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Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition
  1. Li Fong Seet1,2,3,
  2. Yang Fei Tan4,
  3. Li Zhen Toh1,
  4. Stephanie WL Chu1,
  5. Ying Shi Lee1,
  6. Subbu S Venkatraman1,4,5,6,
  7. Tina T Wong1,2,3,4,7
  1. 1 Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, Singapore, Singapore
  2. 2 Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
  3. 3 Duke-NUS Medical School, Singapore, Singapore
  4. 4 School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore
  5. 5 NTU – Northwestern University Institute for Nanomedicine, Singapore, Singapore
  6. 6 MedTech, National Heart Centre, Singapore, Singapore
  7. 7 Glaucoma Service, Singapore National Eye Centre, Singapore, Singapore
  1. Correspondence to Professor Tina T Wong, Glaucoma Service, Singapore National Eye Centre, Singapore 168751, Singapore; tina.wong.t.l{at}singhealth.com.sg; Subbu S Venkatraman, School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore; ASSubbu{at}ntu.edu.sg

Abstract

Background To develop targeted antifibrotic therapy for glaucoma filtration surgery; this study determines the effectiveness of small interfering RNA (siRNA) to reduce in vivo secreted protein acidic and rich in cysteine (SPARC) expression using the mouse model of conjunctival scarring.

Methods Experimental surgery was performed as described for the mouse model of conjunctival scarring. Scrambled (siScram) or Sparc (siSparc) siRNAs, loaded on layer-by-layer (LbL) nanoparticles, were injected into the conjunctiva immediately after surgery. Expression of Sparc, Col1a1, Fn1 and Mmp14 was measured by real-time PCR and immunoblotting on days 7 and 14 postsurgery. Live imaging of the operated eyes was performed using slit lamp, anterior segment-optical coherence tomography and confocal microscopy. Tissue pathology was evaluated by histochemical and immunofluorescent analyses of operated conjunctival cryosections. Tissue apoptosis was quantitated by annexin V assay.

Results siSparc, delivered via expanded LbL nanoparticles, significantly inhibited Sparc transcription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues. Sparc suppression on day 7 was associated with a significant reduction of Col1a1 (2.52-fold), Fn1 (2.89-fold) and Mmp14 (2.23-fold) mRNAs. At the protein level, both SPARC and collagen 1A1 (COL1A1) were significantly reduced at both time points with siSparc treatment. Nanoparticles were visualised within cell-like structures by confocal microscopy, while overt tissue response or apoptosis was not observed.

Conclusions SPARC targeted therapy effectively reduced both SPARC and collagen production in the operated mouse conjunctiva. This proof-of-concept study suggests that targeted treatment of fibrosis in glaucoma surgery is safe and feasible, with the potential to extend to a range of potential genes associated with fibrosis.

  • conjunctiva
  • treatment surgery
  • wound healing
  • Experimental – animal models

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Footnotes

  • LFS and YFT contributed equally.

  • Contributors LFS analysed the data and wrote the manuscript. LFS, YFT, LZT and SCWL performed the experiments. SV and TTW conceptualised the study. TTW edited and approved the manuscript.

  • Funding This work was funded by a Clinician Scientist Award grant (NMRC/CSA-SI/0001/2015) and by the Singapore National Research Foundation under its Translational and Clinical Research (TCR) Programme (NMRC/TCR/002-SERI/2008; NMRC/TCR/008-SERI/2013) to TTW, both administered by the Singapore Ministry of Health’s National Medical Research Council. Animal studies were funded by a SingHealth Foundation Research Grant (SHF/FG583P/2014) to LFS and partially funded by the SERI core grant (NMRC/CG/015/2013). 

  • Competing interests None declared.

  • Patient consent Not required.

  • Ethics approval Institutional Animal Care and Use Committee (IACUC).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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