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Novel Chitosan Nanoparticle-aptamer Complexes Targeting TFG-β ReceptorⅡ Inhibit the Transdifferentiation of Human Tenon’s Capsule Fibroblasts

Author: ChenXia
Tutor: XieLin
School: Third Military Medical University
Course: Ophthalmology
Keywords: nanoparticle aptamer TGF-β anti-scarring human Tenon’s capsulefibroblasts
CLC: R775
Type: Master's thesis
Year: 2013
Downloads: 1
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Abstract

Background:Filtration surgery can effectively decrease the IOP by enhancing the drainage ofaqueous material and is the principal treatment for glaucoma. However, the soaringoccurrence of subconjunctival scarring that blocks the surgically created drainage channellimits the success rates of this surgery. TGF-β is believed to be a pivotal mediator drivingboth normal wound healing and tissue fibrosis. The TGF-β isoform TGF-β₂has beenfound to be the predominant isoform related to ocular scarring diseases. The binding of aTGF-β ligand to a type Ⅱ receptor dimer triggers the transdifferentiation of fibroblastsinto myofibroblasts （MFs） with high contractility at wound sites, which is the key processin scar formation. Thus, it may be an effective strategy to impair TGF-β activity andanti-scarring by interfering with the binding of the TGF-β ligand to the type Ⅱ receptor. Inour previous study, a dominant sequence called aptamer S58, which was selected bySELEX, antagonised TGF-β-induced myofibroblast transdifferentiation in human Tenon’scapsule fibroblasts through sealing the targeting site of TGF-β receptor Ⅱ （TβR Ⅱ）.However, rapid degradation by ubiquitous nucleases limited the aptamer’s efficacy to12h,which was much less than the weeks of time required for scar formation. Therefore,nuclease degradation is the main hindrance to clinical and therapeutic applications ofssDNA and RNA medicine. Although various chemical modifications of aptamers, such asphosphorylation and phosphorothioate modifications, may help to overcome thisshortcoming, these modifications possess disadvantages, such as high cytotoxicity andincreased non-specific effects. Therefore, it is necessary to develop efficient methods foraptamer protection and delivery. Chitosan is a natural biodegradable biopolymer obtainedby the deacetylation of chitin. Owing to its biocompatibility, biodegradability, relativelylow immunogenicity and especially good macro-adhesion, chitosan has been widely studiedas a delivery system for therapeutic macromolecules, nucleotides, and protein molecules since the beginning of the last century. Research has shown that these chitosannanoparticles can protect encapsulated DNA against nuclease degradation. Thus, we chosechitosan as the aptamer carrier to preserve S58and to prolong its efficacy.Purpose:To synthesise novel chitosan nanoparticle–aptamer complexes called CS（S58）-NP thattarget TGF-β receptorⅡ（TβRⅡ） and to evaluate their characteristics and inhibitory effectson TGF-β-induced transdifferentiation of human Tenon’s capsule fibroblasts （HTFs）.Methods:First, we synthesised CS（S58）-NP at various molar ratios of CS-NP to aptamer S58using an ionic gelation method. Second, the CS（S58）-NP were characterised by dynamiclight scattering, gel electrophoresis, and serum incubation. A lactate dehydrogenase （LDH）release assay was conducted to assess the cytotoxicity of the complexes. Finally, theexpression of α-smooth muscle actin （α-SMA） was estimated using western blot analysis.Results:The diameter of the CS（S58）-NP tended to decrease whereas their zeta potential tendedto increase as the CS-NP/S58molar ratio increased. The CS（S58）-NP showed high aptamerencapsulation efficiency, good aptamer binding ability, powerful aptamer protection andstable sustained-release ability. FITC-labelled CS（S58）-NP could successfully bind to TβRⅡ. As a result, TGF-β-induced α-SMA expression was inhibited. Furthermore, the effectiveconcentration of the CS（S58）-NP exhibited low cytotoxicity. Most impressively, theCS（S58）-NP could inhibit TGF-β-induced α-SMA expression for a longer time than nakedS58, even in serum.Conclusions:All of the results demonstrate that the use of CS-NP is promising for aptamer deliverybecause of their powerful protection ability, low cytotoxicity and stable sustained-releaseability. Our results suggest that S58aptamers encapsulated in CS-NP can be a potentialpharmaceutical agent to antagonised TGF-β-induced myofibroblast transdifferentiation inhuman Tenon’s capsule fibroblasts following glaucoma filtration surgery.

Novel Chitosan Nanoparticle-aptamer Complexes Targeting TFG-β ReceptorⅡ Inhibit the Transdifferentiation of Human Tenon’s Capsule Fibroblasts

Abstract

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