Injectable hyperbranched poly(ß-amino ester) hydrogels with on-demand degradation profiles to match wound healing processes.

Adjusting biomaterial degradation profiles to match tissue regeneration is a challenging issue. Herein, biodegradable hyperbranched poly(ß-amino ester)s (HP-PBAEs) were designed and synthesized via "A2 + B4" Michael addition polymerization, and displayed fast gelation with thiolated hyaluronic acid (HA-SH) via a "click" thiol-ene reaction. HP-PBAE/HA-SH hydrogels showed tunable degradation profiles both in vitro and in vivo using diamines with different alkyl chain lengths and poly(ethylene glycol) diacrylates with varied PEG spacers. The hydrogels with optimized degradation profiles encapsulating ADSCs were used as injectable hydrogels to treat two different types of humanized excisional wounds - acute wounds with faster healing rates and diabetic wounds with slower healing and neo-tissue formation. The fast-degrading hydrogel showed accelerated wound closure in acute wounds, while the slow-degrading hydrogel showed better wound healing for diabetic wounds. The results demonstrate that the new HP-PBAE-based hydrogel in combination with ADSCs can be used as a well-controlled biodegradable skin substitute, which demonstrates a promising approach in the treatment of various types of skin wounds.

Brushlike Cationic Polymers with Low Charge Density for Gene Delivery.

Using a combined synthesis approach comprising reversible addition-fragmentation transfer polymerization and ring opening reaction, a series of poly glycidyl methacrylate (polyGMA) polymers were designed and synthesized for gene delivery. These polymers characterized by low cationic charge respective to established gene delivery vectors such as PEI were studied to further elucidate the key structure-activity parameters that mediate efficient and biocompatible gene delivery. Compared to PEI, these brushlike polymers facilitated markedly improved safety and gene delivery efficiency.

Highly Branched Poly(ß-amino esters) for Non-Viral Gene Delivery: High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight.

A successful polymeric gene delivery vector is denoted by both transfection efficiency and biocompatibility. However, the existing vectors with combined high efficacy and minimal toxicity still fall short. The most widely used polyethylene imine (PEI), polyamidoamine (PAMAM) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) suffer from the correlation: either too toxic or little effective. Here, we demonstrate that with highly branched poly(ß-amino esters) (HPAEs), a type of recently developed gene delivery vector, the high gene transfection efficiency and low cytotoxicity can be achieved simultaneously at high molecular weight (MW). The interactions of HPAE/DNA polyplexes with cell membrane account for the favorable correlation between molecular weight and biocompatibility. In addition to the effect of molecular weight, the molecular configuration of linear and branched segments in HPAEs is also pivotal to endow high transfection efficiency and low cytotoxicity. These findings provide renewed perspective for the further development of clinically viable gene delivery vectors.

The transition from linear to highly branched poly(ß-amino ester)s: Branching matters for gene delivery.

Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(ß-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(ß-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(ß-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(ß-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their translation to a clinical environment.

Highly branched poly(ß-amino ester)s for skin gene therapy.

Poly(ß-amino ester)s (PAEs) have emerged as a promising class of gene delivery vectors with performances that can even be compared to viruses. However, all of the transfection studies (over 2350 PAEs) have been limited to linear poly(ß-amino ester)s (LPAEs) despite increasing evidence that polymer structure significantly affects performance. Herein, we describe the development of highly branched poly(ß-amino ester)s (HPAEs) via a new "A2+B3+C2" Michael addition approach demonstrating 2 to 126-fold higher in vitro transfection efficiencies of different cell types in comparison to their linear LPAE counterparts as well as greatly out-performing the leading transfection reagents SuperFect and the "gold-standard" polyethyleneimine (PEI) - especially on skin epidermal cells. More importantly, the ability to correct a skin genetic defect is demonstrated in vivo utilizing a recessive dystrophic epidermolysis bullosa (RDEB) knockout mouse model. Our results provide evidence that the "A2+B3+C2" approach can be controlled and offers sufficient flexibility for the synthesis of HPAEs. The branched structures can significantly improve the transfection efficiency and safety of PAEs highlighting the great promise for the successful application of non-viral gene therapy in skin disease.

A knot polymer mediated non-viral gene transfection for skin cells.

A knot polymer, poly[bis(2-acryloyl)oxyethyl disulphide-co-2-(dimethylamino) ethyl methacrylate] (DSP), was synthesized, optimized and evaluated as a non-viral vector for gene transfection for skin cells, keratinocytes. With recessive dystrophic epidermolysis bullosa keratinocytes (RDEBK-TA4), the DSP exhibited high transfection efficacy with both Gaussia luciferase marker DNA and the full length COL7A1 transcript encoding the therapeutic type VII collagen protein (C7). The effective restoration of C7 in C7 null-RDEB skin cells indicates that DSP is promising for non-viral gene therapy of recessive dystrophic epidermolysis bullosa (RDEB).

Highly Branched Poly(ß-Amino Esters): Synthesis and Application in Gene Delivery.

Highly branched poly(ß-amino esters) (HPAEs) are developed via a facile and controllable "A2+B3/B2" strategy successfully. As nonviral gene delivery vectors, the performance of HPAEs is superior to the well-studied linear counterpart as well as the leading commercial reagent Superfect. When combined with minicircle DNA construct, HPAEs can achieve ultrahigh gene transfection efficiency, especially in keratinocytes.

Tailoring highly branched poly(ß-amino ester)s: a synthetic platform for epidermal gene therapy.

Highly branched poly(ß-amino ester)s (HPAEs) were designed and synthesised for safe and efficient gene delivery to human keratinocytes. HPAEs outperformed commercial transfection reagents: PEI and SuperFect®, for both transfection efficiency and biocompatibility. A 22 and 3.4 fold enhancement of gene transfection was seen coupled with superior biocompatibility.

Gene therapy: pursuing restoration of dermal adhesion in recessive dystrophic epidermolysis bullosa.

The replacement of a defective gene with a fully functional copy is the goal of the most basic gene therapy. Recessive dystrophic epidermolysis bullosa (RDEB) is characterised by a lack of adhesion of the epidermis to the dermis. It is an ideal target for gene therapy as all variants of hereditary RDEB are caused by mutations in a single gene, COL7A1, coding for type VII collagen, a key component of anchoring fibrils that secure attachment of the epidermis to the dermis. RDEB is one of the most severe variants in the epidermolysis bullosa (EB) group of heritable skin diseases. Epidermolysis bullosa is defined by chronic fragility and blistering of the skin and mucous membranes due to mutations in the genes responsible for production of the basement membrane proteins. This condition has a high personal, medical and socio-economic impact. People with RDEB require a broad spectrum of medications and specialised care. Due to this being a systemic condition, most research focus is in the area of gene therapy. Recently, preclinical works have begun to show promise. They focus on the virally mediated ex vivo correction of autologous epithelium. These corrected cells are then to be expanded and grafted onto the patient following the lead of the first successful gene therapy in dermatology being a grafting of corrected tissue for junctional EB treatment. Current progress, outstanding challenges and future directions in translating these approaches in clinics are reviewed in this article.