Yongsheng Gao

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

Highly branched poly(β-amino ester) polymers are developed to carry a gene and to enter cells for the production of protein. 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(β-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(β-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(β-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(β-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.

A rapid crosslinking injectable hydrogel for stem cell delivery, from multifunctional hyperbranched polymers via RAFT homopolymerization of PEGDA

Stem cell therapies have attracted much attention for the last few decades in the field of regenerative medicine and tissue engineering. The 3-dimensional (3D) microenvironment surrounding the transplanted stem cells plays an essential role that influences the cell fate and behaviors. Thus advanced functional biomaterials and extracellular matrix (ECM) replacements with adjustable chemical, mechanical and bioactive properties are requisites in this field. In this study, PEG-based hyperbranched multifunctional homopolymers were developed via RAFT homopolymerization of the divinyl monomer of poly(ethylene glycol) diacrylate (PEGDA). Due to its high degree of multi-acrylate functionality, the hyperbranched polyPEGDA can rapidly crosslink with a thiolated hyaluronic acid under physiological conditions and form an injectable hydrogel for cell delivery. In addition, by simply varying the synthesis conditions such as the reaction time and the ratio of the monomer to the chain transfer agent (CTA), the polymer molecular weight, acrylate functionality degree and the cyclized/hyperbranched polymeric architecture can be finely controlled in a one-step reaction. The gelation speed and the mechanical properties of this hydrogel can be easily adjusted by altering the crosslinking conditions. Rat adipose-derived stem cells (rASCs) were embedded into the in situ crosslinked hydrogels, and their cellular behavior such as the morphology, viability, metabolic activity and proliferation were fully evaluated. The results suggested that the hydrogel maintained good cell viability and it can be easily modified with other bioactive signals, which provide this injectable hydrogel delivery system with good potential for polymeric biomaterials and tissue regeneration applications.

Controlled Polymerization of Multivinyl Monomers: Formation of Cyclized/Knotted Single-Chain Polymer Architectures

Seventy years ago, Flory and Stockmayer predicted that the polymerization of multivinyl monomers (MVMs) would inevitably lead to insoluble cross-linked gel networks. Since then, the use of MVMs has largely been limited to as cross-linking agents. More recently, however, polymerization strategies such as reversible deactivation radical polymerization (RDRP) have paved the way for the exploration of new possibilities in terms of both polymer architectures and functional capabilities. This Minireview provides historical context to the problem of polymerizing MVMs, before highlighting how RDRP has led to the formation of new cyclized/knotted polymer structures. Although the potential of such cyclized/knot polymer architectures is far from being fulfilled, some emerging applications are discussed.

Main-chain degradable single-chain cyclized polymers as gene delivery vectors

Single-chain technology (SCT) allows the manipulation of polymeric architectures at an individual polymer chain level, providing a new platform for the fabrication of nanoscale polymeric objects. However, it remains problematic to apply this newborn technology to the biological and medical fields, since synthesis of single-chain polymeric nanoparticles relies heavily on controlled/living radical polymerization of vinyl based monomers, yielding a persistent non-degradable carbon-carbon based backbone. Moreover, the ultrahigh dilution conditions often required for single-chain polymer nanoparticle synthesis limits large-scale applicability. A versatile approach to achieve backbone degradability in single-chain cyclized polymers was developed by combining ring-opening addition polymerization and intramolecular cyclization into a one-pot RAFT copolymerization of cyclic and mono/multi-vinyl monomers system under concentrated conditions. The in situ intramolecular cyclization of individual propagating chains was achieved by kinetic control and statistical manipulation of mono- and multi-vinyl monomer copolymerization. The cyclic allylsulfide monomer 3-methylidene-1,9-dioxa-5,12,13-trithiacyclopentadecane-2,8-dione (MDTD) was copolymerized via the ring-opening pathway to introduce disulfide groups into the vinyl-based backbone without compromising the single chain propagation nature. Backbone degradable single chain polymeric nanoparticles were obtained with molecular weights of 10kDa and MDTD incorporation ratios of 4.7%. Chemical degradation of the nanoparticles confirmed both their single chain nature, as well as backbone degradability. The single-chain cyclized polymeric nanoparticles were evaluated for their gene transfection capabilities. The backbone degradable nanoparticles displayed high transfection efficiencies and low cytotoxicities in both 3T3 and HeLa cells.

Water soluble hyperbranched polymers from controlled radical homopolymerization of PEG diacrylate

A series of water soluble PEG based hyperbranched polymers were successfully synthesized by homopolymerization of poly(ethylene glycol) diacrylate (PEGDA) (Mn = 575 and 700 g mol−1 respectively) via vinyl oligomer combination. The homopolymerization of diacrylate macromers underwent a slow vinyl propagation combined with a polycondensation by coupling of reactive oligomers. At a high initiator-to-monomer ratio (e.g. 1 : 2), high monomer conversions up to 96% were achieved in concentrated reaction conditions (60% w/v) without gelation. The hyperbranched polymers obtained from homopolymerization of PEGDA575 show concentration-dependent thermoresponsive properties in aqueous solutions.

Is it ATRP or SET-LRP? part I: Cu0&CuII/PMDETA – mediated reversible – deactivation radical polymerization

There is a controversial matter of debate as to the mechanism of the Cu0 catalyzed radical polymerization. Two models exist, one based upon ATRP whilst the other upon SET-LRP. Here we present new experimental results and insights into the nature of this polymerization. A good controlled/living polymerization was eventually obtained by Cu0&CuII/PMDETA-mediated radical polymerization. A comparative analysis shows that the mechanism behind this reaction lies between the competition and equilibrium results of SET-LRP and ATRP.

Highly Branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) for High Performance Gene Transfection

The top-performing linear poly(β-amino ester) (LPAE), poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32), has demonstrated gene transfection efficiency comparable to viral-mediated gene delivery. Herein, we report the synthesis of a series of highly branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (HC32) and explore how the branching structure influences the performance of C32 in gene transfection. HC32 were synthesized by an “A2 + B3 + C2” Michal addition strategy. Gaussia luciferase (Gluciferase) and green fluorescent protein (GFP) coding plasmid DNA were used as reporter genes and the gene transfection efficiency was evaluated in human cervical cancer cell line (HeLa) and human recessive dystrophic epidermolysis bullosa keratinocyte (RDEBK) cells. We found that the optimal branching structure led to a much higher gene transfection efficiency in comparison to its linear counterpart and commercial reagents, while preserving high cell viability in both cell types. The branching strategy affected DNA binding, proton buffering capacity and degradation of polymers as well as size, zeta potential, stability, and DNA release rate of polyplexes significantly. Polymer degradation and DNA release rate played pivotal parts in achieving the high gene transfection efficiency of HC32-103 polymers, providing new insights for the development of poly(β-amino ester)s-based gene delivery vectors.

miRNA delivery for skin wound healing

ABSTRACT The wound healing has remained a worldwide challenge as one of significant public health problems. Pathological scars and chronic wounds caused by injury, aging or diabetes lead to impaired tissue repair and regeneration. Due to the unique biological wound environment, the wound healing is a highly complicated process, efficient and targeted treatments are still lacking. Hence, research‐driven to discover more efficient therapeutics is a highly urgent demand. Recently, the research results have revealed that microRNA (miRNA) is a promising tool in therapeutic and diagnostic fields because miRNA is an essential regulator in cellular physiology and pathology. Therefore, new technologies for wound healing based on miRNA have been developed and miRNA delivery has become a significant research topic in the field of gene delivery.

Hyperbranched PEG-based multi-NHS polymer and bioconjugation with BSA

Star-shaped poly(ethylene glycol)-N-hydroxysuccinimide (star-PEG-NHS) has shown great promise in a variety of biomedical applications owing to its non-toxicity, innate non-immunogenic properties and versatile, multifunctional end groups. However, its complex and sophisticated synthetic methods, as well as high costs, have significantly impeded its wide application. Here, we report the design and synthesis of a hyperbranched PEG-based polymer with multiple NHS functional groups (>12). The hyperbranched PEG-based multi-NHS polymer can react easily with a protein (bovine serum albumin, BSA) to form a PEG-protein hydrogel that displays great potential for biomedical applications.

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.