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

Liao, Xin; Falcon, Noelia Dominguez; Mohammed, Ali A; Paterson, Yasmin Z; Mayes, Andrew G.; Guest, Deborah J.; Saeed, Aram

doi:10.1021/acsomega.9b03216

Cited by 14 publications

(16 citation statements)

References 48 publications

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“…Gene expression in tendon cells has been shown to change with culture (Shukunami et al, 2018; Yao, Bestwick, Bestwick, Maffulli, & Aspden, 2006) and can change rapidly from the expression levels found in the native tissue (Kuemmerle et al, 2016). However, we have previously demonstrated that COL3A1 gene expression levels do not change between passages 0 and 10 during equine tenocyte culture (Liao et al, 2020). Additionally, although there is a significantly higher level of COL1A1 in equine tenocytes at passage 0 than at passage 3, there are no further changes in COL1A1 expression beyond passage 3 (Liao et al, 2020).…”

Section: Discussionmentioning

confidence: 89%

“…However, we have previously demonstrated that COL3A1 gene expression levels do not change between passages 0 and 10 during equine tenocyte culture (Liao et al, 2020). Additionally, although there is a significantly higher level of COL1A1 in equine tenocytes at passage 0 than at passage 3, there are no further changes in COL1A1 expression beyond passage 3 (Liao et al, 2020). Furthermore, there is no significant difference in collagen gel contraction and response to inflammation in equine tenocytes used between passages 4 and 11 (McClellan, Evans, et al, 2019).…”

Section: Discussionmentioning

confidence: 89%

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Cyclical strain improves artificial equine tendon constructs in vitro

Atkinson

Evans

Guest³

et al. 2020

J Tissue Eng Regen Med

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Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo environment, in this study, we have utilised a novel and affordable, custom-made bioreactor to apply a cyclical strain to tendon-like constructs generated in three-dimensional (3D) culture by equine tenocytes. Dynamic shear analysis (DSA), dynamic scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy were used to determine the mechanical and chemical properties of the resulting tendon-like constructs. Our results demonstrate that equine tenocytes exposed to a 10% cyclical strain have an increased amount of collagen gel contraction after 7 and 8 days of culture compared with cells cultured in 3D in the absence of external strain.While all the tendon-like constructs have a very similar chemical composition to native tendon, the application of strain improves their mechanical properties. We envisage that these results will contribute towards the development of improved biomimetic artificial tendon models for the development of novel strategies for equine regenerative therapies. K E Y W O R D S3D culture, dynamic shear analysis, equine, mechanical properties, tendon cells

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 89%

Cyclical strain improves artificial equine tendon constructs in vitro

Atkinson

Evans

Guest³

et al. 2020

J Tissue Eng Regen Med

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“…Previous reports studying the charge reversal of pDMAEA into pAA have identified a pH-independent self-catalyzed hydrolysis mechanism. However, most of these studies utilize block copolymers, homopolymers, ,,, or statistical copolymers containing co-monomers with complementary functionalities such that there is limited spacing between basic residues within the chain . Given this, we were interested in identifying if the relative lipophilicity of the copolymers impacted the rate of pDMAEA hydrolysis and its pH-independent mechanism.…”

Section: Results and Discussionmentioning

confidence: 99%

“…Notable examples include enhanced mRNA delivery utilizing an oligo(carbonate-β–α-amino ester), which rapidly degrades into a neutral diketopiperazine through an ester–amide cyclization reaction, releasing the mRNA . Another key example is the self-catalyzed hydrolysis of poly(dimethylaminoethyl) acrylate (pDMAEA), which degrades from a polycation into polyanionic poly(acrylic acid) (pAA) in the presence of water releasing dimethylaminoethanol (DMAE). − From a gene delivery perspective, this delivery system will efficiently condense nucleic acids in their positively charged state and then hydrolyze, charge invert, repell, and release the genetic cargo using a single material. Systems based on the charge-reversing nature of pDMAEA have already been utilized to deliver siRNA ,, and pDNA; however, no studies have yet examined its potential for mRNA delivery.…”

Section: Introductionmentioning

confidence: 99%

“…23−31 From a gene delivery perspective, this delivery system will efficiently condense nucleic acids in their positively charged state and then hydrolyze, charge invert, repell, and release the genetic cargo using a single material. Systems based on the chargereversing nature of pDMAEA have already been utilized to deliver siRNA 29,31,32 and pDNA; 25 however, no studies have yet examined its potential for mRNA delivery.…”

Section: ■ Introductionmentioning

confidence: 99%

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The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA

et al. 2020

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RNA technology has the potential to revolutionize vaccination. However, the lack of clear structure–property relationships in relevant biological models mean there is no clear consensus on the chemical motifs necessary to improve RNA delivery. In this work, we describe the synthesis of a series of copolymers based on the self-hydrolyzing charge-reversible polycation poly(dimethylaminoethyl acrylate) (pDMAEA), varying the lipophilicity of the additional co-monomers. All copolymers formed stable polyplexes, showing efficient complexation with model nucleic acids from nitrogen/phosphate (N/P) ratios of N/P = 5, with more hydrophobic complexes exhibiting slower charge reversal and disassembly compared to hydrophilic analogues. The more hydrophobic copolymers outperformed hydrophilic versions, homopolymer controls and the reference standard polymer (polyethylenimine), in transfection assays on 2D cell monolayers, albeit with significantly higher toxicities. Similarly, hydrophobic derivatives displayed up to a 4-fold higher efficacy in terms of the numbers of cells expressing green fluorescent protein (GFP+) cells in ex vivo human skin (10%) compared to free RNA (2%), attributed to transfection enrichment in epithelial cells. In contrast, in a mouse model, we observed the reverse trend in terms of RNA transfection, with no observable protein production in more hydrophobic analogues, whereas hydrophilic copolymers induced the highest transfection in vivo. Overall, our results suggest an important relationship between the vector lipophilicity and RNA transfection in vaccine settings, with polymer biocompatibility potentially a key parameter in effective in vivo protein production.

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Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell–derived factors

et al. 2022

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Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1β, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells’ ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1β using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1β stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB.

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

Cited by 14 publications

References 48 publications

Cyclical strain improves artificial equine tendon constructs in vitro

Cyclical strain improves artificial equine tendon constructs in vitro

The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA

Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell–derived factors

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