(175t) An in-Situ Hydrogen Sulfide Synthesis and Delivery Technique for Treatment and Healing of Chronic Wounds

Relevance: Peripheral artery disease (PAD) and small vessel disease (SVD) are common cardiovascular complications prevalent among smokers and diabetic patients with more than one in every 20 Americans over the age of 50 estimated to suffer from these conditions [1]. The projected impact will affect more than 19 million people in the U.S. alone by 2050. There is substantial preclinical and clinical evidence demonstrating a strong correlation between low circulating hydrogen sulfide (H₂S) bioavailability and cardiovascular problems, particularly in the aging population, contributing to the vicious circle of ischemia, diabetes, heart failure, and/or limb loss [2]. Consequences of reduced endogenous H₂S include diminished angiogenic signaling, poor vasculogenesis, and inadequate tissue regeneration [3] leading to clinical vascular disease and in severe conditions, chronic limb threatening ischemia (CLTI) [4]. H₂S is synthesized endogenously within the vascular endothelial layer by the cysthathionine γ-lyase (CSE) enzyme [5] and has known physiological benefits that include angiogenesis, vasodilation, cytoprotection, and reduction in inflammation [6]. The potential of H₂S as a bioactive therapeutic molecule, through supplements and a variety of exogenous donors, have demonstrated marked improvement in VEGF expression, tissue granulation, vascularization, and accelerated healing [7].

Background on H₂S Delivery Techniques: Most H₂S donors and delivery methods currently under investigation lack the potential for clinical translation due to their pharmacokinetic limitations. The ideal H₂S releasing ‘drug’ should work locally without systemic or side effects, have controlled and sustained and long-duration bioavailability and release, and reasonable shelf-life [8]. Studies on solution based H₂S therapy using microfluidic systems (i.e., MEMS) for delivery of small quantities of pre-mixed H₂S donor solution to tissue have revealed several limitations, including system performance (e.g., electromechanical failure), system size, and chemical stability of the H₂S donor compounds. Similar problems plague in-situ mixing of the solution and/or passive H₂S delivery techniques such as H₂S-eluting dressings [9] or GYY4137 impregnated hydrogels (e.g., GelMA) and fibrous material [10]. While the recently developed light-actuated or photoactivatable (alternatively, photoremovable, photoreleasable, or photocleavable) protecting group (PPG) donors show promise of better control, higher stability, and more targeted delivery, they are either too complex and cumbersome or are not suitable for broad applications in treating ischemic wounds [11, 12].

Innovation and Approach: The patent-protected method described in this paper is a viable alternative to solution- and gel-based precursors (e.g., Na₂S, NaHS, and GYY-4137). The innovative electrolytic method safely synthesizes, delivers, and maintains H₂S within a therapeutic window. The core technology employs a controllable, in-situ, real-time process for conversion of a stable metal sulfide compound to H₂S gas for a ‘true sustained delivery’. In principle, as depicted in Figure 1, it uses the inverse of a process that our group uses to amperometrically sense H₂S at low concentrations of <100 ± 5 ppb [13]. In the sensing process, H₂S converts a thin coating of silver (Ag) nanoparticles deposited on a Nafionâ„¢ membrane working electrode to silver sulfide (Ag₂S), thus releasing electrons to generate a current measurable within 1 nA and detection sensitivity of 10 nA/ppb. Conversely, a potentiometric injection of electrons into the reaction at low voltage (<1 VDC) reduces a thin Ag₂S coating, instantaneously releasing H₂S gas for local and on-demand delivery to tissue:

Ag₂S (solid) + 2 H₂O + 2e^— ⇔ 2 Ag + H₂S (gas) + 2 OH^—. (1)

The dosage can be delivered at a low level continuously (e.g., 1 nmol/hour) or large intermittent doses (e.g., two discrete doses of 100 nmol/day). The total dosage is a defined quantity, equal to the integral or sum of doses provided during the treatment period:

n_g = ∫_t=1-N [n_dot (t)] ∙ dt = γ ∑_k i_k ∙ ∆t_k , (2)

where, n_g is total dose (nmol), n_dot(t) is the controllable H₂S emanation rate (nmol/s), t is time (s), i(t) is the instantaneous current provided (nA), γ is generation/synthesis rate constant (nmol/nA), and N is number of dose segments for a treatment protocol.

Results: Typical dose curves for both linear and planar sources show a linear correlation between the charge transferred and the H₂S released or dosage. The generation rate constants have been found to be <1 µAh/nmol, thus requiring extremely low power and battery usage. Results on multiple H₂S generators show high control over the dosage amount, with relatively low pulse-to-pulse variability. Figure 2 shows the ability to deliver as low as 1 nmol±0.5 nmol/dose. This approach is currently being implemented in a disposable and inexpensive miniature H₂S delivering cartridge technology named Genie:Sâ„¢ with capacities exceeding 2 µmol/cartridge and more than 10 µmol per wound treatment application. The cartridge is designed to integrate into smart dressings for non-invasive, topical, and local delivery of H₂S to the wound site. Preclinical treatment of open wounds in animal models such as Sprague-Dawley rats and Yucatan minipigs at two different institutes is currently in progress, requiring dosages well within the range of the designed Genie:Sâ„¢ technology.

Conclusions: This presentation describes a novel electrolytic approach for the development of a wound treatment technology based on H₂S as a bioactive therapeutic molecule. The ability to do in-situ synthesis and delivery of the elusive H₂S molecule will address the shortcomings in shelf-life stability and precision control in dosage/delivery that plague the current passively delivered H₂S donors used in most on-going preclinical and clinical studies. As a result, the technique described herein is expected to meet two treatment requirements that have become barriers to the clinical translational of H₂S wound therapy: i.e., safety and efficacy. Our poster presentation will detail the results of the performance studies on both linear as well as planar H₂S sources, in addition to the results of the in-vivo physiological studies with rats and the positive impact of in-situ delivery of H₂S on wound healing acceleration.

References:

[1] CDC, "Peripheral Arterial Disease (PAD) Fact Sheet," 27 September 2021. [Online]. Available: http://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_pad.htm. [Accessed 29 November 2021].

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[7] G. Wang, W. Li, Q. Chen, Y. Jiang, X. Lu and X. Zhao, "Hydrogen sulfide accelerates wound healing in diabetic rats," Int J Clin Exp Pathol, vol. 8, no. 5, pp. 5097-5104, 2015.

[8] A. R. Jensen, N. A. Drucker, S. Khaneki, M. J. Ferkowicz, M. C. Yoder, E. R. DeLeon, K. R. Olson and T. A. Markel, "Hydrogen Sulfide: A Potential Novel Therapy for the Treatment of Ischemia," Shock, vol. 48, no. 5, pp. 511-524, 2017.

[9] R. Raggio, W. Bonani, E. Callone, S. Dirè , L. Gambari, F. Grassi and A. Motta, "Silk Fibroin Porous Scaffolds Loaded with a Slow-Releasing Hydrogen Sulfide Agent (GYY4137) for Applications of Tissue Engineering," ACS Biomater Sci Eng, vol. 4, no. 8, pp. 2956-2966, 2018.

[10] B. T. Matheson, D. M. Friedrichsen, B. J. Brooks, J. Giacolone, R. M. Clark and R. Shekarriz, HEALSâ„¢: An Active Hydrogen Sulfide Delivery Technique for Accelerated, Effective Wound Healing, Albuquerque, N.M.: Final Report for NIH/NIGMS Phase I SBIR Grant 1R43GM144027-01., 2022.

[11] Y. Ge, F. Rong, W. Li and Y. Wang, "Review Article: On-demand therapeutic delivery of hydrogen sulfide aided by biomolecules," Journal of Controlled Release, vol. 352, pp. 586-599, 2022.

[12] C. M. Levinn, M. M. Cerda and M. D. Pluth, "Activatable Small-Molecule Hydrogen Sulfide Donors," Antioxid Redox Signal, vol. 32, no. 2, pp. 96-109, 2020.

[13] R. Shekarriz and e. al., "Sensor of Transdermal Biomarkers for Blood Perfusion Monitoring," Sensing and Bio-Sensing R., vol. 28, no. 100328, pp. 1-7, 2020.

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