Beyond Branching: Multiknot Structured Polymer for Gene Delivery

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Beyond Branching: Multi-knot Structured Polymer for Gene Delivery Ahmed Aied, Yu Zheng, Ben Edward Newland, and Wenxin Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5013162 • Publication Date (Web): 06 Nov 2014 Downloaded from http://pubs.acs.org on November 20, 2014

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Beyond Branching: Multi-knot Structured Polymer for Gene Delivery Ahmed Aied1, Yu Zheng2, Ben Newland3, and Wenxin Wang1* 1

Charles Institute of Dermatology, University College Dublin, Dublin, Ireland 2

The Melville Laboratory, Cambridge University, Cambridge, UK 3

School of Biosciences, Cardiff University, Cardiff, UK

Corresponding author: Dr Wenxin Wang, Charles Institute of Dermatology, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. Tel.: +35391493131, Fax.: +353091495585 Email. [email protected]

Abstract: Polymer-based transfection vectors are increasingly becoming the preferred alternative to viral vectors thanks to their safety and ease of production, but low transfection potency has limited their application. Many polycationic vectors show high efficiency in vitro, but their excessive charge density makes them toxic for in vivo applications. Herein, we demonstrate the synthesis of new and unique disulfide-reducible polymeric gene nano-carriers that exhibit significantly enhanced transfection potency and low cytotoxicity, particularly in skin cells, surpassing the efficiency of the well-known transfection reagents Polyethylenimine (PEI) and Lipofectamine®2000. The unique 3D ‘multi-knot’ vectors were synthesized from in situ deactivation enhanced atom transfer radical (co-)polymerization (DE-ATRP) of multivinyl monomers (MVMs). The high transfection levels and low toxicity of this multi-knot structured polymer in vitro, combined with its ability to mediate ACS Paragon Plus Environment

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collagen VII expression in 3D skin equivalents made from cells of Recessive dystrophic Epidermolysis Bullosa patients, demonstrates its use as a platform nanotechnology which should be investigated further for dermatological disease therapies. Our findings suggest that the marked improvements stem from the dense multi-knot architecture and degradable property, which facilitate both the binding and releasing process of the plasmid DNA.

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Introduction Gene therapy is a promising treatment strategy for a variety of inherited and sporadic diseases, but safe and efficient delivery remains a challenge. Though gene therapy mediated by viral vectors has recently made great clinical progress, limitations associated with their use persist, such as the possibility of adverse immune reactions and the difficulty of repeat dosing1. Therefore, non-viral gene delivery nanoparticles have attracted much attention in the past few decades because of their potential for limited immunogenicity, their ability to accommodate and deliver large-size genetic materials, and the potential for modification of their surface structures2-5. Nonetheless, the diversity of synthetic materials offers potential for the identification and incorporation of functional motifs that confer not only efficient gene transfection, but also formulation stability and biocompatibility. It is well known that the transfection by non-viral vectors usually requires materials with excess charge, resulting in stable nanoparticles formed between cationic polymers and negatively charged DNA via electrostatic interaction. Furthermore, compared with low molecular weight (Mw) cationic polymers, high molecular weight polymers can afford more entropy loss, and thus are theoretically more efficient in condensing DNA.6, 7 For those polymeric vectors, a further chain entanglement effect, which depends on polymer molecular weight, might also be required. As a result, the current polymeric gene vectors require a high charge density and high molecular weight to compensate the low transfection efficiency. However, these aspects can be a double-edged sword. Most of the cationic vectors with a high charge density and high molecular weight developed so far, exhibit substantial toxicity, which has limited their clinical applicability. This too appears to depend on charge, since the excess positive charges on the surface of the complexes can interact with cellular components, inhibit normal cellular processes and cell-survival signaling. Most of the current studies focus on the systematic screening of linear polymers from different monomers and composition.


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However, these studies cannot resolve the issue of target polymer dependency on high charge density and low cytotoxicity - they seem mutually exclusive. To overcome this issue, we hypothesize that molecular architecture is another key factor for gene delivery, particularly for cationic polymers with a high charge density and low cytotoxicity. Recently, we synthesized a family of a new class of polymers, called ‘knot polymers’ by the control polymerisation of multi-vinyl monomers8, 9. Compared with most linear polycations, the knot polymer has a compact molecular conformation which facilitates the interaction with DNA forming nanoscale particles (polyplexes). Thus, relatively higher binding between the knot polymer and DNA are anticipated. In this study, we are going further to achieve unique multi-knot polymers with multi vinyl functional groups by DE-ATRP of two simple starting monomers: dimethyl amino ethyl methacrylate (DMAEMA) and propenoyloxy ethyl disulfanyl ethyl propenoate (PEEDEPE). DMAEMA provides the cationic unit and PEEDEPE acts as knot and connecting points (Figure 1). Because of the post-modification of residue vinyl groups with 1, 3-diaminopropane, the binding ability could be further enhanced by additional amine groups. Under physiological conditions inside the cells, the PEEDEPE units can be easily broken and the multi-knot polymers degrade into small molecules (Mw=5 kDa), allowing release of DNA and avoiding the toxicity of high molecular weight polymers. To this end, we designed and utilized multi-knot polymer for the correction of collagen type VII-null skin cells of Recessive Dystrophic Epideromylis Bullosa (RDEB). RDEB is characterized by a mutation in the collagen type VII alpha 1 gene (COL7A1) leading to impaired formation of structural fibrils required for skin adherence. As a result, severe wounding and blistering of the skin occurs after mild trauma. The open wound and ease of access make this disease an ideal platform for the analysis of gene therapy.


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Experimental Procedures The monomers were purchased from Sigma Aldrich and used as received. The propenoyloxy ethyl disulfanyl ethyl propenoate (PEEDEPE) crosslinker was synthesized as previously reported by Li et al.10. COL7A1 plasmid DNA (subcloned into pcDNA 3.1 (+) Invitrogen) was obtained from Dr. Andrew South (Surgery and Molecular Oncology department, University of Dundee, Ninewells, Dundee, UK) and amplified in E-coli DH5α treated with ampicillin for positive selection and then prepared by a Giga-plasmid preparation kit (QAIGEN Plasmid Plus Giga kit, QAIGEN HOUSE, west Sussex, UK). COL7A1 cDNA is 9.2Kb, total plasmid size 14.3Kb. The cervical cancer cell line (HeLa) was maintained in DMEM media with 10% fetal bovine serum and 1% penicillin/streptomycin. The type VII collagen null-RDEB keratinocytes and type VII collagen null-RDEB fibroblasts were kindly supplied by Ferrnado Larcher (Madrid). These cells were harvested from a patient homozygous for mutations 6527ins in exon 80 (RDEB keratinocytes) and mutations 706ins in exon 6 (RDEB fibroblasts) of the COL7A1 gene and immortalized with Lenti-HPV-16 E6/E7 virus (ABM inc. Canada). Keratinocytes were cultured in DMEM (Sigma-Aldrich) with Keratinocyte Growth Medium 2 Supplement Pack (Promocell). RDEB fibroblasts were cultured in DMEM: F-HAM’s 12 (1:1) (Sigma-Aldrich) and 10% Fetal Bovine Serum (Sigma-Aldrich). Fibroblasts make up the dermal component of the 3D skin substitutes. Polyplex formation: The polyplexes were formed in a weight : weight ratio of transfection agent to DNA, 2:1 for branched poly(ethylene imine) (PEI) and 3:1 for Lipofectamine®2000. The multi-knot polymer and PEI were complexed with the DNA for 1 hour at room temperature in distilled H2O. For Lipofectamine®2000, all procedures were carried out according to manufacturer’s guidelines. Size and Zeta potential: The hydrodynamic diameters and zeta-potential (charge) of the multi-knot polymer/COL7A1 plasmid complexes were determined by light scattering and zeta-potential ACS Paragon Plus Environment

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analyser (Malvern instruments, Zetasizer Nano-ZS90). Polyplex solutions (1ml) containing 1µg of plasmid DNA (COL7A1) were prepared at various weight ratios ranging from 1:1 to 100:1 after incubation at 25°C for 1 hour. Measured sizes and charge were presented in the results as the average values of 5 runs. Transmission Electron Microscopy: Confirmation of polyplex size was obtained by examining the polyplexes under a transmission electron microscope (Hitachi H-7500 Transmission Electron Microscope) that utilizes 80kV accelerating voltage, US1000 high resolution digital camera and Gatan, Inc. Digital micrograph acquisition software v1.82.366. Ten microlitres of polyplex solution at weight ratios of 5:1 and 10:1 containing 0.5µg of COL7A1 plasmid were pipetted on to Graphene grids (Agar scientific, UK) and visualized after drying. PicoGreen® Assay: Quant-iT™ PicoGreen® dsDNA Assay Kit was used to determine the DNA release from the multi-knot polymer after bio-reduction (Figure 3 d). A 0.1µg of COL7A1 plasmid solution was prepared as control or mixed with the multi-knot polymer, PEI or Lipofectamine®2000 (in 96-well plate) at optimal ratios to make the nanoparticles/polyplexes as described earlier. The recommended dilutions of PicoGreen® (PG) were made according to the manufacturer’s protocol and mixed with the DNA alone or with nanoparticle solution for 5 minutes before taking the fluorescence measurements (Ex.480nm, Em.520nm). In the bio-reduction step, glutathione is added to the nanoparticles solution and incubated for 5 minutes after the nanoparticles are made. PG is then added and measurements are taken like in the previous step. Cell transfection: Cells were seeded at a density of 1x104 cells/well of 96-well plate and grown to reach 60-90% confluence prior to transfection. This percentage of confluence yields best transfection efficiency for fast proliferating cells. The cells were washed before adding polyplex solutions containing 0.5µg of pCMV-Gluc or pCMV-GFP, coding for the secreted Gaussia luciferase reporter protein or Green fluorescent protein (GFP), at a range of different multi-knot/DNA weight ratios in ACS Paragon Plus Environment

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serum free medium. After a 4 hour incubation period at 37°C, supplemented medium was added to the cells which were incubated for a further 48 hours at 37°C with 5.0% CO2. The amount of Gaussia luciferase protein expressed in each well was measured by luminescence intensity according to the manufacturer’s protocol (BioLux® Gaussia luciferase assay kit, New England BioLabs®, UK). The number of positive GFP cells was quantified using flow cytometry. For COL7Α1 gene delivery, the growth media was removed from the RDEB keratinocytes (60-70% confluency) and replaced with DMEM with 1%Pencillin/streptomycin (Sigma-Aldrich). Complexes were combined with 500µl of DMEM alone and then added to the cells. After 4 hours incubation (37oC with 5.0% CO2), the complexes were removed and fresh growth media added. Twenty-four hours after transfection, ascorbic acid (50µg/ml) was added to the RDEB keratinocytes and normal human keratinocytes. Cells were either fixed 48 hours after transfection for immunofluorescence or the media was collected for immunoblot. The cell metabolic activity was measured after 4 hour incubation (replaced with fresh media after the 4 hour incubation period) with the polyplexes using the alamarBlue® assay (Invitrogen). Fifteen microlitres/well (96-well plate) of alamarBlue® was added to transfected and untreated (control) cells in 150µl/well of media and incubated at 37°C for 4 hours. Microplate (Thermo Scientific, Varioskanflash multimode reader) fluorescence measurements were then taken at 560EX nm/590EM nm filter settings. Results were obtained as the mean and standard deviation from triplicate values and displayed as percentage relative to untreated control cells. Green Fluorescent Protein (GFP) Expression: The expression of the internally expressed pCMV-GFP green fluorescent protein plasmid (New England Biolabs UK, also obtained as described earlier) was used to assess polymer transfection in terms of the percentage of cells transfected. RDEB keratinocytes were either seeded in 4-well chamber slides (for fluorescent microscopy analysis) or in 6-well plates (for flow cytometry analysis) at 100,000 cells/ml, 24 hours ACS Paragon Plus Environment

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prior to the same treatment as outlined earlier, but with 2µg of plasmid GFP instead of luciferase. For flow cytometry analysis, the cells were trypsinized at 48 hours post treatment. Subsequently, the cells were re-suspended in 1% bovine serum albumin in PBS. Flow cytometry was performed using a BD FACS Canto with FITC (GFP) filter. Skin equivalent culture and maintenance: Fibrinogen, 1 ml fibroblasts in DMEM/1%PS (38,000 c/ml), 100 µl trasylol, and 100 µl thrombine were mixed in that order gently by pipetting up and down, avoiding bubble formation, then allowed to polymerize for 1h at 37°C (incubator). RDEB Keratinocytes were then seeded at 60,000 cells per well onto the dermal matrix (in 1 ml Green media or DMEM/1%PS) the media was changed 48 h later into Green medium and allowed to grow to confluence for 6-7 days. After one week of culture in green medium, the media was changed to special media (DMEM HyClone) 130 ml, Ham F-12 (HyClone):60 ml, FCII (HyClone): (filtered, 0.22um) 1 ml, 10% BSA in PBS: (filtered, 0.22um) 3.2 ml, Glutamine stock: 200 mM 4 ml, NaPyruvate stock: 100mM 1.3 ml, Hydrocortisone stock 500x 400 µl, Insuline stock 1000x 200 µl, Vitamin C, 50 mg/ml). Vitamin C was added separately every day. Transfection of skin equivalents: The polyplexes (ratio 3:1) were added to the air-exposed surface of the skin equivalents by pipetting 50ml of the solution (containing 10µg of COL7A1 plasmid and 30µg of the polymer). The treatment was applied after 30 days of air-liquid interface culture and left for 48 hours before the skin equivalents were cryosectioned for analysis.


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Results and Discussion Until recently, the majority of polymerizations involving multi-vinyl monomers (MVMs) were carried out by using the co-polymerization system containing only a low percentage of MVMs. To overcome this bottle-neck, we have developed a deactivation enhanced strategy for ATRP that efficiently delayed the point of gelation in the homopolymerization of ethylene glycol dimethacrylate (EGDMA) to over 60% monomer conversion in a concentrated polymerization system8-11. With this method, termed in situ deactivation enhanced ATRP (in situ DE-ATRP), we produced a new 3D ‘Single knot’ molecule architecture that consisted of a single polymer chain cyclized within itself at the early stage of reaction8,9. Furthermore, we reported the use of ‘Single knot’ polymer for gene delivery applications through the preparation of a series of cationic transfection agents. We observed that the single knot gene vector interacts differently with plasmid DNA compared to conventional vectors and has a superior transfection profile in terms of both transfection capability and preservation of cell viability8. This seeding work has driven us to continue designing and exploring this new gene vector, as we believe that it can, in principle, be improved by higher cationic density and lowering the cytotoxicity. As the non-degradable single knot vector demonstrates that a high transfection performance can be achieved, it can be reasonably hypothesized that the advanced degradable ‘multi-knot’ polymer chain will out-perform currently available polymer structures in terms of efficacy, cell viability, and scalability.


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Figure 1: Formation of the multi-knot polymer via in situ DE-ATRP. The reaction initiates when the free radical (asterisk) reacts with neighboring free monomers to form a small linear chain (a). In the meantime, cyclisation (or intramolecular crosslinking) begins to happen. This leads to the formation of the single knot (b). However, if the reaction is continued, the radical will react with vinyl groups of other single knot molecules and assemble into the multi-knot polymer (c). Termination of the vinyl groups with diamines using Michael addition (d).

The multi-knot vectors were synthesized via in situ DE-ATRP copolymerization of DMAEMA and PEEDEPE, and post-functionalized by 1, 3-diaminopropane. The feed ratio of these two monomers was 90:10. The disulfide monomer, PEEDEPE, was synthesized by chlorine substitution of acroylyl chloride and hydroxy ethyle disulfide in the presence of triethylamine as reported previously12. First, the polymer chains display a linear-like growth, which is the increase of molecular weight (The molecular weight is obtained from refractive index (RI) detector which using linear PMMA standard as calibration), is linear with monomer conversion and PDI remaining low with unimodal molecular distribution (Figure 1 a and c). When the reaction reaches high monomer conversion, the single knot molecules start to combine into multi-knot molecules, along with a significant molecular weight increase (Figure 1 c). After 7 hours of reaction time at 60˚C, the ACS Paragon Plus Environment

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DE-ATRP reaction was stopped resulting in a polymer with 42 kDa Mw (Table 1). The remaining vinyl groups were end-functionalized with 1, 3-diaminopropane by Michael Addition (Figure 1 d). It could reasonably be assumed that these vinyl groups could be subject to other post modifications for precise cell targeting or crossing in vivo or intracellular gene delivery barriers. Gel permeation chromatography (GPC) and proton nuclear magnetic resonance (1H NMR) were used to determine the polymer molecular weight and composition, respectively (Figure 2 a and c). To elucidate the bio-reduction potential of the gene carrying multi-knot nanoparticles, the polymer was dissolved in 5mM glutathione solution, a disulfide reducing compound found at roughly that concentration in the intracellular space13. As a result, the polymer molecular weight reduced by 10 times within 20 minutes (Figure 2 d). This degradable property could be the sole factor for the reduced cytotoxicity of the polymer (Figure 2 e).

Table 1: Increase in number average molecular weight (Mn), weight average molecular weight (Mw), polydispersity index and percentage monomer to polymer conversion over the reaction lifetime of the polymer. The reaction was stopped after 8 hours to obtain a polymer with final Mw of 42 kDa. Entry

Time (hrs)

Mn (kDa)

Mw (kDa)

PDI

Conversion (%)

H1

1

9.3

12.9

1.4

76.3

H2

2

10.6

15.0

1.4

78.0

H5

5

17.1

30.3

1.8

91.0

H8

8

19.9

42.1

2.1

92.2


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a

b

c

d

e

Figure 2: a. GPC trace of polymer synthesis. Each peak represents a sample taken at different time points of the reaction. The formation of the multi-knot polymer occurs when a number of knotted polymer molecules combine during a reaction lasting 8 hours. b. Gel Permeation Chromatography graph of molecular weight plotted against conversion of the monomer units into polymer. c. 1H NMR spectrum showing the peaks, which identify the multi-knot polymer atomic structure after diaminopropane termination. d. Decrease in Mw over time as the polymer is incubated with a 5mM of


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the disulfide reducing agent, glutathione. e. Cell metabolic activity of RDEB keratinocytes after application of increasing polymer concentrations of the multi-knot polymer and PEI.

The electrostatic interaction between cationic polymers and negatively charged nucleic acids condenses the genetic material into nanoscale complexes (polyplexes). These nanosized polyplexes formed with knot polymers typically show high resistance to DNase degradation8 and were characterized here in terms of electrophoretic mobility, size and charge. Size/charge analysis (Figure 3 a) showed that nanoparticles increased in charge with increasing polymer/plasmid ratio (between 15 and 60 mV) but showed a variety of sizes (between 100 and 320 nm). Figure 3 b shows that nanoparticles could successfully be formed at a 0.5:1 polymer to plasmid weight ratio, or higher, as shown by the hindrance of mobility through the agarose gel. By using the highly sensitive PicoGreen® dye (PG) to detect DNA, it is possible to determine the amount of DNA released after the degradation of the multi-knot polymer. This is particularly advantageous for intracellular release of DNA cargo. PG was chosen because it selectively binds double stranded DNA and remains relatively non-fluorescent when unbound14. When bound to double stranded DNA, fluorescence enhancement of PG is exceptionally high; little background occurs since the unbound dye has virtually no fluorescence15. The chemical is very stable to photo-bleaching, allowing longer exposure times and assay flexibility16, 17. PG can detect the DNA bound to the polymer, although the signal is significantly reduced in comparison to naked DNA. PEI polyplexes completely quenched the fluorescence of PG at all ratios (Figure 3 d). This could be due to the hyperbranched structure of PEI that prevents the dye from excessing the DNA18. In figure 3 d, it is also possible to argue that the multi-knot polymer forms more tightly packaged polyplexes than lipofetacmine®2000 because PG cannot access the plasmid DNA bound by the multi-knot vectors, resulting in lowered fluorescence signal. The more noticeable advantage of the multi-knot polymer, is its fast degradation in glutathione containing solution resulting in almost 80% ACS Paragon Plus Environment

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DNA

release,

100-fold

more

efficient

than

lipofectamine®2000.

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Neither

PEI

nor

Lipofectamine®2000 degrade in glutathione and thus little to no DNA is released by these agents. This could have contributed to the elevated transfection levels and reduced toxicity of the multi-knot nanoparticles seen in follow up transfection experiments (Figure 4). Whilst improving the transfection efficiency of non-viral vectors remains a necessity, analysis of the material toxicity is of equal importance. Nanoscale materials may have safety issues due to a high aspect ratio19 or an inherent charge density, causing toxicity20. Thus, the toxicity of the multi-knot polymer was specifically considered and compared to a relatively non-toxic liposomal based commercial agent. Reporter gene expression levels of gaussia luciferase were used to study the transfection efficiency and cytotoxicity of the multi-knot polymer in HeLa cells. Different polyplex ratios showed different transfection properties, of which, 3 showed the highest protein expression with the lowest toxicity (Figure 4). In addition, the DNA dose played a crucial role in determining the expression level of the reporter protein. Between 1 and 2 µg of DNA showed the best results in terms of expression. The same amount used with lipofectamine®2000 however, resulted in high cytotoxicity and cell death.


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a

b

c

d e

Figure 3: Multi-knot polymer binds to DNA to form nanoparticles. a. Average charge and average size of nanoparticles formed by the multi-knot polymer with luciferase plasmid (polymer : plasmid) at different ratios. b. The polyplexes are immobilized in Agrose gel at ratios above 1:1. c. TEM image of the nanoparticles (10:1 ratio). d. PicoGreen® assay of DNA release profiles before and after treatment of the polyplexes with glutathione. e. PicoGreen® labelled polyplexes interact with keratinocytes cellular membrane (arrows) before internalization. Statistical analysis was carried out using one-way Anova. Error bars represent ± standard deviation, n = 3.


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a

b

Figure 4: a. Three ratios, 3, 4, and 5 were tested with different amounts of DNA (0.1µg-2µg) on HeLa cells. Gaussia luciferase protein expression was analyzed 48 hours after transfection. b. Cell viability (alamarBlue®) of the same transfected cells relative to 100% control viable cells shows significant enhancement over lipofectamine®2000. Statistical analysis was carried out using one-way Anova. Error bars represent ± standard deviation, n = 3.

The high charge density and fast bio-reducibility are important characteristics in gene delivery to cells especially in the case of polymer based gene nano-carriers21, 22. The structure of the multi-knot polymer of dense cyclized chains creates high concentration of amine groups providing the elevated charge density required upon protonation. The bio-reduction is achieved through disulfide-bond breakage leading to polymer disassembly into small fragments ten times it original size (Figure 2 d). We found this polymer capable of transfecting a range of cell types with high efficiency particularly in RDEB skin keratinocytes, fibroblasts and adipose derived stem cells. As a result, we examined its efficiency for gene therapy in the skin disease RDEB. Epidermolysis Bullosa is a group of heritable skin diseases, defined by chronic fragility and blistering of the skin and mucosal membranes. One of the most severe variants, RDEB, is characterized by a lack of adhesion of the epidermis to the dermis23. All variants of RDEB are caused by mutations in the COL7A1 gene, encoding for collagen VII protein, which is a key component of anchoring fibrils that secure attachment of the epidermis to the dermis24. This condition has a high ACS Paragon Plus Environment

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personal, medical and socio-economic impact as people with RDEB require a broad spectrum of medications and specialized care. Gene therapy offers a great potential for the treatment, or at least amelioration of RDEB phenotype which is why we used this non-immunogenic approach to test the feasibility of the multi-knot vectors. Keratinocytes were used to study the in vitro transfection of the nanoparticles because they produce more than 90% of skin’s collagen VII25 . Collagen VII null keratinocytes from RDEB patients were transfected with the multi-knot polymer and PEI showing no statistical difference between the two. However, it is safe to say that the multi-knot polymer is more efficient because it shows 2-fold GFP positive cells than other commercial vectors (Figure 5 b). We have also seen previously how cells rapidly die in the presence of concentrated PEI and lipofectamine®2000, while the multi-knot polymer preserves more than 80% of cells at the same concentration (Figure 2 e and figure 4 b). Note that the discrepancies between the GFP and Gaussia luciferase data relate to the method of detection and the nature of the protein. While GFP provides information regarding the number of cells transfected, Gaussia luciferase is used to estimate the amount of protein expressed. Total protein was extracted from the transfected cells (or controls) and analyzed by immunoblotting. Figure c shows bands for collagen VII just above the 250 kDa marker in multi-knot transfected cells. Lower levels were seen in normal human keratinocytes with no trace of the protein band being detected in the untreated RDEB keratinocytes. Although the β-actin levels are slightly higher for the multi-knot treated cells, the bands for collagen VII protein are notably much thicker than those of the normal human keratinocytes. These results were supported by indirect immunofluorescence images which show high expression of the protein 48 hours post transfection (Figure 5 d).


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a

c

d

b e

Figure 5: a. Reporter gene delivery efficiency and cytotoxicity of the multi-knot polymer. Gaussia luciferase and alamarBlue® were used to test the transfection efficiency and cytotoxicity of the multi-knot polymer. b. Percentage of GFP transfected cells of various commercial agents compared to the multi-knot polymer as determined by flow cytometry. c. Western blot detecting collagen VII media extracted from normal human keratinocytes, RDEB keratinocytes transfected with multi-knot polymer carrying the COL7A1 gene. Only β-actin was detected in RDEB keratinocytes. d. Immunofluorescence images of normal and RDEB keratinocytes before and after transfection showing clear patterns of collagen VII expression in the latter. e. Immunofluorescence images of cryosections from 3D skin equivalents. Collagen VII: green, actin filaments: red and DAPI: blue. Scale bar represent 50µm and 100µm. Statistical analysis was carried out using one-way Anova. Error bars represent ± standard deviation, n = 3. Asterisk represents significant difference between multi-knot polymer and commercial agents. ACS Paragon Plus Environment

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In the final step of preliminary tests, the polymer was used to restore collagen VII in three dimensional skin equivalents. The 3D skin cultures were generated using fibrin as a scaffold. Type VII collagen-deficient fibroblasts were embedded into the matrix and immersed in growth media. On the surface of the scaffold, type VII collagen-null keratinocytes were seeded. The keratinocytes were in air-liquid interphase where they form a crest representing the upper layer of the epidermis after 28 days. These cultures were then transfected by topical application of the polyplex solution containing the multi-knot polymer and COL7A1 plasmid. Figure 5 e shows a cross section of the 3D skin cultures. Expression of collagen VII in transfected cells was visualized using an affinity-purified antibody to the NC-2 domain of the type VII collagen. Normal keratinocytes show a green stain for collagen VII at the dermal-epidermal junction (DEJ) where it normally resides23, 26. Although the amount of protein released is low, these results are still very encouraging and prove the feasibility of using non-viral gene therapy for treating RDEB patients. These preliminary results demonstrate the early potential of highly sophisticated, bio-reducible and polymer-based delivery nanoparticles for clinical therapy of RDEB wounds and possibly other wounds such as diabetic wound ulcers.


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Conclusion In conclusion, we went beyond the single cyclized knot polymer to a unique higher and more complex multi-knot structure. This was achieved by delaying the onset of gelation in a one-pot in situ DE-ATRP reaction. This polymer displayed high charge density but fast bio-reduction which is driven by biological reducing agents that cleave the disulfide bridge of the MVM. Its unique structure combines these two properties in one vector for efficient DNA packaging and swift DNA release, both of which raise the transfection traits of the current polymer above the ‘gold’ standard PEI and Lipofectamine®2000. As a proof of concept, RDEB was selected for a number of 2D and 3D in vitro studies. Successful transfection of RDEB keratinocytes with COL7A1 lead to the expression of recombinant protein faithful to the native protein showing signs of a possible treatment of the disease. The benign nature of this potential gene therapy make it a valid option, allowing the reparative gene to be repeatedly applied. The collagen VII protein (known to have a long half-life) would hopefully build up in the tissue and over time provide structural integrity to reverse the RDEB phenotype.


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Associated Content Supporting Information Experimental details of the polymer synthesis, characterization, 1H NMR, GPC, transfection and protein expression analysis. This information is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgements The authors would like to acknowledge the help and support of Dr. Fernado Larcher who provided the RDEB cells and Dr. Andrew South who contributed with his scientific expertise to the project and for allowing team members to work in his lab. The authors would also like to thank the financial support of DEBRA Ireland and DEBRA international, Science Foundation Ireland, and the National University of Ireland, Galway. The technical support of Dr. Oliver Carroll and Dr. Eva Murauer is also greatly appreciated.

Competing financial interests The authors declare no competing financial interests Author contribution W.W. proposed and supervised the project and came up with the multi-knot principle. A.A carried out the polymer synthesis and characterization, and the 2D and 3D in vitro analysis. Y.Z. and B.N. assisted in polymer synthesis and NMR analysis.


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351x104mm (150 x 150 DPI)


Beyond Branching: Multiknot Structured Polymer for Gene Delivery

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