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Efficient and Robust Highly Branched Poly(#-Amino Ester)/ Minicircle COL7A1 Polymeric Nanoparticles for Gene delivery to Recessive Dystrophic Epidermolysis Bullosa Keratinocytes Ming Zeng, Fatma Alshehri, Dezhong Zhou, Irene Lara-Sáez, Xi Wang, Xiaolin Li, Sigen A, Qian Xu, Jing Zhang, and Wenxin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b13135 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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ACS Applied Materials & Interfaces

Efficient and Robust Highly Branched Poly(β-Amino Ester)/Minicircle COL7A1 Polymeric Nanoparticles for

Gene

delivery

to

Recessive

Dystrophic

Epidermolysis Bullosa Keratinocytes Ming Zeng†,‡,⊥, Fatma Alshehri†,§,⊥, Dezhong Zhou*,†,∥, Irene Lara-Sáez†, Xi Wang†, Xiaolin Li†, Sigen A†, Qian Xu†, Jing Zhang† and Wenxin Wang*,† †Charles

Institute of Dermatology, University College Dublin, Dublin, D04 V1W8, Ireland of Dermatology, the First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China §Princess Nourah bint Abdulrahman University, Riyadh, 11671, Saudi Arabia ∥School of Chemical Engineering and Technology, Xi'an Jiaotong University, Xi'an, 710049, Shaanxi, China ‡Department

ABSTRACT: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe congenital skin fragility disease caused by COL7A1 mutations that result in type VII collagen (C7) deficiency. Herein, we report a synergistic polyplex system that can efficiently restore C7 expression in RDEB keratinocytes. A highly branched multifunctional poly(β-amino ester) (HPAE), termed as HC32122, was optimized systematically as the high-performance gene delivery vector for keratinocytes, achieving much higher transfection capability than PEI, SuperFect and lipofectamine 2000 without inducing obvious cytotoxicity. Concurrently, a 12 kb length minicircle DNA encoding ~9 kb fulllength COL7A1 (MCC7) devoid of bacterial sequence was biosynthesized as the therapeutic gene.

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Combining the highly potent polymer and the miniaturized gene structure, HC32-122/MCC7 polyplexes achieve 96.4% cellular uptake efficiency, 4019-fold COL7A1 mRNA enhancement and robust recombinant C7 expression. Structure-property investigations reveal that HC32-122 can effectively condense MCC7 to form small, uniform, compact and positively-charged spherical nanoparticles with high DNA release flexibility. Moreover, formulation study shows that sucrose is conductive to lyophilized HC32-122/DNA polyplexes for maintaining the transfection capability. Direct frozen polyplexes can maintain the full gene transfection capability after oneyear storage. The high efficiency, biocompatibility, facile manipulation and long-term stability make the HC32-122/MCC7 system a promising bench-to-bed candidate for treating the debilitating RDEB.

KEYWORDS: non-viral gene therapy, polymeric nanoparticles, highly branched poly(β-amino ester)s, minicircle DNA, RDEB, keratinocyte transfection, type VII collagen; polyplex formulation

Epidermolysis bullosa (EB) represents a rare heterogeneous group of blistering disorders characterized by mutations in genes that encode for extracellular matrix proteins. There are more than 30 subtypes of EB,1 recessive dystrophic epidermolysis bullosa (RDEB), one of the most severe forms, is attributed to biallelic loss-of-function mutations in the COL7A1 gene.2 The fulllength of COL7A1 messenger RNA (mRNA)/complementary DNA (cDNA) encoding for the 290kDa type VII collagen (C7) is about 9 kb.3,4 In healthy skin, procollagen VII secreted from keratinocytes and dermal fibroblasts is processed into C7 and assembled into the anchoring fibril (AF) which provides a “Biologic Velcro” at the dermal-epidermal junction (DEJ).5 In RDEB


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patients, loss or diminished presence of functional C7 results in the structural architecture ruin of DEJ and mucosal membrane. Consequently, the compromised skin integrity in RDEB produces the characteristic clinical manifestations of severe and chronic blistering, mitten deformities of the hands and feet, and mucosa and organ lesions with an increased risk for aggressive form of squamous cell carcinoma.2,6 Over the last three decades, significant efforts have been made and several therapeutic strategies have been proposed to ameliorate the debilitating skin fragility diseases aiming at transient or life-long relief. Due to the relatively long physiological half-life of C7, the recombinant protein therapy has shown some benefits to restore C7 in several preclinical investigations, intradermally or intravenously.7,8 However, the possibility of anti-C7 antibody formation may limit the translational application of protein therapy to clinical settings. Allogeneic cellular therapies under development include the fibroblast injection,9 mesenchymal stem cell (MSC) infusion10 and bone marrow transplantation (BMT).11 To some extent, these studies provided proof-of-concept for reversing the disease phenotype by introducing wild-type donor cells that can be the secretion sources of C7. However, the transient and limited efficiency with abnormal AF morphology from the fibroblast injection, the short-lived persistence of MSC cells and severe transplant-related adverse events from HCT along with potential immunologic complications from allogeneic therapies would hamper their treatments in practice.2 From the early 2000s onwards, gene therapy has been developed for ectopic C7 expression in patient-derived RDEB cells. Viral gene therapy has historically been the delivery approach for RDEB gene therapy, by primarily transducing the autologous RDEB keratinocyte (RDEBK) or/and fibroblast ex vivo and then transplanting the gene-corrected autograft.12 Although the benchmark retroviral (RV) and lentiviral (LV) vectors are able to integrate into transcriptionally active regions of the host genome and thus allowing for stable and long-term gene expression,13


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the risks of inducing carcinogenesis,14 immunogenicity,15 and broad tropism16 lead to significant concerns and a more stringent regulatory environment when viral vectors are employed. Besides, limited gene cargo capacity,2 difficulty of vector production17 and uncontrolled vector mobility18 impede the viral gene therapy to translational applications. In contrast, the number of non-viral gene therapy in clinical trials increased dramatically due to its relatively high safety profile, large genetic payload, localized gene expression, flexible vector design, easy vector synthesis and costeffective manufacturing.19–21 Since 2010, at least 40 nanoparticle-based non-viral gene therapies have entered various stages of clinical trials, targeting over 15 different disease indications.21 These clinical trials have been centered on polyethylenimine (PEI) or its modified variants and lipid-based systems. Although the off-the-shelf cationic polymer PEI has shown some potential, it has been widely known to induce substantial cytotoxicity.22 As an alternative to PEI, linear structured poly(β-amino ester)s (LPAEs) were developed by Langer and co-workers in 2000.23 After systematic structure and function optimization,24–26 versatile LPAEs that can navigate multiple extra- and intra-cellular barriers associated with gene delivery have been identified. Notably, the top-performing LPAE can even rival viral counterparts in gene delivery studies.27 To further improve the gene transfection performance and functionalization ability, highly branched poly(β-amino ester)s (HPAEs) with three-dimensional (3D) structure and multiple terminal groups have been developed by our group via a facile “A2+B3+C2” Michael addition strategy since 2015.28,29 Studies have shown that HPAEs outperform LPAEs across diverse cell transfections, highlighting the critical role of branching strategy in enhancing the gene transfection efficiency of poly(β-amino ester)s.30–33 Especially, inspired by the top-performing LPAE - C32 (Poly(5-amino1-pentanol-co-1,4-butanediol diacrylate)),24,25,27,34 highly branched C32 (HC32) series have


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provided a powerful tactic for the development of effective non-viral gene therapy systems due to their adjustable branching structure design and high gene transfection potency.30,33,35 To date, RDEB is still without curative therapies. The 9-kb transcript length of COL7A1 exceeds the gene cargo capacity of the majority viral systems.2 Additionally, a large gene construct size above 6 kb is detrimental for nucleic acid transfer efficiency, with a substantial decrease in gene delivery efficiency.36 The challenge is, the requirement of a non-integrating and efficient vector with high gene cargo capacity sets a very high technical barrier for the COL7A1 gene delivery. Here, for the first time, by rationally designing both the polymeric vector and DNA construct, we have successfully developed an efficient and robust gene delivery system for highperformance C7 restoration in COL7A1-null RDEBKs. First, a multifunctional HPAE of HC32122 was synthesized via the “A2+B3+C2” Michael addition strategy (Figure 1a). It was further optimized in order to achieve the maximum gene transfection efficiency and favorable cell viability. Concurrently, an optimized and miniaturized gene construct, the 12 kb-length minicircle DNA encoding ~9 kb full-length COL7A1 (MCC7) devoid of bacterial sequence was prepared as the therapeutic gene. Combining the versatile polymer and the optimized gene construct, ultrahigh MCC7 cellular uptake efficiency (96.4%), high transcriptional level (4019-fold COL7A1 mRNA enhancement) and robust recombinant C7 expression were obtained from the optimized HC32-122/MCC7 system for reversing C7 defects in RDEBK cells. Furthermore, HC32122/MCC7 polyplexes exhibited excellent physiochemical properties to facilitate the navigation of multiple extra- and intra-cellular barriers associated with keratinocyte gene transfections. Moreover, considering the requirement for a new candidate medication, apart from the efficiency and safety, the manipulation, process and storage stability have far more practical significance in its bench-to-bedside translation. Our formulation studies further demonstrated that HC32-


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122/MCC7 polyplexes can be facilely manipulated, lyophilized and stored for RDEBK gene delivery. This work offers a promising polymeric nanoparticle-based system, which enables the application of a biocompatible, efficient, easily fabricated and applicable approach for RDEB gene therapies. RESULTS AND DISCUSSION HC32-122 Synthesis and Characterization The facile “A2+B3+C2” Michael addition strategy provides a versatile platform for the synthesis of HPAEs with tailorable compositions, structures and functionalities.30,31,33 Previously, HC32-122 polymers with weight molecular weight (Mw) around 14-17 kDa were synthesized via the “A2+B3+C2” strategy and used for gene transfection, showing high level of gene transfection efficiency.33 However, as one of the most critical structural parameters, effects of HC32-122 molecular weight (MW) on the gene transfection efficiency and cell viability have not been investigated. Therefore, further optimization of HC32-122, especially its MW, to achieve both high efficiency and safety is of great significance and highly desirable. To this end, HC32-122 polymers were synthesized (Figure 1a). As shown in Figure 1b, by simply increasing the polymerization time of the base polymers, four HC32-122 polymers with increased Mw from 11 kDa to 41 kDa were synthesized and no gelation was occurred, demonstrating high flexibility of the “A2+B3+C2” Michael addition strategy in controlling the Mw of HAPEs. MH plot alpha values of all HC32-122 polymers are below 0.5 (Figure 1c, Table S1), indicating their highly branched structures.28,37 Proton nuclear magnetic resonance (1H NMR) characterization confirms the chemical compositions of HC32-122 (Figure S1).

Effects of MW of HC32-122 on Reporter Gene Transfection in RDEBK Cells


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Anderson and co-workers reported that MW of LPAEs is a key factor that affects DNA complexation, nanoparticle size and thus gene transfection efficiency.38 For HPAEs, due to the branched structure and multiple terminal groups, the effects of MW on gene transfection may be different from that of LPAEs. In our preliminary studies, HC32-122 with Mw of 5.6 kDa was synthesized. It was found that the polymer only showed limited gene transfection efficiency due to the relatively weak DNA binding affinity. To identify the most favorable Mw for gene delivery, four HC32-122 polymers were used to transfect RDEBK cells using green fluorescent protein (GFP)-encoding DNA as the reporter gene. As shown in Figure S2 and Figure S3, although no obvious cytotoxicity is observed at the polymer/DNA weight/weight (w/w) ratio of 10:1, the transfection efficiency from all HC32-122 polymers is low. When the w/w ratio is increased to 30:1 or greater, among all the polymers, 11 kDa HC32-122 at 30:1 achieves the highest transfection efficiency with the strongest GFP expression, while preserving high cell viability of 98%. Generally, GFP expression decreases with the increasing Mw of HC32-122 polymers. On the other hand, the cytotoxicity correlates very well with the increasing polymer Mw. For example, at the w/w of 50:1, as the Mw increases, cell viability decreases from 91% to 58%, 42% and 15%, respectively. Transfection efficiency is compromised with the increasing cytotoxicity which might be attributed to the incremental main chain of the polymer. These results demonstrate that MW has significant effects on the transfection performance of HC32-122, and a ~10 kDa Mw is more favorable for RDEBK gene transfection to achieve both high transfection efficiency and low cytotoxicity. The high gene transfection ability of the 11 kDa HC32-122 was further verified by comparing with that of the commercial gene transfection reagent PEI (Mw = 25 kDa). As shown in Figure 2a, at all three tested w/w ratios, the relative Gaussia luciferase (Gluc) activity of RDEBK cells after


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transfection by HC32-122/DNA polyplexes was much higher than that mediated by the PEI/DNA counterparts. The highest Gluc activity achieved by HC32-122/DNA polyplexes at 30:1 w/w ratio was 17-fold higher than the PEI/DNA counterparts at the w/w ratio of 2:1. Importantly, the HC32122/DNA polyplexes did not induce obvious cytotoxicity and preserved almost 100% cell viability. By contrast, PEI showed evident dose-dependent cytotoxicity, the cell viability decreased significantly from 90% at 1:1 w/w ratio to 38% at 3:1 w/w ratio (Figure 2b). Due to the considerable cytotoxicity, PEI with 1:1 w/w ratio was used for following studies. To further validate the high performance of HC32-122/DNA polyplexes, GFP-encoding DNA was used for transfection. HC32-122/DNA polyplexes mediated a much higher level of GFP expression than PEI, evidenced by the substantially stronger green fluorescence observed (Figure 2c). Correspondingly, flow cytometry quantification analysis shows that more cells are shifted corresponding to the GFP-determining channel (Figure 2d). 75% of the RDEBK cells were GFPpositive after transfection by the HC32-122/DNA polyplexes, in contrast to 39% achieved by the PEI/DNA polyplexes. Moreover, the median fluorescence intensity (MFI) of the RDEBK cells transfected by the HC32-122/DNA polyplexes is 13-fold higher than that mediated by the PEI/DNA counterparts (Figure 2e), indicating that much higher gene expression was achieved in individual cells. All these results demonstrate that HC32-122 is far more efficient and biocompatible than PEI for gene transfection in RDEBK cells.

Biosynthesis of MCC7 and Cellular Uptake of HC32-122/MCC7 Polyplexes To circumvent the limited nucleic acid payload of viral vectors and virus-integrating risks, tremendous efforts have been made to modify plasmid DNA construct, design of bacterial sequence-free minicircle DNA is one of the most promising strategies.21 Due to the smaller size


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and lack of bacterial sequences, minicircle DNA usually shows superior bioavailability, immunocompatibility and safety to the regular counterpart.39,40 In addition, minicircle DNA mediates higher transgene expression and more stable ectopic transgene expression compared to regular plasmid (RP).41 Utilization of a phiC31 integrase based minicircle technology,42 MCC7 encoding the ~9 kb full-length COL7A1 was biosynthesized (Figure 3a). Among all the three COL7A1-tagged DNA, gel electrophoresis shows that MCC7 only has 3 kb length of backbone, which is 2 kb and 5 kb shorter than RP pcDNA3.1COL7A1 and parental plasmid (PP) MN511A1-COL7A1 (Figure 3b), respectively, indicating the MCC7 biosynthesis with miniaturized derivative devoid of bacterial sequences from the traditional PP vector. Cellular uptake efficiency of HC32-122/MCC7 polyplexes was explored in RDEBK cells. MCC7 was labelled with the red fluorescent dye Cy3 for intracellular tracking. 4 h post transfection, very strong red fluorescence was observed around the nucleus in the cells (Figure 3c). In comparison, PEI/MCC7 polyplexes show much lower cellular uptake efficiency with far weaker red fluorescence. Flow cytometry measurements further demonstrate that although the percentage of Cy3-positive cells is similar (96.4% versus 93%, Figure 3d), MFI of the cells incubated with the HC32-122/MCC7 polyplexes was around 2-fold higher than that treated by the PEI/MCC7 counterparts (Figure 3e), indicating a higher number of DNA copies was taken up by RDEBK cells. The maximum DNA sizes that RV and adeno-associated virus (AAV) vectors can carry are 7-8 kb and 5 kb, respectively.2 The fact that HC32-122 is capable of delivering 12 kblength MCC7 into RDEBK cells in an efficient manner, highlighting its potential to achieve high C7 expression for RDEB treatment.

High Levels of COL7A1 mRNA and Recombinant C7 Expression


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Following internalization, vector/DNA polyplexes will be further challenged by a set of intracellular barriers, including endo/lysosomal escape, transport through cytoplasm, DNA release and nucleus entry.43 Cell cycle is another obstacle that could affect the nuclear uptake efficiency. Cells undergoing mitosis exhibit greater than 10 times higher nuclear uptake efficiency than those in the growth phase of the cell cycle.44 To further evaluate the transcript COL7A1 mRNA and C7 protein expression mediated by the HC32-122/MCC7 polyplexes, quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunofluorescence staining and western blotting studies were performed. Figure 4a and Figure 4b outline the RT-qPCR amplification plots of the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and COL7A1 mRNA expression, respectively. After normalized to the endogenous control, it is shown that the HC32122/MCC7 polyplexes mediated a 4019-fold upregulation of COL7A1 mRNA expression in comparison with the untreated cells (Figure 4c), and a 2.2-fold enhancement relative to the PEI/MCC7 polyplexes. Immunofluorescence staining studies further reveal that null-C7 expression was detected in the untreated RDEBK cells (Figure 4d). In contrast, after transfection with the HC32-122/MCC7 polyplexes, much higher level of cellular C7 expression around the nucleus was observed in the cyto-immunofluorescence images. Once again, in agreement with the results of COL7A1 mRNA expression, HC32-122/DNA polyplexes mediated more efficient C7 expression than the PEI/MCC7 counterparts. Meanwhile, western blotting results show that no C7 secretion was detected from untreated RDEBK cells (Figure 4e). On the contrary, a very clear 290-kDa protein band of C7 is visible after transfection with HC32-122/polyplexes, the C7 band is even stronger than that of the wild-type normal human keratinocyte (NHK) cells, suggesting that the high efficiency of HC32-122 leads to the overexpression of C7. It is noted that although PEI/MCC7 polyplexes achieved high level of COL7A1 mRNA expression, C7 production is


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limited. It is believed that the well-known toxicity from PEI would reduce its efficiency with persistent protein expression. A mechanism study demonstrated that the compact DNA structure increased cellular uptake, intracellular vector copy numbers, nuclear localization and mRNA transcription levels.45 Herein, taking advantages of the optimized HPAE and miniaturized gene construct, HC32-122 can effectively deliver COL7A1 gene into RDEBK cells, promote subsequent mRNA transcription and ultimate recombinant C7 expression which is considered to strength the skin integrity.

Physicochemical Parameters of HC32-122/MCC7 Polyplexes DNA condensation, binding, polyplex size, zeta potential, morphology and DNA release are key parameters that synergistically dictate the transfection performance.31,35 To better understand the possible mechanisms of the high gene transfection efficiency mediated by HC32-122/MCC7 polyplexes, these crucial physicochemical parameters were investigated. The cationic HC32-122 is believed to condense the negatively-charged MCC7 to form polyplexes via electrostatic selfassembly (Figure 5a). To confirm this, agarose gel electrophoresis was conducted to evaluate the DNA condensation ability of HC32-122 2 h post polyplex preparation. As shown in Figure 5b, naked MCC7 DNA shifted on the gel, whereas HC32-122 was capable of condensing DNA on the well without obvious DNA shifting. Heparin competition assay further showed that HC32-122 polyplexes with low heparin concentrations (0.1-0.3 IU/μL) still condensed the majority of DNA, suggesting strong DNA condensation ability and highly stable property of HC32-122 polyplexes. Correspondingly, as quantified by PicoGreen Assay (Figure 5c), HC32-122 showed stable and high DNA binding affinity of 96.3%, indicating only 3.7% of DNA unpacking. When 3 and 6 IU/μL concentrations of heparin were applied, 68.2% and 99.6% of DNA unpacking were detected


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respectively. These results demonstrate that HC32-122 can condense, bind and release DNA efficiently in a controlled manner in the presence of adjustable negatively-charged heparin. At the optimal w/w ratio employed for efficient C7 expression (30:1), nanoparticles exhibited a mean size of 110 nm and a mode size of 81 nm, respectively (Figure 5d), with the zeta potential of +37.4 mV (Figure 5e), indicating a compact nanoparticle formation with positive surface charge. It is known that polyplexes are most commonly formed of spherical, toroidal or doughnut-shaped morphology.19,43 Here, uniform and spherical HC32-122/MCC7 polyplexes were observed by transmission electron microscopy (TEM) (Figure 5f), which are favorable for keratinocyte transfection. The enhancement of transfection efficiency of LPAEs is benefited from the diamine end-group modification, which increases the polymers’ cationic charge, leading to the improvement of the polymer/DNA binding dynamics, the condensation of DNA into nanoparticles and the DNA protection from degradation.27 In addition to the end-modification with diamine 122, multiple DNA-binding/condensation moieties - including primary, secondary, tertiary amines - reside in HC32 backbone and terminal groups. Generally, LPAE/DNA complexes are less than 250 nm and particles of smaller size were found to be more efficiently internalized by cells.25,27 It is conceivable that the small, compact, uniform and cationic properties of HC32/MCC7 polyplexes are vital to high cellular uptake efficiency of RDEBK cells. In addition, multiple protonatable secondary and tertiary amines can buffer a wide range of protons which can facilitate the endo/lysosomal escape via the “proton sponge effect”.43 Afterwards, the stable gene packaging stability of HC32-122 suggests that it can assist the intracellular transport of polyplexes through the cytoplasm toward the nucleus. An efficient vector must balance sufficient binding strength to protect DNA with the ability to release DNA.43 We speculate that the moderate electrostatic


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interaction between HC32-122 and MCC7 and the biodegradable property of HC32-122 can facilitate gene release from polyplexes inside nucleus to start the transcription steps. Collectively, gene transfection involves multiple mechanistic steps, the high-performance gene transfection is believed to be benefited from the abovementioned favorable parameters.

Formulation of HC32-122/DNA Polyplexes Over the past decades, numerous efforts have been made to improve the efficiency and safety of gene delivery systems, however, relatively little attentions have been paid to their formulation work that plays a decisive role on clinical applications. To date, the majority polymeric formulation work in terms of storage, lyophilization and/or up-scaled production mainly focuses on LPAEs46–49 and PEI.50 Here, we further optimized the HC32-122/DNA polyplex formulation by varying the storage condition and protectant concentration in the lyophilization process. Figure 6a illustrated the scheme of polyplex lyophilization fabrication and further gene transfection studies in RDEBK cells using the GFP reporter assay. Except the freshly prepared polyplex control group, all polyplexes were employed in gene transfection 1 day post preparation. As shown in Figure 6b, by varying the storage temperature, except the one stored at room temperature (RT), fresh polyplexes and stored polyplexes at 4°C, -20 °C and -80 °C exhibit the same high level of GFP expression, with significant shifts of the cell population in the flow cytometry histogram distributions (Figure 6c). The efficiency from these top-performing polyplexes is higher than 70%, and their normalized MFIs were all around 10-fold higher than untreated group (Figure 6d and 6e). These results demonstrate that refrigeration storage is more advantageous for retaining the high gene transfection ability of HC32-122/DNA polyplexes. Next, using sucrose as the protectant during the freeze-drying process, the effects of protectant concentration on the gene transfection


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ability of HC32-122/DNA polyplexes were evaluated. Cells treated with the freeze-drying polyplexes without any protectant (0% sucrose) lost the majority of transfection ability, exhibiting only 20% efficiency and 1.4-fold higher MFI compared to the untreated group. When 1%, 3% and 5% of sucrose were added into the polyplex solution prior to lyophilization, transfection efficiency was increased to 54%, 61% and 52%, respectively. These results demonstrate that 3% of sucrose is more efficient for maintaining the gene transfection ability of HC32-122/DNA polyplexes. It should be noted that although compromised to a certain degree in comparison with that of the freshly prepared counterparts, gene transfection ability of the polyplexes freeze-dried with sucrose is still much higher than that of the commercial reagents SuperFect and Lipofectamine, which only displayed 10% and 32% efficiency respectively. Meanwhile, polyplex lyophilization also has its unique advantages: 1) it enables subsequent reconstruction of polyplexes at a higher concentration, which is particularly beneficial for in vivo injection that requires a limited administration volume;49 2) easily adjustable solute (sucrose) can make the reconstructed polyplex solution isotonic during formulation;49 3) lyophilized polyplexes with sucrose would be more stable in the presence of serum compared with freshly prepared polyplexes;46 4) lyophilized polyplexes can be stored for years without losing efficacy.47 Furthermore, gene transfection study of the top-performing HC32-122/DNA polyplexes (4 °C, -20 °C and -80 °C groups) with different storage time was carried out to evaluate the shelf life. As shown in Figure 7, after storage at 4 °C for 0.5 and 1 month, Gluc activity of the polyplexes was two to three magnitudes lower than that mediated by the freshly prepared counterparts. After 2 months, efficiency of the polyplexes became negligible. In contrast, even after one year, polyplexes that stored at -20 °C and -80 °C mediated the same level of Gluc activity as the freshly formulated polyplexes. These results demonstrate that the HC32-122/DNA polyplexes are very


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stable and can retain their full function of gene transfection by simply storing at -20 °C or -80 °C, making them highly feasible for clinical applications. To our knowledge, this is the first demonstration of HPAE/DNA polyplex formulation in gene transfection studies. The facile manipulation, lyophilization and storage method shed substantial light on their future translational applications for RDEB.

Conclusion In summary, this work introduces a nanobiotechnology with a robust, efficient, biocompatible and stable HC32-122/MCC7 polyplex system for RDEBK gene delivery. It is demonstrated that HC32-122 with the Mw around 10 kDa far outperforms PEI in both Gluc and GFP reporter assays, demonstrating orders-of-magnitude higher transfection efficiency without inducing obvious cytotoxicity. In addition, with the focus on delivering a large therapeutic gene for C7 restoration, an optimized MCC7 encoding the full-length COL7A1 gene devoid of bacterial sequence is biosynthesized. Taking advantages of the potent HC32-122 and the miniaturized COL7A1 gene construct, HC32-122 is far superior to PEI in gene delivery mechanism steps including the cellular uptake, COL7A1 mRNA expression and recombinant C7 expression. Furthermore, in-depth mechanism studies reveal that due to multiple DNA binding moieties, endosome-buffering amines and hydrolytically degraded ester groups resided in the polymer, HC32-122 can efficiently condense and bind MCC7 to small, compact, positively-charged, uniform and spherical nanoparticles, exhibiting stable gene packaging ability with the flexibility to release DNA. These favorable properties are considered to promote the cellular uptake, enable the DNA transport to nucleus and thus increase the mRNA and C7 expressions. Moreover, the formulation study shows that lyophilized nanoparticles are benefited from the lyoprotectant sucrose with the majority of


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efficiency retained, possessing much higher transfection efficiency than commercial transfection reagents SuperFect and Lipofectamine 2000. More importantly, frozen storage method maintains the full function of nanoparticles across a 1-year span storage. Given that skin lesions have easy accessibility of topical administration, and the high efficiency and biocompatibility, facile formulation and excellent stability of this system, HC32-122/MCC7 polyplexes provide a promising translational gene therapy for the debilitating skin disease RDEB. MATERIALS AND METHODS Materials Monomers 5-amino-1-pentanol (32, 99%), trimethylolpropane triacrylate (TMPTA, 99%), 1,11diamino-3,6,9-trioxaundecane (122, 98%) were purchased from Sigma-Aldrich, and 1,4butanediol diacrylate (C, 98%) was purchased from VWR. Chemicals lithium bromide (LiBr, 99%), tris-buffered saline and tween 20 (TBST), paraformaldehyde (PFA) and triton X-100 were purchased from Sigma-Aldrich. Solvents dimethyl sulfoxide (DMSO, Sigma-Aldrich, 99%), dimethylformamide (DMF, Fisher Scientific, 99%), diethyl ether (Sigma-Aldrich, 99%) and deuterated chloroform (CDCl3, Sigma-Aldrich, 99.9%) were used as received. Branched PEI, (Mw = 25 kDa, Sigma-Aldrich), SuperFect (QIAGEN), Lipofectamine 2000 (Invitrogen) were used as the commercial reagent controls. Keratinocyte cell basal medium (Clonetics KBM-Gold) with the supplement pack (Clonetics KGM-Gold SingleQuots) was purchased from Lonza. Cell secreted Gluc plasmid and BioLux Gaussia luciferase assay kits were obtained from New England Biolabs UK. GFP plasmid was purchased from Aldevron. Hank’s balanced salt solution (HBSS), sodium acetate (SA, pH 5.2±0.1, 3 M) buffer, tris acetate-EDTA (TAE) buffer and RadioImmunoprecipitation assay (RIPA) buffer, agarose, bovine serum albumin (BSA), goat serum, monoclonal anti-C7 antibody produced in mouse, Protease Inhibitor Cocktail (PIC) and Bradford


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Reagent were purchased from Sigma-Aldrich. 1× Dulbecco’s phosphate buffered saline (PBS), Gibco OPTI-MEM I reduced serum medium, 4’,6-diamidino-2-phenylindole (DAPI) and PicoGreen assay kits were purchased from Life Technologies. Penicillin-steptomycin (PS), EcoRI, Alexa-568 goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody and Pierce ECL plus Western Blotting substrate were purchased from Thermo Fisher Scientific. Alamarblue assay kits, SYBR safe DNA gel stain and SuperScript III First-Strand Synthesis SuperMix were purchased from Invitrogen. Collagen type VII alpha 1 (Fam-MGB, primer & probe), human GAPDH endogenous control (VIC/MGB probe, primer limited) and TaqMan gene expression master mix were purchased from Applied Biosystems. TE buffer (QIAGEN), Cy3 DNA labelling kit (Mirus), RNeasy Mini Kit (QIAGEN), Fluoroshield mounting medium with DAPI (Abcam) were used as per manufacturers’ protocols. Polycolonal anti-C7 rabbit primary antibody (Merck Millipore), anti-beta actin mouse primary antibody (Abcam), anti-rabbit IgG HRP-linked antibody (Cell Signaling) and anti-mouse IgG HRP-linked antibody (Cell Signaling) were used as received. Polymer Synthesis and Characterization Four HC32-122 polymers with different MWs were synthesized via the “A2+B3+C2” Michael addition strategy. HC32 base polymers were first synthesized. Briefly, the A2 monomer 32 (9.0 mmol, 0.923 g), B3 monomer TMPTA (0.5 mmol, 0.148 g) and C2 monomer C (10.0 mmol, 1.98 g) were dissolved in 3.1 mL DMSO, and then reacted at 90℃. Gel permeation chromatography (GPC) was used to monitor the growth of MW and polydispersity index (PDI). 20 μL of reaction sample was taken at different time points, followed by diluting in 1 mL DMF and filtering through a 0.2 μm filter prior to GPC measurement on an Agilent 1260 Infinite GPC equipped with a triple detector: a 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-


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M, 7.5 × 300 mm, two in series) at a flow rate of 1 mL/min at 60 °C. GPC columns were calibrated with the linear poly(methyl methacrylate) (PMMA) standards. When the weight average molecular weight (Mw) of the based polymer was approaching target values (around 10, 20, 30 and 40 kDa, respectively), the reaction was stopped by diluting the reaction solution in DMSO to 100 mg/mL. Afterwards, the end-capping agent 122 (10.0 mmol, 1.92 g) dissolved in DMSO (100 mg/ mL) was used to end-cap the HC32 base polymers through Michael addition at RT for 48 h to obtain the HC32-122 polymers, which were purified by precipitation with diethyl twice to remove the excess monomers, oligomers and end-capping agent. The final HC32-122 products were dried in a vacuum oven for 24 h and then freeze-dried for another 24 h to remove the residual solvents. To measure the MW and PDI of the final products, 10 mg sample was dissolved in 1 mL DMF and GPC measurements were carried out as mentioned above. 1H NMR was utilized to confirm chemical compositions and purity of the HC32-122 polymers, which were dissolved in CDCl3 and 1H

NMR spectra was acquired on a 400 MHz Varian Inova spectrometer. Sample was reported in

parts per million (ppm) relative to the solvent (7.24 ppm) or internal control (tetramethylsilane 0.00 ppm). MCC7 Biosynthesis RP pcDNA3.1COL7A1 was kindly provided by Dr. Andrew P. South at Thomas Jefferson University (USA). MCC7 was biosynthesized by inserting the COL7A1 sequence originated from the pcDNA3.1COL7A1 to the MN511A-1 cassette offered from System Biosciences with cytomegalovirus promoter, induction and production of minicircle DNA were carried out according to the user’s manual of System Bioscience and the published phiC31 integrase system of minicircle technology.42 To confirm the biosynthesis of MCC7, DNA digestion study was carried out. To this end, 0.5 μg pcDNA3.1COL7A1, parental plasmid (MN511A-1-COL7A1) and


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MCC7 were digested by 1 μL EcoRI and then subjected to agarose gel electrophoresis at 100 V for 40 minutes. Then images were visualized using a Syngene's G:BOX. Polyplex Preparation and Formulation HC32-122 was dissolved in DMSO to a 100 μg/μL stock solution which was stored at -20 ℃ for the following studies. DNA was dissolved in TE buffer and stored at -20 ℃ as well. SA buffer was diluted to 0.025 M prior to use. For standard polyplex preparation, according to the polymer/DNA weight ratio (w/w), DNA and polymer were dissolved in the SA buffer to equal volume, respectively. And then the polymer solution was added to the DNA solution, mixed for 10 seconds using a vortex and incubated for another 10 minutes at RT to allow the polyplex formation. For the formulation study, typically, 5 μg GFP plasmid DNA and 150 μg HC32-122 were dissolved in 200 μL SA, respectively, to formulate the HC32-122/DNA polyplexes (w/w = 30:1). The polyplexes were either immediately used as the fresh ones, or stored at RT, 4 ℃, -20 ℃ and -80 ℃, or lyophilized prior to transfection. For lyophilization, sucrose was added to the polyplex solution to final sucrose concentrations of 0%, 1%, 3% and 5%, respectively. All samples were frozen at -80 ℃ for 1 h and then immediately subjected to freeze dry with a Christ Alpha 12 LDplus Freeze Dryer at -55 °C for 24 h. Afterwards, the polyplexes were reconstituted with the original volume of SA and used for transfection. After optimization of the polyplex formulation procedure, HC32-122 complexed with Gluc-encoding DNA stored at different conditions was used to evaluate the feasibility for long-term storage of polyplexes prior to transfection applications. Here, 0.5 μg DNA at 30:1 polymer/DNA w/w ratio was used for each well in 96-well plates. DNA Condensation and Heparin Release Studies


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To assess the DNA condensation ability of HC32-122 and the physical stability of the HC32122/DNA polyplexes, DNA condensation assay and heparin release assay were performed using agarose gel electrophoresis. 0.5 μg DNA (MCC7) was used for each sample and polyplexes were prepared at the w/w ratio of 30:1. Aqueous heparin solution was added in the polyplex solution with concentration increasing from 0.1-6 IU/μL. Naked DNA and HC32-122/MCC7 polyplexes without heparin were used as the controls. All samples were incubated at RT for 2 h and then loaded on a 1% agarose gel stained with 10 μL SYBR safe DNA stain. Electrophoresis was performed in 1 × TAE buffer at 100 V for 1 h. PicoGreen Assay PicoGreen assay was used to quantify the DNA binding affinity of HC32-122 and DNA release in the presence of heparin. HC32-122/MCC7 polyplexes were prepared with 0.2 μg DNA at the 30:1 w/w ratio, and then heparin was introduced to the polyplex solution at the concentration of 0.3 IU/μL, 3 IU/μL and 6 IU/μL, respectively. Naked DNA and HC32-122/MCC7 polyplex without heparin treatment were used as the controls. After 2 h incubation, all the samples were mixed with 10 μL PicoGreen working solution and incubated for another 5 minutes. Afterwards, the mixture solution was diluted by deionized water to a final concentration of 1 μg/mL in a black 96-well plate. Fluorescence measurements were carried out using a SpectraMax M3 plate reader with the excitation at 490 nm and the emission at 535 nm in quadruplicate. DNA release efficiency was quantified by normalizing the fluorescence intensity of samples to naked DNA control. Size and Zeta Potential Measurement of Polyplexes Polyplex size was measured by nanoparticle tracking analysis (NTA) using a Nanosight NS300. Polyplexes were prepared using 0.5 μg DNA with 30:1 w/w ratio in 10 μL SA. Next, the polyplex solution was diluted to 1 mL distilled water and then subjected to NTA analysis. A 60 second


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movie containing the Brownian motion tracking of the particles was recorded using the NTA software (Version 3.2). 10 tracks were assessed for each sample. Zeta potential measurements of polyplexes was conducted using a Malvern Instruments Zetasizer (Nano-ZS90) at a 90° scattering detector angle. TEM Observation Morphology of polyplexes was characterized by TEM. 80 μL polyplex solution with 2 μg MCC7 at the w/w ratio of 30:1 was centrifuged and the supernatant was discarded, and then polyplexes were further washed with 80 μL distilled water twice to remove excess salts. Afterwards, polyplexes were resuspended to a final volume of 10 μL distilled water. Then 2.5 μL polyplex solution was placed onto Formvar support films on 200 mesh copper grids and lyophilized immediately. Images were captured on a FEI Tecnai 120 TEM at 120 kV in UCD Conway Imaging Core Center. Cell Culture The RDEBK cell line (A6) was kindly obtained from Dr. Fernando Larcher in Cutaneous Diseases Modelling Unit, Division of Biomedicine, CIEMAT, Madrid, Spain. NHK cells were purchased from Lonza. RDEBK and NHK cells were cultured in keratinocyte cell basal medium (KBM-Gold) with the supplement pack (KGM-Gold SingleQuots) and 1% PS in a humid incubator with 5% CO2 at 37 ℃ under standard cell culture conditions. GFP Expression and Cell Viability GFP reporter gene transfection was first performed to evaluate the gene transfection efficiency of four HC32-122 polymers and screen out the best-performing candidate. RDEBKs were seeded in 96-well plates at a density of 2 × 104 cells per well. Next day, 0.5 μg plasmid DNA encoding


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GFP was used for each well. HC32-122 polyplexes with different Mws were prepared at polymer/DNA w/w ratios of 10:1, 30:1 and 50:1 in 20 μL SA, which was mixed with 80 μL fresh culture medium as the transfection medium. 4 h post transfection, transfection medium was replaced with fresh medium. 48 h post transfection, GFP expression of cells were visualized under a fluorescence microscope (Olympus IX81). Cell viability was measured with Alamarblue assay. Cell supernatants were removed and then cells were incubated with 10% Alamarblue reagent in HBSS for another 1 h at 37 ℃. Afterwards, the Alamarblue solution was transferred to a flat bottomed 96-well plate. Fluorescence intensity was read by a SpectraMax M3 plate reader with an excitation at 570 nm and emission at 590 nm. Fluorescence intensity of the untreated cell group was plotted as 100% viable. Cell viability was measured in quadruplicate and calculated by normalizing the fluorescence intensity of sample to that of the untreated group. HC32-122 showing the highest GFP expression and cell viability was used for the following studies. In the formulation studies, SuperFect/DNA polyplexes were prepared at the w/w ratio of 3:1 according to the publication.35,51 Lipofectamine 2000/DNA lipoplexes were prepared according to manufacturer’s protocol (2:1 volume/weight ratio). The MFI and GFP-positive cells were quantified by flow cytometry on an Accuri C6 system in triplicate and further analyzed with FlowJo V10 software. 1 × 104 cells were counted for each run. Gluc Reporter Gene Transfection and Cell Viability The optimized HC32-122 was further evaluated in Gluc reporter gene transfection studies. Using 0.5 μg plasmid DNA encoding Gluc for each well, HC32-122/DNA polyplexes were prepared at the w/w ratios of 20:1, 30:1 and 40:1, respectively. According to previous publications,27,52,53 PEI/DNA polyplexes were prepared at w/w ratios of 1:1, 2:1 and 3:1, respectively. RDEBKs were seeded and gene transfection was carried out as mentioned above. 48


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h post transfection, to quantify the gene transfection efficiency, 50 μL of the cell supernatant was mixed with equal volume of Gluc assay working solution. Fluorescence intensity of the mixture was measured using a SpectraMax M3 plate reader with an excitation at 485 nm and emission at 525 nm. Gluc activity results were plotted in terms of relative light units (RLU). Cell viability were measured as mentioned above. Both Gluc activity and cell viability experiments were determined in quadruplicate. Cellular Uptake of Polyplexes Cy3 DNA labelling kits were used to label MCC7 according to standard protocol. RDEBKs were seeded in 96-well plates at a density of 1 × 104 cells per well. Next day, using 0.25 μg MCC7 for each well, cells were transfected with HC32-122/MCC7 polyplexes (w/w = 30:1) and PEI/MCC7 (w/w = 1:1) for 4 h, and then fixed with 4% PFA, permeabilized with 0.1% triton-100 and incubated with DAPI at a working concentration of 1 µg/mL in HBSS. Fluorescent images were taken with a microscope (Olympus IX81). The MFI and Cy3-positive proportion of cells were quantified by an Accuri C6 system in triplicate. Results were further analyzed with Flowjo V10 software with 1 × 104 cells counted for each measurement. RT-qPCR RT-qPCR was performed to quantify the COL7A1 mRNA expression. RDEBKs were seeded on 6-well plates at a density of 2.5 × 105 cells per well one day prior to transfection. Cells were transfected with HC32-122/MCC7 and PEI/MCC7 polyplexes complexed with 5 μg DNA at w/w ratios of 30:1 and 1:1. Three days post treatment, both treated and untreated cells were harvested and subjected to the purification of total RNA. RNA Extraction work was carried out according to the protocol of RNeasy Mini Kit. Next, 0.5 μg of total RNA from each group was used to synthesize the first-strand cDNA. The reverse transcription was performed with the primer 50 μM


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Oligo(dT)20 according to the protocol of SuperScript III First-Strand Synthesis SuperMix. Afterwards, 1 μL of the final complementary DNA (cDNA) product was added to 9 μL of reaction mix (0.5 μL TaqMan primer, 5 μL TaqMan PCR mix, 3.5 μL RNase free water) which was loaded to one well of 384-well plates. Each sample was measured in triplicate. For COL7A1 quantitative gene expression, GAPDH was used as the endogenous control. Comparative CT values and TaqMan reagents, QuantStudio 7 Flex System were set up for the experiments. Results were analyzed with the QuantStudio Real-Time PCR Software. Cyto-Immunofluorescence Staining of C7 Cyto-immunofluorescence staining was used to determine C7 restoration of RDEBKs after treatment with HC32-122/MCC7 and PEI/MCC7 polyplexes. 1.5 × 104 cells were seeded on each coverslip in an 8-well chamber (Ibidi). 1 μg MCC7 was used for each well, HC32-122/MCC7 and PEI/MCC7 polyplexes were prepared with the w/w ratio of 30:1 and 1:1, respectively. 3 days post transfection, cells were fixed with 4% PFA, permeated with 0.1 % Triton X-100 and blocked in 5% goat serum in 1 × DPBS for 1 h at RT, and then incubated with primary antibody (monoclonal anti-collagen, Type VII antibody produced in mouse) at 4 °C overnight at an antibody dilution of 1:200 in blocking buffer. Afterwards, cells were incubated with the secondary antibody (Alexa568 goat anti-mouse IgG (H+L) at a dilution of 1: 800 in blocking buffer). After final washes, the coverslips were mounted with Fluoroshield mounting medium with DAPI. Finally, cell images were captured with a fluorescence microscope (Olympus IX81). Western Blotting RDEBKs were seeded in a T-75 flask at a density of 1.5 × 106 cells per flask one day prior to transfection. HC32-122/MCC7 and PEI/MCC7 polyplexes at the w/w ratio of 30:1 and 1:1, respectively, were used for the transfection with 39 μg MCC7 for each flask. Four days post


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transfection, cells were harvested and treated with RIPA lysis buffer which enables efficient cell lysis and solubilization of cellular proteins. 1 μL PIC was added to the cell lysis to a final volume of 50 μL and stored at -80 °C. Bradford Assay was used to quantify the concentration of protein by normalizing the sample concentrations to the known BSA concentration. 40 μg denatured protein samples were loaded into the SDS-Page gel (4%-10%), and then electrophoresis was run at 75 V for 20 minutes followed by 120 V for 1 h. Protein samples were then transferred onto nitrocellulose membrane at 80 V for 1 h at RT followed by 90 V for 30 minutes at 4 °C. Membrane blocking was carried out in the blocking buffer (5% BSA in TBST buffer) at RT for 1 h. β-Actin was used as the endogenous control. Then primary antibodies (polycolonal anti-C7 rabbit antibody and anti-actin mouse antibody at 2500 dilution in blocking buffer) were respectively added to the membrane and incubated at 4 °C overnight. Following washing steps, secondary antibodies (antirabbit HRP and anti-mouse HRP at 5000 dilution in blocking buffer) were respectively added to the membrane and incubated for 1 h at RT. After 3 times of TBST washing, the membrane was visualized with the Pierce ECL Plus Substrate. Statistics SPSS Statistics for windows version 24 (IBM Corp., Armonk, N.Y., USA) was used for statistics. Student's t-test was used to analyze all the gene transfection data, which were expressed as mean ± standard deviation (SD). For all analyses, p value < 0.05 was considered statistically significant. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Chemical compositions of HC32-122 by 1H NMR characterization (Figure S1); MW optimizations


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of HC32-122 polymers in RDEBK transfection (Figure S2); Cell viability tests after the gene transfection with HC32-122 polymers (Figure S3); PDI and MH plot alpha values of all HC32122 polymers (Table S1) (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] ORCID Ming Zeng: 0000-0001-5919-6237 Dezhong Zhou: 0000-0002-5783-8707 Wenxin Wang: 0000-0002-5053-0611 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ⊥These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We are deeply grateful for Dr. Andrew P. South and Dr. Fernando Larcher for providing the pcDNA3.1COL7A1 plasmid and RDEBK cells, respectively. We thank the technical support of Dr. A. Blanco in the Conway Flow Cytometry Core and Dr. D. Scholz in the Conway Electron Microscopy Core of University College Dublin. This work is supported by Science Foundation Ireland (SFI) Principal Investigator Award (13/IA/1962), Investigator Award (12/IP/1688), Health Research Board (HRA-POR2013-412) and DEBRA Ireland.


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FIGURES

Figure 1. (a) Schematic illustration of HC32-122 synthesis via the “A2+B3+C2” Michael addition strategy. (b) GPC curves and calculated Mw of HC32-122. (c) MH Alpha curves of HC32-122.


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Figure 2. Reporter gene transfection studies in RDEBK cells. (a) Relative Gluc activity of RDEBK cells 48 h post transfection by HC32-122/DNA and PEI/DNA polyplexes. Data presented as the percentage normalized to the Gluc activity of RDEBK cells transfected by the HC32122/DNA polyplexes at the w/w of 30:1. *Significant difference from the HC32-122/DNA group (w/w = 30:1) (p ＜ 0.05, Student’s t-test). (b) Viability of RDEBK cells after transfection with HC32-122/DNA and PEI/DNA polyplexes. (c) GFP images of untreated (UT) cells, cells treated with HC32-122/DNA (w/w = 30:1) or PEI/DNA (w/w = 1:1) polyplexes. Scale bar, 200 μm. (d) Representative histogram distributions of UT and transfected cell population. (e) Percentage of GFP-positive RDEBK cells and MFI quantified with flow cytometry. Significant difference from PEI in the *percentage of GFP-positive cells and #cell MFI (p ＜ 0.05, Student’s t-test).


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Figure 3. MCC7 biosynthesis and cellular uptake of HC32-122/MCC7 polyplexes. (a) Scheme of MCC7 biosynthesis with phiC31 integrase system. (b) Agarose gel electrophoresis of three COL7A1-encoding plasmid DNA after EcoR1 digestion. Regular plasmid (RP) of pcDNA3.1COL7A1, parental plasmid (PP) of MN511A-1-COL7A1 and MCC7 have 5 kb, 8 kb and 3 kb backbone lengths, respectively. (c) Fluorescent images of RDEBK cells after transfection with different polyplexes. The nucleus was stained with DAPI (blue), DNA was labeled with Cy3 (red). Scale bar, 20 μm. (d) Polyplex cellular uptake efficiency quantified with flow cytometry. (e) Percentage of Cy3-positive cells and MFI. *Significant difference from the PEI/MCC7 group in cell MFI (p ＜ 0.05, Student’s t-test).


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Figure 4. COL7A1 mRNA and recombinant C7 expression after transfection. (a) Amplification plot of endogenous control GAPDH obtained by RT-qPCR. (b) Amplification plot of COL7A1 mRNA of RDEBK cells after transfection obtained by RT-qPCR. (c) COL7A1 mRNA quantification, *Significant difference from PEI group (p ＜ 0.05, Student’s t-test). (d) Cytoimmunofluorescence images of C7 staining (red fluorescence), scale bar, 20 μm. (e) Western blotting results of C7 expression. The 42-kDa β-Actin was used as the loading control.


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Figure 5. Physicochemical properties of HC32-122/MCC7 polyplexes at the w/w ratio of 30:1. (a) Schematic illustration of HC32-122/MCC7 polyplex formation. (b) Agarose gel results of DNA condensation and heparin competition assay 2 h post polyplex preparation. (c) DNA binding ability test by PicoGreen assay with or without the presence of heparin 2 h post polyplex preparation. (d) Size of HC32-122/MCC7 polyplexes measured by NTA. (e) Zeta potential distribution of HC32122/MCC7 polyplexes. (f) TEM image of HC32-122/MCC7 polyplexes. Scale bar, 500 nm.


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Figure 6. Comparison of gene transfection performance of HC32-122/DNA polyplexes with different formulation conditions. (a) Schematic illustration of polyplex lyophilization and further transfection studies in RDEBK cells. (b) GFP images of cells after transfection with polyplexes from different storage methods and lyophilization conditions. FZ: freeze-drying; Suc: sucrose. Scale bar, 200 μm. (c) Representative histogram distributions of UT and transfected cell population. (d) GFP expression efficiency of cells after transfection quantified by flow cytometry. *Significant difference from the freshly prepared polyplex group (p ＜ 0.05, Student’s t-test). (e) Normalized MFI quantified by flow cytometry. *Significant difference from the freshly prepared polyplex group (p ＜ 0.05, Student’s t-test).


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Figure 7. Long-term storage of HC32-122/DNA Polyplexes in the transfection of RDEBK cells. HC32-122/DNA polyplexes were stored at 3 conditions (-80 °C, -20 °C and 4 °C) from 0.5 month to 12 months, freshly prepared polyplexes were used as the control.


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GRAPHICAL ABSTRACT


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