Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_896_Библиотеки_им_академика_М_И_Перельмана

To optimize EV functionality and therapeutic impact, vari­ous approaches have been proposed, aiming to manipulate their cargo or their surface protein expression. These engi­neering methods can be either focused on directly modifying the EVs or indirectly altering the EV through manipulation of the parental cell [51].

Messenger ribonucleic acid (mRNA) functions as the tran­sient intermediator between deoxyribonucleic acid (DNA) and proteins. These single-stranded RNAs transcribed from the protein-coding regions in DNA can be further translated to produce functional protein molecules. By the develop­ment of in vitro-transcribed (IVT) mRNA that was demon­strated in the late 1990s [52], the mRNA-based therapeutics received considerable attention and numerous researches have been investigated to optimize IVT mRNA transfection efciency, instability, immunogenicity, and delivery [53].

One of the rst experiments using liposomes to deliver exogenous mRNA into mouse lymphocytes dates back to 1978 [61]. The successful translation of mRNA encoding rabbit globin introduced by liposome opened the door to advancing in ionizable lipid and RNA entrapment techniques [55, 58, 62]. Over decades, a variety of materials, mainly lipids-based, have been formulated for mRNA delivery, such as lipids, polymer derivatives, lipid nanoparticles (LNPs), and lipid-like materials. Above all, LNPs have been exten­sively studied. As a well-established delivery vehicle, LNPs rst received US Food and Drug Administration (FDA) approval in 2018 for delivering small molecular: siRNA [63]. By 2020, two authorized mRNA-based COVID-19 vac­cines validated the efciency of lipid nanoparticles in deliv­ering base-modied antigen mRNA in humans. They are BNT162b2 from Pzer-BioNTech and mRNA-1273 from Moderna [64].

During the SARS-CoV-2 pandemic, modied mRNA thera­peutic applications in viral infection and vaccination gained global attention. The investigation of mRNA-based thera­peutics on how to treat DFU is also progressing. Many researches have focused on identifying and targeting the genes that are associated with diabetic/chronic wound healing.

Small interfering RNAs (siRNA)-based drug/treatment is one other major class in RNA therapies that can manipulate gene/protein expression. The main biochemical mechanism of action of siRNA-based therapeutics currently under research, clinical trials, or FDA approval is to mediate gene silencing [72]. Unlike mRNA (~340–2300kDa), siRNAs are smaller in size (~ 13kDa) with a double-stranded structure. To sufciently suppress protein translation (gene knock­down), siRNA interferes with the mature mRNA; thus, the

Enhancing the stability of hypoxia-inducible factor-α (HIF-1α) via the siRNA mechanism to regulate cell behavior and promote tissue formation has been studied for over 15years [102, 103]. This critical transcription factor is pri­marily involved in cellular responses, including cell viability, motility, and adhesion, to lower O2 levels (Hypoxia) [104]. Oxygen is required for the normal function of most cells; however, during the course of wound healing, hypoxia can be benecial for the production of growth factors and cyto­kines that promote angiogenesis and tissue repair [105]. Studies have demonstrated that mice with constitutive expression of the HIF-1α protein developed 66% more blood vessels compared to mice without HIF-1α expression [106]. The overexpression of HIF-1α upregulated VEGF and FGF2 protein expression, and vascular density. Hyperglycemia has been shown to negatively impact the hypoxic response, lead­ing to less stabilization of HIF-1α in diabetic wound environ­ments when compared to non-diabetic wounds. Berra etal. reported that HIF prolyl-hydroxylases domain-2 (PHD2) contributes to hydroxylate proline residues on HIF-1α in normoxia. Silencing PHD2 via PHD2 siRNA was found to sufciently improve the stability of HIF-1α in normoxia [103]. Thus, targeting expression of HIF-1α via siRNA­based therapy would be benecial for promoting angiogen­esis DFU healing.

Various delivery systems for PHD2 siRNA have been validated in different animal models. Diabetic db/db mice wounds treated by agarose matrix loaded PHD2 siRNA topi­cally upregulated the protein levels of HIF-1α, mRNA levels of VEGF and FGF-2, and vascular density compared to the control group which was treated with nonsense siRNA [105]. To promote siRNA delivery efciency, Nelson etal. devel­oped biodegradable scaffolds (polyester triol prepolymers) loaded with siRNA-nanoparticles (di-block copolymer poly [DMAEMA71-b-(BMA103-co-PAA68-co-DMAEMA57)] self-assembled micellar) to achieve a sustained release of siRNA and facilitate exosome escape. The scaffold-loaded PHD2 siRNA-nanoparticle-treated balb/c mice wounds demonstrated an increased number of vessels within scaf­folds [107]. A similar PHD2 siRNA delivery method has

Matrix metalloproteinases (MMPs) are a family (24 different MMPs in human) of zinc-dependent endopeptidases that are overexpressed in many diseases, leading to physiological and pathological disorders [109]. MMPs play critical roles in the pathology of diabetic foot ulcers, the most common forms of chronic wounds [110]. The persistence of elevated levels of MMP-9 was detected in prolonged chronic cutane­ous wounds where healing was signicantly impaired [111]. MMP-9 was found to be detrimental to tissue remodeling and regeneration in the healing process due to its capability of degrading various kinds of extracellular matrix. The study of diabetic MMP-9 knockout mice provided further conr­mation of the detrimental impact of MMP-9 on diabetic wound pathology, as these mice were observed to have impaired wound healing [110]. Thus, a siRNA-based thera­peutic approach of targeting MMP-9 for the treatment of DFU has been increasingly investigated.

One of the major challenges for gene therapy strategies is to build an efcient and safe delivery platform. Due to the anionic phosphate groups in the MMP-9-siRNA, cationic polymers are often selected as the carriers for this siRNA. Compared to naked MMP-9 siRNA, the β-cyclodextrin (β-CD) core and poly (amidoamine) Dendron arms (β-CD-[D3]7)/MMP-9-siRNA complexes promoted wound healing in diabetic rats on Day 7 [112]. Repeated administration of (β-CD-[D3]7)/MMP-9-siRNA, however, may result in the accumulation of siMMP-9 in the liver, which could cause thrombocytopenia or peripheral neuropa­thy. A controlled, sustained, and localized delivery system for RNAi therapy is needed to maximize the MMP-9 siRNA therapeutic effect in diabetic wound healing. Many research labs have focused on using cationic polysaccharide deriva­tives, including chitosan, dextran, pectin, and pullulan, or a hybrid hydrogel dressing to achieve controlled MMP-9 siRNA release [113–118]. Using electrostatic assembly, a layer-by-layer (layer of siRNA lm and layer of chitosan) self-assembled hydrogel incorporated with siRNA was able to achieve localized delivery into wound beds with sustained release. This approach resulted in a signicant reduction of MMP-9 expression through sufcient knockdown [113]. Unlike linear cationic polymers, hyperbranched cationic polysaccharide derivatives (HCP) have been found to be effective as nonviral gene delivery vectors due to their com­pact and globular structures. Studies have demonstrated that these vectors exhibit higher transfection efciency while causing minimal cytotoxicity [119]. The ethylenediamine (EDA)—or diethylenediamine (DETA)-modied glycogen can form complexes with siMMP-9 with a diameter of 100-

There are other types of emerging siRNA that have demon­strated excellent results in regulating gene expression in impaired diabetic animal wound models, including Keap1 and GM3S siRNA.While these siRNAs of interest have not been heavily studied, the remarkable results they have obtained are noteworthy.

imbalanced ROS production may result in dysregulated reduction-oxidation (redox) state in the wounds. Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway plays a crucial role in regulating antioxidants and maintain­ing redox homeostasis by reducing the oxidative stress [120]. Upregulation of Kelch-like ECH-associated protein 1 (Keap1) is observed in diabetes, which functions as a repres­sor of Nrf2 and leads to a high level of ROS accumulation. Topical siKeap1 therapy can effectively restore Nrf2 signal­ing in diabetic mouse models, leading to a reduction in ROS accumulation, and higher Nrf2 protein expression in the wound area. The wound healing outcomes in db/db mice were improved with increased granulation tissue, newly gen­erated vessel counts, and reduced epithelial gap [120]. The improved siKeap1 delivery efcacy was demonstrated using lipoproteoplex nanoparticles composed of cationic lipid nanoparticles and coiled-coil protein, or milk-derived exo­somes as carriers [121, 122]. Both topical delivery systems successfully restored Nrf2 antioxidant function, promoted diabetic tissue regeneration, and enhanced collagen forma­tion in db/db mice and STZ-induced diabetic mice models, respectively [121, 122].

The siRNA-based treatment has exhibited great potential in treating diabetic wounds, as it has the capability of target­ing/silencing specic undesired mRNA expression. However, there are still limitations and challenges, such as efcient gene selection from databases, target delivery, endosomal escape, and prolonged RNA stability. The future approach for RNA-based therapeutic is a combination of appropriate gene selection and their nano carriers (LNP), with the ulti­mate objective of maximizing the efcacy of RNA delivery. Advanced RNA-based therapies have shown promising results in treating diabetic wound healing and may become a standard treatment option in the future. However, further research and clinical trials are necessary to fully understand their safety and efcacy before they can be widely adopted in clinical settings.