(217cr) Delivery of Diblock Copolymer/Plasmid DNA Polyplexes From Polyurethane Scaffolds

Introduction

Local delivery of nonviral vectors from biomaterial scaffolds has potential for use in tissue regeneration and treatment of diseases. Plasmids (pDNA) are cheaper and easier to produce than proteins and lack the immunogenic risk of viral vectors. However, many traditional transfection reagents such as polyethylenimine (PEI) suffer from instability, especially when lyophilized for storage or incorporation into biomaterial scaffolds. Previously, a novel library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were developed and screened for improved colloidal stability and nucleic acid transfection following lyophilization. These polymers were designed such that DMAEMA initiates nucleic acid electrostatic interactions to trigger formation of polyplexes that are further stabilized by the hydrophobic interactions of the BMA in the polyplex core. The PEG corona was incorporated in order to shield the polyplex charge in the core and provide enhanced steric stabilization. The BMA content was also finely tuned so that the polyplexes exert pH-dependent membrane disruption to promote endosome escape. The polymer with the best performance had a 5,000 Da PEG block and a 21,000 Da DMAEMA/BMA block with 40% BMA. In this study, the stability and in vitro transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))/pDNA and PEI/pDNA polyplexes were investigated. Furthermore, polyplexes were incorporated into polyurethane (PUR) scaffolds. The release kinetics of polyplexes from the scaffolds and the ability to transfect cells seeded on the scaffolds were investigated.

Methods

Poly(EG-b-(DMAEMA-co-BMA)) with a 5,000 Da PEG block and a 21,000 Da DMAEMA/BMA block with 40% BMA was synthesized by reversible addition fragmentation chain transfer polymerization. Polyplexes were formed by mixing equal volumes of polymer and luciferase pDNA solutions. The amine/phosphate ratio was 10 for poly(EG-b-(DMAEMA-co-BMA)) polyplexes and 30 for PEI polyplexes. For in vitro transfection experiments, polyplexes with a dose of 150 ng pDNA were delivered to MDA-MB-231 tumor cells in 96-well plates, and bioluminescence was measured after 24 h using a Xenogen IVIS 200. PUR scaffolds were synthesized by reacting a lysine triisocyanate-poly(ethylene glycol) prepolymer with lyophilized complexes and a hardener component comprising a polyester triol with a backbone composed of 60% caprolactone, 30% glycolide, and 10% lactide; triethylenediamine catalyst; and water. Scaffolds were sectioned into 6x2 mm discs, and 100,000 cells were seeded on each disc. Bioluminescence was measured at various time points up to 21 days. To determine polyplex release kinetics, pDNA was fluorescently labeled, and scaffolds were incubated in phosphate buffered saline. Releasates were collected and replaced with fresh PBS at various time points up to 21 days, and fluorescence in the scaffolds and releasates was measured.

Results

Size measurements performed using dynamic light scattering and transmission electron microscopy showed that the diameter of PEI/pDNA polyplexes significantly increased after lyophilization (p < 0.05) while the diameter of poly(EG-b-(DMAEMA-co-BMA))/pDNA polyplexes did not. Cells transfected with lyophilized poly(EG-b-(DMAEMA-co-BMA)) polyplexes produced 25 fold higher luminescence than cells transfected with lyophilized PEI polyplexes. Luminescence produced by cells seeded on PUR scaffolds incorporating poly(EG-b-(DMAEMA-co-BMA))/pDNA polyplexes was significantly higher than for PEI/pDNA polyplexes at each time point investigated. During days 1-7, scaffolds incorporating poly(EG-b-(DMAEMA-co-BMA))/pDNA polyplexes had 10-80 fold higher luminescence than scaffolds incorporating PEI/pDNA polyplexes. Transfection for both treatment groups peaked at day 2 and then slowly declined. The release profile of poly(EG-b-(DMAEMA-co-BMA))/pDNA polyplexes showed a burst release of 50% of the polyplexes within the first 24 h followed by a sustained release resulting in 90% released after 21 days. The release profile of PEI/pDNA polyplexes showed a burst release of 35% within the first 24 h followed by a sustained release resulting in 90% released after 21 days. The Weibull model was fit to both profiles. The models for both poly(EG-b-(DMAEMA-co-BMA)) and PEI polyplexes had good fits (R²= 0.98 – 0.99) and values less than 0.75 for fitting parameter b, indicating diffusion-controlled release from the scaffolds.

Conclusion

In conclusion, a novel poly(EG-b-(DMAEMA-co-BMA)) polymer was developed to enhance stability and transfection efficiency of pDNA when lyophilized and/or incorporated into biomaterial scaffolds. Poly(EG-b-(DMAEMA-co-BMA))/pDNA polyplexes had less aggregation and higher transfection efficiency than PEI/pDNA polyplexes after lyophilization. When incorporated into polyurethane scaffolds, poly(EG-b-(DMAEMA-co-BMA)) polyplexes had similar release kinetics but higher transfection activity than PEI polyplexes. Future work will involve tuning the release profile of the polyplexes from PUR scaffolds, measuring release kinetics and transfection efficiency in vivo using a mouse subcutaneous model, and delivering genes for regenerative factors from PUR scaffolds in vivo.