# Precise Delivery of Nitric Oxide Controlled by Bioorthogonal Endocellulase Ameliorates Hindlimb Ischemia

**Authors:** Yating Zhang, Meng Qian, Ruowen Chu, Shengyu Li, Jiawen Yuan, Jian Zhao, Zhixin Xu, Mengmeng Xing, Huan Jiang, Bo He, Chao Chai, Guangyu Yang, Sen Yang, Yongzhen Wei, Qiang Zhao

PMC · DOI: 10.3390/bioengineering13020128 · Bioengineering · 2026-01-23

## TL;DR

A new method for delivering nitric oxide (NO) precisely to treat peripheral artery disease in a way that avoids side effects and improves blood flow.

## Contribution

A bioorthogonal enzyme-prodrug system for controlled and localized nitric oxide delivery in treating hindlimb ischemia.

## Key findings

- The NO delivery system enhanced neovascularization and tissue perfusion in a porcine model.
- Targeted NO delivery reduced inflammation and muscle fibrosis in ischemic tissue.
- The system demonstrated optimal translational potential for treating peripheral artery disease.

## Abstract

Peripheral artery disease (PAD) remains a great threat to the health of older people globally. Nitric oxide (NO), as an important signaling molecule, is integral to processes such as angiogenesis, inflammation, and tissue regeneration, making it a potential candidate for PAD treatment. Nevertheless, NO—based therapies are frequently limited in clinical utility, primarily due to the lack of effective strategies for fine-tuning the release of exogenous NO. In this study, we developed an enzyme—prodrug pair based on endocellulase (Cel5A-h38), which ensured complete bioorthogonality, thus avoiding interference with endogenous enzymes and eliciting an inflammatory response. This delivery system enables localized and controlled NO release, thus preventing side effects induced by systemic exposure. The therapeutic efficacy of the NO delivery system was systematically evaluated in a porcine model of hindlimb ischemia. Our results confirmed the benefits of targeted NO delivery in hindlimb ischemia, which include enhanced neovascularization and tissue perfusion, reduced inflammation, and alleviated muscle fibrosis, demonstrating its optimal translational potential.

## Linked entities

- **Chemicals:** nitric oxide (PubChem CID 145068), NO (PubChem CID 24822)

## Full-text entities

- **Genes:** NOS3 (nitric oxide synthase 3) [NCBI Gene 4846] {aka EC-NOS, ECNOS, MYMY8, NOSIII, cNOS, eNOS}, Pecam1 (platelet/endothelial cell adhesion molecule 1) [NCBI Gene 18613] {aka Cd31, PECAM-1, Pecam}, VEGFA (vascular endothelial growth factor A) [NCBI Gene 397157] {aka VEGF}, TNF (tumor necrosis factor) [NCBI Gene 397086] {aka TNFSF2, TNFa}, Glb1 (galactosidase, beta 1) [NCBI Gene 12091] {aka Bge, Bgl, Bgl-e, Bgl-s, Bgl-t, Bgs}, Nos2 (nitric oxide synthase 2, inducible) [NCBI Gene 18126] {aka MAC-NOS, NOS-II, Nos-2, Nos2a, i-NOS, iNOS}, Igh-V7183 (immunoglobulin heavy chain (V7183 family)) [NCBI Gene 16059] {aka B9-scFv, IgG, IgH, IgVH1(VSG), VH7183, VI24H}, Tnf (tumor necrosis factor) [NCBI Gene 21926] {aka DIF, TNF-a, TNF-alpha, TNFSF2, TNFalpha, Tnfa}, Il6 (interleukin 6) [NCBI Gene 16193] {aka Il-6}, IL6 (interleukin 6) [NCBI Gene 399500] {aka IL-6}, IL4 (interleukin 4) [NCBI Gene 397225], GAPDH (glyceraldehyde-3-phosphate dehydrogenase) [NCBI Gene 396823] {aka GAPD}, IL10 (Interleukin 10 level) [NCBI Gene 103158318], interleukin 6 [NCBI Gene 100628202], TGFB1 (transforming growth factor beta 1) [NCBI Gene 397078] {aka TGF-BETA-1}, Mrc1 (mannose receptor, C type 1) [NCBI Gene 17533] {aka CD206, MR}, NOS2 (nitric oxide synthase 2) [NCBI Gene 396859] {aka INOS, NOS2a}, PECAM1 (platelet and endothelial cell adhesion molecule 1) [NCBI Gene 396941] {aka CD31, PECAM-1}, Acta2 (actin alpha 2, smooth muscle, aorta) [NCBI Gene 11475] {aka 0610041G09Rik, Actvs, SMAalpha, SMalphaA, a-SMA, alphaSMA}, Vegfa (vascular endothelial growth factor A) [NCBI Gene 22339] {aka L-VEGF, Vegf, Vpf}, ARG1 (arginase 1) [NCBI Gene 397115], NOS3 (nitric oxide synthase 3) [NCBI Gene 397557] {aka EC-NOS, NOS, NOSIII, cNOS, eNOS}
- **Diseases:** functional impairment (MESH:D003072), necrosis (MESH:D009336), PAD (MESH:D058729), ischemic injury (MESH:D017202), infection (MESH:D007239), atherosclerotic (MESH:D050197), arterial occlusion (MESH:D001157), hypertension (MESH:D006973), skeletal muscle myopathy (MESH:D005207), claudication (MESH:D007383), HLI (MESH:D007511), skeletal muscle abnormalities (MESH:D009139), swelling (MESH:D004487), CLTI (MESH:D000089802), ischemic (MESH:D002545), diabetes (MESH:D003920), ischemic muscle (MESH:D019042), rest pain (MESH:D010146), loss (MESH:D016388), Fibrosis (MESH:D005355), platelet aggregation (MESH:D001791), muscle damage (MESH:D009133), Inflammation (MESH:D007249), injury to (MESH:D014947)
- **Chemicals:** ROS (MESH:D017382), argon (MESH:D001128), Cy5 (MESH:C085321), DAPI (MESH:C007293), heparin (MESH:D006493), CD (MESH:D002104), PVDF (MESH:C024865), PBS (MESH:D007854), agarose (MESH:D012685), iodine (MESH:D007455), sucrose (MESH:D013395), adamantane (MESH:D000218), paraformaldehyde (MESH:C003043), citric acid (MESH:D019343), acetone (MESH:D000096), galactoside (MESH:D005697), ceftriaxone sodium (MESH:D002443), Na2SO4 (MESH:C012036), sodium phosphate (MESH:C018279), pyridine (MESH:C023666), H&amp;E (MESH:D006371), ammonium ferric sulfate (MESH:C049467), ethyl acetate (MESH:C007650), AD (-), HA-AD (MESH:D006820), NO (MESH:D009569), kanamycin (MESH:D007612), TEMPO (MESH:C003959), ethanol (MESH:D000431), DAC-S (MESH:D000077209), acetic acid (MESH:D019342), SDS (MESH:D012967), Gal (MESH:C101993), TRIzol (MESH:C411644), isoflurane (MESH:D007530), HBr (MESH:D018054), imidazole (MESH:C029899), water (MESH:D014867), beta-cyclodextrin (MESH:C031215), nitrogen (MESH:D009584), Xylazine hydrochloride (MESH:D014991), FITC (MESH:D016650), xylene (MESH:D014992), cellobiose (MESH:D002475), DHE (MESH:C067883), rhodamine (MESH:D012235), oxygen (MESH:D010100), NTA (MESH:D009571), midazolam (MESH:D008874), NaCl (MESH:D012965), gold (MESH:D006046), methanol (MESH:D000432), iodixanol (MESH:C044834), Paraffin (MESH:D010232)
- **Species:** Homo sapiens (human, species) [taxon 9606], Escherichia coli BL21(DE3) (strain) [taxon 469008], Sus scrofa (pig, species) [taxon 9823], Mus musculus (house mouse, species) [taxon 10090]
- **Mutations:** (CTA) at 28, H363A
- **Cell lines:** /6J — Homo sapiens (Human), Cutaneous melanoma, Cancer cell line (CVCL_W797)

## Full text

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## Figures

6 figures with captions in the complete paper: https://tomesphere.com/paper/PMC12937777/full.md

## References

52 references — full list in the complete paper: https://tomesphere.com/paper/PMC12937777/full.md

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Source: https://tomesphere.com/paper/PMC12937777