Synthesis and Formulation of Four-Arm PolyDMAEA-siRNA Polyplex for Transient Downregulation of Collagen Type III Gene Expression in TGF-β1 Stimulated Tenocyte Culture

Xin Liao; Noelia D Falcon; Ali A Mohammed; Yasmin Z Paterson; Andrew Geoffrey Mayes; Deborah J Guest; Aram Saeed

doi:10.1021/acsomega.9b03216

Synthesis and Formulation of Four-Arm PolyDMAEA-siRNA Polyplex for Transient Downregulation of Collagen Type III Gene Expression in TGF-β1 Stimulated Tenocyte Culture

ACS Omega. 2020 Jan 15;5(3):1496-1505. doi: 10.1021/acsomega.9b03216. eCollection 2020 Jan 28.

Authors

Xin Liao¹, Noelia D Falcon¹, Ali A Mohammed¹, Yasmin Z Paterson^{2

3}, Andrew Geoffrey Mayes⁴, Deborah J Guest², Aram Saeed¹

Affiliations

¹ School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K.
² Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, U.K.
³ Department of Veterinary Medicine, University of Cambridge, Cambridgeshire CB3 0ES, U.K.
⁴ School of Chemistry, University of East Anglia, Norwich NR4 7TJ, U.K.

Abstract

The natural healing process for tendon repair is associated with high upregulation of collagen type III, leading to scar tissue and tendon adhesions with functionally deficient tendons. Gene delivery systems are widely reported as potential nanotherapeutics to treat diseases, providing a promising approach to modulate collagen type III synthesis. This work investigates a proof-of-concept four-arm cationic polymer-siRNA polyplex to mediate a transient downregulation of collagen type III expression in a tendon cell culture system. The tendon culture system was first supplemented with TGF-β1 to stimulate the upregulation of collagen type III prior to silencing experiments. The four-arm poly[2-(dimethylamino) ethyl acrylate] (PDMAEA) polymer was successfully synthesized via RAFT polymerization and then mixed with siRNA to formulate the PDMAEA-siRNA polyplexes. The formation of the polyplex was optimized for the N:P ratio (10:1) and confirmed by agarose gel electrophoresis. The size and solution behavior of the polyplex were analyzed by dynamic light scattering and zeta potential, showing a hydrodynamic diameter of 155 ± 21 nm and overall positive charge of +30 mV at physiological pH. All the polyplex concentrations used had a minimal effect on the metabolic activity of cultured cells, indicating good biocompatibility. The dose and time effects of the TGF-β1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL^-1 TGF-β1 and 3-fold increase of COL3α1 expression at 48 h in cultured tenocytes. The PDMAEA-siRNA polyplex concept observed a limited yet successful and promising efficiency in silencing collagen type III at 48 h compared to PEI-siRNA. Therefore, this concept is a promising approach to reduce tissue scarring and adhesion following injuries.