Polyplex-Loaded Hydrogels for Local Gene Delivery to Human Dermal Fibroblasts

Impaired cutaneous healing leading to chronic wounds affects between 2 and 6% of the total population in most developed countries and it places a substantial burden on healthcare budgets. Current treatments involving antibiotic dressings and mechanical debridement are often not effective, causing severe pain, emotional distress, and social isolation in patients for years or even decades, ultimately resulting in limb amputation. Alternatively, gene therapy (such as mRNA therapies) has emerged as a viable option to promote wound healing through modulation of gene expression. However, protecting the genetic cargo from degradation and efficient transfection into primary cells remain significant challenges in the push to clinical translation. Another limiting aspect of current therapies is the lack of sustained release of drugs to match the therapeutic window.

Graphical Abstract

Herein, they have developed an injectable, biodegradable, and cytocompatible hydrogel-based wound dressing that delivers poly(β-amino ester)s (pBAEs) nanoparticles in a sustained manner over a range of therapeutic windows. They also demonstrate that pBAE nanoparticles, successfully used in previous in vivo studies, protect the mRNA load and efficiently transfect human dermal fibroblasts upon sustained release from the hydrogel wound dressing. This prototype wound dressing technology can enable the development of novel gene therapies for the treatment of chronic wounds.

Schematic diagram of the composite hydrogel wound dressing designed herein for applications involving human dermal fibroblasts transfection, based on a pBAE-PEG injectable hydrogel doped with mRNA-loaded polyplexes. (A) Human dermal fibroblast culture. (B) pBAE-PEG hydrogel containing gene-loaded pBAE polyplexes. (C) Release of pBAE nanoparticles. (D) Human dermal fibroblasts transfected using mRNA-GFP-loaded pBAE polyplexes.

Wound healing is a complex process involving four highly orchestrated phases. Failure to complete these normal stages in a coordinated fashion leads to impaired cutaneous healing, such as delayed acute wounds and chronic wounds. In the United States alone, more than 6 million people suffer from chronic wounds, typically due to underlying conditions like obesity, diabetes, or ischemia. In 2014, wound care products accounted for $2.8 billion of the global healthcare budget, and by 2024, the advanced wound care market for surgical wounds and chronic ulcers is expected to exceed $22 billion. Current clinical approaches to chronic wound care are quite limited given the societal impact and consist of approaches such as antibiotic dressings, mechanical debridement and offloading, and negative pressure therapy.

When these treatments fail to work for wounds such as diabetic ulcers, many times, amputation becomes necessary.

(A) General chemical structure of pBAE polymers, where the ratio and chemical identity of R (R1, alkyl alcohol; R2, alkyl; or R3, thiopyridyl ester) define the nomenclature of the final product (C6, C32, or C32Tx). (B) Chemical structure of arginine (CR3) and histidine (CH3) oligopeptides used to modify the terminal acrylates of pBAE polymers. (C) Chemical structure of 4-arm PEG-SH used to cross-link C32Tx polymers to form the hydrogel network. (D) Protecting groups used during the synthesis of the various pBAE custom polymers.

Impaired wound healing has been associated with alterations in the expression of genes that mediate healing, positioning mRNA delivery as an attractive therapeutic approach to restore normal protein expression and promote healing.mRNA therapies can also be exploited to promote cells to synthesize therapeutic proteins efficiently and safely. However, the delivery of nucleic acids is challenging, because of their susceptibility to rapid degradation, clearance in biological fluids, and inability to cross cytoplasmatic membranes. Numerous vehicles have been developed over the past decade, each with its own limitations and challenges. For example, viral vectors are capable of high transduction efficiency and sustained transgene expression, but they cause high levels of immunogenicity, limiting their translation to human use. In contrast, nonviral vectors show lower transfection efficiencies than viruses but are usually cheaper to synthesize, present better loading capacities for both DNA and RNA, and are safer for the host.

(A) Hydrodynamic diameter, PdI and Z-potential of the different pBAE formulations (C32CR3, C6CR3, or C6RH) containing GFP-coding mRNA obtained by DLS technique. (B) Bright-field and fluorescence microscopy images of HDF cell line expressing GFP after transfection with commercially available jetMESSENGER or different pBAE formulations containing mRNA. (C) FACS graphs showing the percentage of the events counted emitting radiation at FITC wavelength. (D) Quantification of transfected cells (in %) by FACS with the different formulations encapsulating GFP-coding mRNA. Imaging and quantification assays were performed 24 h after transfection. (E) Cell viability (in %) after 24 h transfection using the mRNA-GFP-loaded polyplexes formulations studied. Scale bar: 100 μm. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Cationic polymers, such as poly(β-amino ester)s (pBAEs), are a type of nonviral vectors able to neutralize negatively charged oligonucleotides and form discrete particles, also known as polyplexes, through electrostatic interactions. Polyplexes’ positive overall net charge allows them to bind to cell membranes and enter the cytoplasm via endosomal transport. In addition, amines and terminal acrylates in these polymers confer the versatility of incorporating chemical groups into their structure to tune their functions and properties, such as improving transfection efficiencies by conjugating endosmotic moieties. Moreover, pBAEs are biodegradable and biocompatible.

In recent years, they have developed many oligopeptide-modified pBAEs polyplexes (OM-pBAEs), showing high transfection efficiency and excellent biocompatibility in different cancer cell lines, as well as efficient in vivo transfection, making these nanoparticles a highly promising candidate for clinical translation of new cancer therapies. However, efficient transfection of primary human cells remains a challenge, hampering the progress of new gene therapies for numerous noncancerous pathologies, such as chronic wounds.

(A) Image of hydrogel formulation HG11. (B) Confocal microscopy images of 50 μm thickness slices from the HG11 hydrogel tagged with FITC. (C) Image of hydrogel formulation HG14. (D) Confocal microscopy images of 50 μm thickness slices from the HG14 hydrogel tagged with FITC. (E, F) 3D reconstruction of formulations HG11 and HG14, respectively. (G, H) G′-strain curve for HG11 and HG14 hydrogel formulations, respectively; n = 3. (I, J) SEM images (SEM HV: 1 kV) of bulk lyophilized HG11 and HG14, respectively. (K) Degradation of hydrogels HG11 and HG14 was tracked using fluorescently labeled pBAE, which was converted to weight% of pBAE in the hydrogel as a measure of hydrogel integrity; n = 2. (L) Viability of HDFs after 24 h in contact with medium containing the degradation byproducts released from the hydrogels during three time intervals (0–24 h, 24–72 h, and 72–168 h). Confocal microscopy images scale bar: 100 μm; SEM scale bar: 50 μm. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Local delivery of therapeutics, and particularly nucleic acids is often preferred over systemic delivery, as it allows for reduced dosages, enhanced stability, and increased biocompatibility. Moreover, the smart design of local delivery platforms allows for sustained and controlled release of therapeutics to injured or diseased cells, a critically important aspect in the highly dynamic process of wound healing. The structure and properties of hydrogels make them optimal candidates to release therapeutic nanoparticles for wound healing, maintaining a warm moist environment, and allowing the absorption of wound exudates and adequate oxygen circulation, necessary to promote healing and prevent bacterial infections. Moreover, hydrogels’ hydrophilic nature, capable of absorbing up to 90% of water or fluids, confers their high porosity and mechanical properties resembling those of human tissues. Other characteristics such as biodegradability, biocompatibility, low immunogenicity, and ease of usage have propelled their translation to the clinic.

(A) Confocal microscopy images of C6RH polyplexes loaded into hydrogel HG11 (cy5-tagged pBAE shell: blue channel; cy3-tagged DNA core: red channel. Purple results from the pBAE and DNA signal overlap). (B) Confocal microscopy images of C6RH polyplexes loaded into hydrogel HG14 (same tags and channels than that used in A). Scale bar for A and B: 100 μm. (C) Three-dimensional reconstruction of a 79 μm thick section of HG11 doped with C6RH-cy5 encapsulating pGFP-cy3. (D) Three-dimensional reconstruction of a 57 μm thickness section of HG14 doped with C6RH-cy5 encapsulating pGFP-cy3. E) Degradation of hydrogels HG11 and HG14 loaded with polyplexes was tracked using fluorescently labeled pBAE, which was converted to weight percent of pBAE in the hydrogel as a measure of hydrogel integrity; n = 2. (F, G) Confocal microscopy images of 25 and 50 μm slices of FITC-tagged C6RH-loaded HG11 and HG14, respectively. Scale bar for F and G: 50 μm. (H, I) SEM images of lyophilized bulk C6RH-loaded hydrogels HG11 and HG14, respectively; scale bar: 50 μm. (J) Release of fluorescently labeled C6RH nanoparticles from the hydrogels HG11 and HG14. C* corresponds to nanoparticles with cy3-tagged pBAE (shell) and D* to nanoparticles with cy3-tagged DNA (core); n = 2. (K) Cytotoxicity (in % cell viability) of C6RH-loaded HG11 and HG14 after 24 h of transfection.

(A) FACS graphs showing the percentage of single HDF cells counted expressing GFP after transfection using the C6RH-loaded polyplexes encapsulating mRNA-GFP or a scrambled RNA. (B) Percentage of transfected cells after 24 h. HDFs were seeded on 48-well plates and the hydrogel formulations studied were placed on top of the cells. (C) Bright-field and fluorescence microscopy images of HDFs after 24 h contact with the hydrogel formulations used in the transfection experiment. Scale bar: 100 μm. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

In the present work, they describe and characterize a new local gene delivery platform for cutaneous wound healing based on a composite synthetic hydrogel, made of pBAE and PEG polymers, doped with polynucleotide-loaded pBAE nanoparticles to enable efficient transfection of human dermal fibroblasts (HDFs). Efficient transfection of HDFs is essential for developing new gene therapies for wound healing owing to their extensive involvement in the process of wound healing, and their reported altered gene expression profile in chronic wounds. The hydrogel developed herein is injectable, enabling in situ polymerization and high surface contact area in deep wounds with irregular topography, a typical feature of chronic wounds like diabetic foot ulcers. In the future, the versatility of pBAEs will allow for further modifications of the hydrogel network and/or the polyplexes to incorporate new and improved features to this novel wound dressing platform, such as smarter control over the release or tissue- and cell-specific transfection1.

  1. Polyplex-Loaded Hydrogels for Local Gene Delivery to Human Dermal Fibroblasts Jose Antonio Duran-Mota, Júlia Quintanas Yani, Benjamin D. Almquist, Salvador Borrós, and Nuria Oliva ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.1c00159