.; Nishie, W.; Sakai, K.; Shibaki, A.; Akiyama, M.; Shimizu, H. J. Invest. Dermatol. 2006, 126, 766–772. (46) Chen, Z.-Y.; He, C.-Y.; Ehrhardt, A.; Kay, M. A. Mol. Ther. 2003, 8, 495–500. (47) Jia, F.; Wilson, K. D.; Sun, N.; Gupta, D. M.; Huang, M.; Li, Z.; Panetta, N. J.; Chen, Z. Y.; Robbins, R. C.; Kay, M. A.; Longaker, M. T.; Wu, J. C. Nat. Methods 2010, 7, 197–199. (48) Perdoni, C.; Osborn, M. J.; Tolar, J. Transl. Res. 2016, 168, 50–58. (49) Georgiadis, C.; Syed, F.; Petrova, A.; Abdul-Wahab, A.; Lwin, S. M.; Farzaneh, F.; Chan, L.; Ghani, S.; Fleck, R. A.; Glover, L.; McMillan, J. R; Chen, M.; Thrasher, A. J.; McGrath, J. A.; Di, W.L.; Qasim, W. J. Invest. Dermatol. 2016, 136, 284–292.


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Manipulation of Transgene Expression in Fibroblast Cells by a

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