Ben Newland

Mussel-inspired hyperbranched poly(amino ester) polymer as strong wet tissue adhesive

Current medical adhesives based on cyanoacrylates typically exhibit cellular toxicity. In contrast, fibrin adhesives are non-toxic but have poor adhesive properties. To overcome these drawbacks we designed a simple and scalable adhesive precursor inspired by marine mussel adhesion that functioned with strong adhesion in wet conditions and with low cytotoxicity. Dopamine, an-amine derivative of an amino acid abundantly present in mussel adhesive proteins, was co-polymerised with a tri-functional vinyl monomer, to form a hyperbranched poly(β-amino ester) polymer termed poly(dopamine-co-acrylate) (PDA). A variety of molecular weights and crosslinking methods were analysed using an ex vivo porcine skin model and an almost 4 fold increase in wet adhesion strength was observed compared to TISSEEL(®) fibrin sealant. With a fast curing time, degradable properties and low cytotoxicity, PDA is highly attractive for medical purposes and could have a broad impact on surgeries where surgical tissue adhesives, sealants, and haemostatic agents are used.

A highly effective gene delivery vector – hyperbranched poly(2-(dimethylamino)ethyl methacrylate) from in situ deactivation enhanced ATRP

A hyperbranched 2-(dimethylamino)ethyl methacrylate (DMAEMA) based polymer has been synthesised by a one-pot in situ deactivation enhanced atom transfer radical polymerisation (DE-ATRP); it exhibits much higher transfection ability than linear poly(DMAEMA) and is comparable to the well known branched poly(ethylene imine) (PEI) and the SuperFect dendrimer but with lower cytotoxicity.

3D Single Cyclized Polymer Chain Structure from Controlled Polymerization of Multi-Vinyl Monomers: Beyond Flory–Stockmayer Theory

Controlled/living radical polymerization (CRP) is a widely used technique that allows the synthesis of defined polymer architectures through precise control of molecular weights and distributions. However, the architectures of polymers prepared by the CRP techniques are limited to linear, cross-linked, and branched/dendritic structures. Here, we report the preparation of a new 3D single cyclized polymer chain structure from an in situ deactivation enhanced atom transfer radical polymerization of multivinyl monomers (MVMs), which are conventionally used for the production of branched/cross-linked polymeric materials as defined by P. Flory and W. Stockmayer nearly 70 years ago. We provide new evidence to demonstrate that it is possible to kinetically control both the macromolecular architecture and the critical gelling point in the polymerization of MVMs, suggesting the classical Flory-Stockmayer mean field theory should be supplemented with a new kinetic theory based on the space and instantaneous growth boundary concept.

The neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow spheres

Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.

The reduction in immunogenicity of neurotrophin overexpressing stem cells after intra-striatal transplantation by encapsulation in an in situ gelling collagen hydrogel.

Delivery of neurotrophic factors to the brain via genetically modified bone marrow-derived mesenchymal stem cells (MSCs) offers a promising neuroprotective strategy for neurodegenerative diseases. However, MSCs delivered to the CNS typically show poor survival post-transplantation, which is accompanied by microglial activation and astrocyte recruitment at the graft site. Recent studies have shown the potential of biomaterials to provide a supportive matrix for transplanted cells which may assist in the grafting process. In this study, an in situ gelling type I collagen hydrogel was evaluated as an intracerebral transplantation matrix for delivery of glial cell line-derived neurotrophic factor (GDNF)-overexpressing MSCs to the rat brain (GDNF-MSCs). In vitro analyses demonstrated that this collagen hydrogel did not affect the viability of the GDNF-MSCs nor did it prevent GDNF secretion into the surrounding medium. In vivo analyses also confirmed that the collagen hydrogel did not negatively impact on the survival of the cells and permitted GDNF secretion into the striatal parenchyma. Importantly, this study also revealed that transplanting GDNF-MSCs in a collagen hydrogel significantly diminished the host brains response to the cells by reducing the recruitment of both microglia and astrocytes at the site of delivery. In conclusion, this hydrogel, which is composed of the natural extracellular matrix, collagen, was shown to be a well-tolerated cell delivery platform technology which could be functionalised to further aid cell support and graft integration.

Improved axonal regeneration of transected spinal cord mediated by multichannel collagen conduits functionalized with neurotrophin-3 gene

Functionalized biomaterial scaffolds targeted at improving axonal regeneration by enhancing guided axonal growth provide a promising approach for the repair of spinal cord injury. Collagen neural conduits provide structural guidance for neural tissue regeneration, and in this study it is shown that these conduits can also act as a reservoir for sustained gene delivery. Either a G-luciferase marker gene or a neurotrophin-3-encoding gene, complexed to a non-viral, cyclized, PEGylated transfection vector, was loaded within a multichannel collagen conduit. The complexed genes were then released in a controlled fashion using a dual release system both in vitro and in vivo. For evaluation of their biological performance, the loaded conduits were implanted into the completely transected rat thoracic spinal cord (T8–T10). Aligned axon regeneration through the channels of conduits was observed one month post-surgery. The conduits delivering neurotrophin-3 polyplexes resulted in significantly increased neurotrophin-3 levels in the surrounding tissue and a statistically higher number of regenerated axons versus the control conduits (P<0.05). This study suggests that collagen neural conduits delivering a highly effective non-viral therapeutic gene may hold promise for repair of the injured spinal cord.

GDNF Gene Delivery via a 2-(Dimethylamino)ethyl Methacrylate Based Cyclized Knot Polymer for Neuronal Cell Applications

Nonviral genetic therapeutic intervention strategies for neurological disorders hold great promise, but a lack of vector efficacy, coupled with vector toxicity, continue to hinder progress. Here we report the application of a newly developed class of polymer, distinctly different from conventional branched polymers, as a transfection agent for the delivery of glial cell line derived neurotrophic factor (GDNF) encoding gene. This new 2-(dimethylamino)ethyl methacrylate (DMAEMA) based cyclized knot polymer was studied for neuronal cell transfection applications, in comparison to branched polyethyleneimine (PEI). While showing a similar transfection profile over multiple cell types, the cyclized knot polymer showed far lower toxicity. In addition, transfection of Neu7 astrocytes with the GDNF encoding gene was able to cause neurite outgrowth when cocultured with dorsal root ganglia (DRGs). The cyclized knot polymer assessed here (PD-E 8%PEG), synthesized via a simple one-pot reaction, was shown to have great potential for neuronal gene therapy applications.

Biomaterial approaches to gene therapies for neurodegenerative disorders of the CNS

Neurodegeneration gives rise to a wide range of disorders which represent a growing health burden to both western societies and developing countries. Whilst for many disorders such as Alzheimers and Parkinsons disease the cause is unknown, gene therapy is becoming the forefront of novel potential therapies described in the literature and has entered clinical trials. Furthermore, although in somewhat an earlier stage, biomaterials offer means of enhancing gene therapy strategies either through new delivery methods or provision of support for genetically manipulated cells. This review outlines recent uses of biomaterials in the CNS and captures recent advances in non-viral gene delivery to the brain. Three dimensional scaffolding systems for ex vivo gene delivery to the brain are also discussed highlighting the progress of hydrogel mediated cell delivery. This review also addresses the difficulties and safety considerations of these approaches; illustrating the ability of biomaterial strategies to significantly improve outcomes of gene therapies for neurodegenerative disorders.

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.

Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene therapy applications.

Nonviral methods for gene delivery are becoming ever more prevalent along with the need to design new vectors that are highly effective, stable in biological fluids, inexpensive, and facile to produce. Here, we synthesize our previously reported monomer N-ethyl pyrrolidine methacrylamide (EPA) and evaluate its effectiveness in gene vector applications when copolymerized with 1-vinylimidazole (VI). A range of these novel linear cationic copolymers were synthesized via free radical polymerization with low molecular weights (oligomers) and low polydispersities showing two pK(a) values as the two co-monomers are cationic. DNA-polymer polyplexes had average sizes between 100 and 250nm and zeta-potentials between 10 and 25mV, and a strong dependence of composition on the size on the zeta-potential was observed. The cytotoxicity of the homopolymers, oligomers, and polyplexes toward human fibroblasts and 3T3 mouse fibroblasts was evaluated using the MTT and AlamarBlue™ assays, proving that formulations could be made with toxicity as low as low molecular weight linear poly (dimethylaminoethyl methacrylate) (PDMAEMA). The transfection capability of the polyplexes measured using the G-luciferase marker gene far superseded PDMAEMA when evaluated in biological conditions. Furthermore, blood compatibility studies showed that these new oligomers exhibit no significant hemolysis or platelet activation above PBS controls. These new EPA based oligomers with low toxicity and ease of scalability show high transfection abilities in serum conditions, and blood compatibility showing its potential for systemic gene delivery applications.