Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Cell Culture
2.3. Nanoparticle Formulations
2.4. Nanoparticle Size and Zeta Potential
2.5. Transmission Electron Microscopy (TEM)
2.6. Encapsulation Assay
2.7. In Vitro Transfection
2.8. Real-Time Quantitative PCR
2.9. Cell Viability Assay
2.10. Collagen Contraction Assay
2.11. Statistical Analysis
3. Results
3.1. Cell Morphology of Human Trabecular Meshwork (TM) and Conjunctival Fibroblast (FF) Cells Assessed by Phase Contrast Microscopy
3.2. Biophysical Characteristics and Encapsulation Efficiencies of Lipid Nanoparticles (LNPs)
3.3. Gene Expression of MRTF-B, ACTA2, and COL1A2 in Human TM and FF Cells
3.4. Cell Viability of Human TM and FF Cells following Treatment with LNP-siRNAs
3.5. Contractibility of Human TM and FF Cells Transfected with LNP-siRNAs
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Quigley, H.A.; Broman, A.T. The number of people with glaucoma worldwide in 2010 and 2020. Br. J. Ophthalmol. 2006, 90, 262–267. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Allison, K.; Patel, D.; Alabi, O. Epidemiology of Glaucoma: The Past, Present, and Predictions for the Future. Cureus 2020, 12, e11686. [Google Scholar] [CrossRef] [PubMed]
- Coleman, A.L. Glaucoma. Lancet 1999, 354, 1803–1810. [Google Scholar] [CrossRef]
- Schlunck, G.; Meyer-ter-Vehn, T.; Klink, T.; Grehn, F. Conjunctival fibrosis following filtering glaucoma surgery. Exp. Eye Res. 2016, 142, 76–82. [Google Scholar] [CrossRef]
- Sunaric Megevand, G.; Bron, A.M. Personalising surgical treatments for glaucoma patients. Prog. Retin. Eye Res. 2021, 81, 100879. [Google Scholar] [CrossRef] [PubMed]
- Hübner, L.; Schlötzer-Schrehardt, U.; Weller, J.M.; Hohberger, B.; Mardin, C.Y.; Lämmer, R. Ultrastructural analysis of explanted CyPass microstents and correlation with clinical findings. Graefes Arch. Clin. Exp. Ophthalmol. 2022, 260, 2663–2673. [Google Scholar] [CrossRef] [PubMed]
- Capitena Young, C.E.; Ammar, D.A.; Seibold, L.K.; Pantcheva, M.B.; SooHoo, J.R.; Kahook, M.Y. Histopathologic Examination of Trabecular Meshwork Changes After Trabecular Bypass Stent Implantation. J. Glaucoma 2018, 27, 606–609. [Google Scholar] [CrossRef]
- Wang, D.Z.; Li, S.; Hockemeyer, D.; Sutherland, L.; Wang, Z.; Schratt, G.; Richardson, J.A.; Nordheim, A.; Olson, E.N. Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc. Natl. Acad. Sci. USA 2002, 99, 14855–14860. [Google Scholar] [CrossRef] [Green Version]
- Yamanaka, O.; Kitano-Izutani, A.; Tomoyose, K.; Reinach, P.S. Pathobiology of wound healing after glaucoma filtration surgery. BMC Ophthalmol. 2015, 15 (Suppl. 1), 157. [Google Scholar] [CrossRef] [Green Version]
- Masszi, A.; Speight, P.; Charbonney, E.; Lodyga, M.; Nakano, H.; Szászi, K.; Kapus, A. Fate-determining mechanisms in epithelial-myofibroblast transition: Major inhibitory role for Smad3. J. Cell Biol. 2010, 188, 383–399. [Google Scholar] [CrossRef]
- Johnson, L.A.; Rodansky, E.S.; Haak, A.J.; Larsen, S.D.; Neubig, R.R.; Higgins, P.D. Novel Rho/MRTF/SRF inhibitors block matrix-stiffness and TGF-β-induced fibrogenesis in human colonic myofibroblasts. Inflamm. Bowel Dis. 2014, 20, 154–165. [Google Scholar] [CrossRef] [PubMed]
- Keller, K.E.; Aga, M.; Bradley, J.M.; Kelley, M.J.; Acott, T.S. Extracellular matrix turnover and outflow resistance. Exp. Eye Res. 2009, 88, 676–682. [Google Scholar] [CrossRef] [Green Version]
- Tektas, O.Y.; Lütjen-Drecoll, E. Structural changes of the trabecular meshwork in different kinds of glaucoma. Exp. Eye Res. 2009, 88, 769–775. [Google Scholar] [CrossRef] [PubMed]
- McKee, C.T.; Wood, J.A.; Shah, N.M.; Fischer, M.E.; Reilly, C.M.; Murphy, C.J.; Russell, P. The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents. Biomaterials 2011, 32, 2417–2423. [Google Scholar] [CrossRef] [Green Version]
- Pattabiraman, P.P.; Maddala, R.; Rao, P.V. Regulation of plasticity and fibrogenic activity of trabecular meshwork cells by Rho GTPase signaling. J. Cell Physiol. 2014, 229, 927–942. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pattabiraman, P.P.; Rao, P.V. Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells. Am. J. Physiol. Cell Physiol. 2010, 298, C749–C763. [Google Scholar] [CrossRef] [Green Version]
- Rao, P.V.; Deng, P.F.; Kumar, J.; Epstein, D.L. Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632. Investig. Ophthalmol. Vis. Sci. 2001, 42, 1029–1037. [Google Scholar]
- Elbashir, S.M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411, 494–498. [Google Scholar] [CrossRef]
- Dana, H.; Chalbatani, G.M.; Mahmoodzadeh, H.; Karimloo, R.; Rezaiean, O.; Moradzadeh, A.; Mehmandoost, N.; Moazzen, F.; Mazraeh, A.; Marmari, V.; et al. Molecular Mechanisms and Biological Functions of siRNA. Int. J. Biomed. Sci. 2017, 13, 48–57. [Google Scholar]
- Yin, H.; Kanasty, R.L.; Eltoukhy, A.A.; Vegas, A.J.; Dorkin, J.R.; Anderson, D.G. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 2014, 15, 541–555. [Google Scholar] [CrossRef]
- Kulkarni, J.A.; Myhre, J.L.; Chen, S.; Tam, Y.Y.C.; Danescu, A.; Richman, J.M.; Cullis, P.R. Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine 2017, 13, 1377–1387. [Google Scholar] [CrossRef] [PubMed]
- Sayed, N.; Allawadhi, P.; Khurana, A.; Singh, V.; Navik, U.; Pasumarthi, S.K.; Khurana, I.; Banothu, A.K.; Weiskirchen, R.; Bharani, K.K. Gene therapy: Comprehensive overview and therapeutic applications. Life Sci. 2022, 294, 120375. [Google Scholar] [CrossRef] [PubMed]
- Begum, A.A.; Toth, I.; Hussein, W.M.; Moyle, P.M. Advances in Targeted Gene Delivery. Curr. Drug Deliv. 2019, 16, 588–608. [Google Scholar] [CrossRef] [PubMed]
- Angelov, B.; Angelova, A.; Drechsler, M.; Garamus, V.M.; Mutafchieva, R.; Lesieur, S. Identification of large channels in cationic PEGylated cubosome nanoparticles by synchrotron radiation SAXS and Cryo-TEM imaging. Soft Matter 2015, 11, 3686–3692. [Google Scholar] [CrossRef] [Green Version]
- Ewert, K.; Slack, N.L.; Ahmad, A.; Evans, H.M.; Lin, A.J.; Samuel, C.E.; Safinya, C.R. Cationic lipid-DNA complexes for gene therapy: Understanding the relationship between complex structure and gene delivery pathways at the molecular level. Curr. Med. Chem. 2004, 11, 133–149. [Google Scholar] [CrossRef] [Green Version]
- Alabi, C.A.; Love, K.T.; Sahay, G.; Yin, H.; Luly, K.M.; Langer, R.; Anderson, D.G. Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery. Proc. Natl. Acad. Sci. USA 2013, 110, 12881–12886. [Google Scholar] [CrossRef] [Green Version]
- Zhao, Y.; Huang, L. Lipid nanoparticles for gene delivery. Adv. Genet. 2014, 88, 13–36. [Google Scholar]
- Sanghani, A.; Kafetzis, K.N.; Sato, Y.; Elboraie, S.; Fajardo-Sanchez, J.; Harashima, H.; Tagalakis, A.D.; Yu-Wai-Man, C. Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts. Pharmaceutics 2021, 13, 382. [Google Scholar] [CrossRef]
- Fernando, O.; Tagalakis, A.D.; Awwad, S.; Brocchini, S.; Khaw, P.T.; Hart, S.L.; Yu-Wai-Man, C. Development of Targeted siRNA Nanocomplexes to Prevent Fibrosis in Experimental Glaucoma Filtration Surgery. Mol. Ther. 2018, 26, 2812–2822. [Google Scholar] [CrossRef] [Green Version]
- Sato, Y.; Hashiba, K.; Sasaki, K.; Maeki, M.; Tokeshi, M.; Harashima, H. Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo. J. Control. Release 2019, 295, 140–152. [Google Scholar] [CrossRef]
- Kennedy, S.M.; Sheridan, C.; Kearns, V.R.; Bilir, E.K.; Fan, X.; Grierson, I.; Choudhary, A. Thrombospondin-2 is up-regulated by TGFβ2 and increases fibronectin expression in human trabecular meshwork cells. Exp. Eye Res. 2019, 189, 107820. [Google Scholar] [CrossRef] [PubMed]
- Sato, Y.; Hatakeyama, H.; Sakurai, Y.; Hyodo, M.; Akita, H.; Harashima, H. A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo. J. Control. Release 2012, 163, 267–276. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Premarket Studies of Implantable Minimally Invasive Glaucoma Surgical (MIGS) Devices. Available online: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/premarket-studies-implantable-minimally-invasive-glaucoma-surgical-migs-devices (accessed on 1 September 2022).
- Shalaby, W.S.; Lam, S.S.; Arbabi, A.; Myers, J.S.; Moster, M.R.; Kolomeyer, N.N.; Razeghinejad, R.; Shukla, A.G.; Hussein, T.R.; Eid, T.M.; et al. iStent versus iStent inject implantation combined with phacoemulsification in open angle glaucoma. Indian J. Ophthalmol. 2021, 69, 2488–2495. [Google Scholar] [CrossRef] [PubMed]
- Ahmed, I.I.K.; Fea, A.; Au, L.; Ang, R.E.; Harasymowycz, P.; Jampel, H.D.; Samuelson, T.W.; Chang, D.F.; Rhee, D.J. A Prospective Randomized Trial Comparing Hydrus and iStent Microinvasive Glaucoma Surgery Implants for Standalone Treatment of Open-Angle Glaucoma: The COMPARE Study. Ophthalmology 2020, 127, 52–61. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Omoto, T.; Sugiura, A.; Fujishiro, T.; Asano-Shimizu, K.; Sugimoto, K.; Sakata, R.; Murata, H.; Asaoka, R.; Honjo, M.; Aihara, M. Twelve-month surgical outcome and prognostic factors of stand-alone ab interno trabeculotomy in Japanese patients with open-angle glaucoma. PLoS ONE 2021, 16, e0245015. [Google Scholar] [CrossRef]
- Wolters, J.E.J.; van Mechelen, R.J.S.; Al Majidi, R.; Pinchuk, L.; Webers, C.A.B.; Beckers, H.J.M.; Gorgels, T. History, presence, and future of mitomycin C in glaucoma filtration surgery. Curr. Opin. Ophthalmol. 2021, 32, 148–159. [Google Scholar] [CrossRef]
- Khaw, P.; Grehn, F.; Holló, G.; Overton, B.; Wilson, R.; Vogel, R.; Smith, Z. A phase III study of subconjunctival human anti-transforming growth factor beta(2) monoclonal antibody (CAT-152) to prevent scarring after first-time trabeculectomy. Ophthalmology 2007, 114, 1822–1830. [Google Scholar]
- Na, J.H.; Sung, K.R.; Shin, J.A.; Moon, J.I. Antifibrotic effects of pirfenidone on Tenon’s fibroblasts in glaucomatous eyes: Comparison with mitomycin C and 5-fluorouracil. Graefes Arch. Clin. Exp. Ophthalmol. 2015, 253, 1537–1545. [Google Scholar] [CrossRef]
- Yu-Wai-Man, C.; Khaw, P.T. Developing novel anti-fibrotic therapeutics to modulate post-surgical wound healing in glaucoma: Big potential for small molecules. Expert Rev. Ophthalmol. 2015, 10, 65–76. [Google Scholar] [CrossRef] [Green Version]
- Naik, S.; Shreya, A.B.; Raychaudhuri, R.; Pandey, A.; Lewis, S.A.; Hazarika, M.; Bhandary, S.V.; Rao, B.S.S.; Mutalik, S. Small interfering RNAs (siRNAs) based gene silencing strategies for the treatment of glaucoma: Recent advancements and future perspectives. Life Sci. 2021, 264, 118712. [Google Scholar] [CrossRef]
- Revia, R.A.; Stephen, Z.R.; Zhang, M. Theranostic Nanoparticles for RNA-Based Cancer Treatment. Acc. Chem Res. 2019, 52, 1496–1506. [Google Scholar] [CrossRef] [PubMed]
- Sahoo, S.K.; Dilnawaz, F.; Krishnakumar, S. Nanotechnology in ocular drug delivery. Drug Discov. Today 2008, 13, 144–151. [Google Scholar] [CrossRef] [PubMed]
- Rizk, M.; Tüzmen, Ş. Update on the clinical utility of an RNA interference-based treatment: Focus on Patisiran. Pharmgenomics Pers. Med. 2017, 10, 267–278. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Clogston, J.D.; Patri, A.K. Zeta potential measurement. Methods Mol. Biol. 2011, 697, 63–70. [Google Scholar]
- Bhattacharjee, S.; Ershov, D.; Gucht, J.; Alink, G.M.; Rietjens, I.M.; Zuilhof, H.; Marcelis, A.T. Surface charge-specific cytotoxicity and cellular uptake of tri-block copolymer nanoparticles. Nanotoxicology 2013, 7, 71–84. [Google Scholar] [CrossRef]
- Tagalakis, A.D.; He, L.; Saraiva, L.; Gustafsson, K.T.; Hart, S.L. Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery. Biomaterials 2011, 32, 6302–6315. [Google Scholar] [CrossRef]
- Yu-Wai-Man, C.; Tagalakis, A.D.; Manunta, M.D.; Hart, S.L.; Khaw, P.T. Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis. Sci. Rep. 2016, 6, 21881. [Google Scholar] [CrossRef]
- Bini, L.; Schvartz, D.; Carnemolla, C.; Besio, R.; Garibaldi, N.; Sanchez, J.C.; Forlino, A.; Bianchi, L. Intracellular and Extracellular Markers of Lethality in Osteogenesis Imperfecta: A Quantitative Proteomic Approach. Int. J. Mol. Sci. 2021, 22, 429. [Google Scholar] [CrossRef]
- Chen, M.; Liu, J.; Yang, W.; Ling, W. Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling. Autophagy 2017, 13, 1813–1827. [Google Scholar] [CrossRef] [Green Version]
- Hetzler, P.T., 3rd; Dash, B.C.; Guo, S.; Hsia, H.C. Targeting Fibrotic Signaling: A Review of Current Literature and Identification of Future Therapeutic Targets to Improve Wound Healing. Ann. Plast. Surg. 2019, 83, e92–e95. [Google Scholar] [CrossRef]
- Hinz, B.; Mastrangelo, D.; Iselin, C.E.; Chaponnier, C.; Gabbiani, G. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am. J. Pathol. 2001, 159, 1009–1020. [Google Scholar] [CrossRef] [Green Version]
- Zhang, Q.; Wang, P.; Fang, X.; Lin, F.; Fang, J.; Xiong, C. Collagen gel contraction assays: From modelling wound healing to quantifying cellular interactions with three-dimensional extracellular matrices. Eur. J. Cell Biol. 2022, 101, 151253. [Google Scholar] [CrossRef] [PubMed]
- Ni, R.; Feng, R.; Chau, Y. Synthetic Approaches for Nucleic Acid Delivery: Choosing the Right Carriers. Life 2019, 9, 59. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Grosse, S.M.; Tagalakis, A.D.; Mustapa, M.F.; Elbs, M.; Meng, Q.H.; Mohammadi, A.; Tabor, A.B.; Hailes, H.C.; Hart, S.L. Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers. FASEB J. 2010, 24, 2301–2313. [Google Scholar] [CrossRef]
- Shirley, M. Bimatoprost Implant: First Approval. Drugs Aging 2020, 37, 457–462. [Google Scholar] [CrossRef] [PubMed]
- Belamkar, A.; Harris, A.; Zukerman, R.; Siesky, B.; Oddone, F.; Verticchio Vercellin, A.; Ciulla, T.A. Sustained release glaucoma therapies: Novel modalities for overcoming key treatment barriers associated with topical medications. Ann. Med. 2022, 54, 343–358. [Google Scholar] [CrossRef]
- Tagalakis, A.D.; Madaan, S.; Larsen, S.D.; Neubig, R.R.; Khaw, P.T.; Rodrigues, I.; Goyal, S.; Lim, K.S.; Yu-Wai-Man, C. In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis. J. Nanobiotechnol. 2018, 16, 97. [Google Scholar] [CrossRef] [Green Version]
- Kim, H.M.; Ha, S.; Hong, H.K.; Hwang, Y.; Kim, P.; Yang, E.; Chung, J.Y.; Park, S.; Park, Y.J.; Park, K.H.; et al. Intraocular Distribution and Kinetics of Intravitreally Injected Antibodies and Nanoparticles in Rabbit Eyes. Transl. Vis. Sci. Technol. 2020, 9, 20. [Google Scholar] [CrossRef]
- Shi, Z.; Li, S.K.; Charoenputtakun, P.; Liu, C.Y.; Jasinski, D.; Guo, P. RNA nanoparticle distribution and clearance in the eye after subconjunctival injection with and without thermosensitive hydrogels. J. Control. Release 2018, 270, 14–22. [Google Scholar] [CrossRef]
- Keller, K.E.; Bhattacharya, S.K.; Borrás, T.; Brunner, T.M.; Chansangpetch, S.; Clark, A.F.; Dismuke, W.M.; Du, Y.; Elliott, M.H.; Ethier, C.R.; et al. Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Exp. Eye Res. 2018, 171, 164–173. [Google Scholar] [CrossRef]
Function | Name | Structure/Sequence |
---|---|---|
Lipid | 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6) | |
Lipid | 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) | |
Lipid | 1,2-dimirystoyl-rac-glycero, methoxyethylene glycol 2000 ether (PEG-DMG) | |
Peptide | Cleavable (c) | K16RVRR-GACYGLPHKFCG |
siRNA | MRTF-B | Sense: GGAUGGAACUUUACCCUCA Antisense: UGAGGGUAAAGUUCCAUCC |
siRNA | Irrelevant Control | Sense: UGGUUUACAUGUCGACUAA Antisense: UUAGUCGACAUGUAAACCA |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Luo, J.; Tan, G.; Thong, K.X.; Kafetzis, K.N.; Vallabh, N.; Sheridan, C.M.; Sato, Y.; Harashima, H.; Tagalakis, A.D.; Yu-Wai-Man, C. Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery. Pharmaceutics 2022, 14, 2472. https://doi.org/10.3390/pharmaceutics14112472
Luo J, Tan G, Thong KX, Kafetzis KN, Vallabh N, Sheridan CM, Sato Y, Harashima H, Tagalakis AD, Yu-Wai-Man C. Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery. Pharmaceutics. 2022; 14(11):2472. https://doi.org/10.3390/pharmaceutics14112472
Chicago/Turabian StyleLuo, Jinyuan, Greymi Tan, Kai Xin Thong, Konstantinos N. Kafetzis, Neeru Vallabh, Carl M. Sheridan, Yusuke Sato, Hideyoshi Harashima, Aristides D. Tagalakis, and Cynthia Yu-Wai-Man. 2022. "Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery" Pharmaceutics 14, no. 11: 2472. https://doi.org/10.3390/pharmaceutics14112472
APA StyleLuo, J., Tan, G., Thong, K. X., Kafetzis, K. N., Vallabh, N., Sheridan, C. M., Sato, Y., Harashima, H., Tagalakis, A. D., & Yu-Wai-Man, C. (2022). Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery. Pharmaceutics, 14(11), 2472. https://doi.org/10.3390/pharmaceutics14112472