(653a) Development of Spray-Dried Carriers for in-Situ Production of Volatile Phytochemicals

The emergence of MDR bacterial strains and the lack of new antibiotics discovered in the past 30 years, known as the discovery void, are major risks to modern society. Less than 100 years after the breakthrough discovery of penicillin, we are facing the grim scenario that even common superficial injuries or routine surgical procedures may become dangerous and life-threatening again. Many strategies have been developed, but most fail due to the continual adaptation of bacteria. Therefore, an alternative approach and out-of-the-box solutions are needed. For instance, new antibiotics were discovered with the assistance of AI machine learning [1]. Nevertheless, due to practical and financial difficulties associated with developing new antibiotics, most pharmaceutical companies already shifted their attention to cost-effective and more lucrative products. On the other hand, the development of bacterial resistance against potent but highly reactive and unstable thiosulfinates found in Allium plants is more than 1000 times slower compared to commonly used antibiotics [2]. A combination of the short half-life, high reactivity (oxidation of thiol groups), and non-specificity to particular proteins are reasons why most bacteria cannot effectively cope and develop defensive mechanisms.

Garlic (Allium sativum) is a typical example of a compartmentalized natural system, where the highly potent but unstable compound allicin is enzymatically formed from its precursor alliin only when the inner cellular structure is compromised. This concept makes allicin an everlasting bactericide since it is locally formed when needed, leaving no permanent traces of its active form in the environment after application. Alliin, a substance found in intact garlic cells, is not the only sulfoxide involved in the natural self-defence of the genus Allium. Other plants like onion or leek have chemical analogues to alliin, e.g., isoalliin, methiin, propiin, etc. These plant-specific compounds with similar chemical backbones share identical reaction mechanisms leading to the enzymatic production of various thiosulfinates. It has been reported that allicin-like analogues exhibit similar properties. However, their volatility and partition coefficient depend on a particular sulfoxide's molecular weight and character [3]. Moreover, allicin analogues' physicochemical properties, biological activity on various bacterial strains, and cytotoxicity towards different human cell lines are yet to be explored.

Allicin, the substance responsible for garlic's beneficial properties, is one of the most bioactive compounds found in nature. However, allicin's high instability, which is simultaneously responsible for its persistent biological activity, has always been considered a critical weakness preventing further practical and broader use. On the other hand, it has been reported that allicin in very low concentrations can increase the therapeutic effect of FDA-approved antibiotics, causing temporal permeabilization of bacterial membranes [4]. For all these reasons, many research groups around the world are focusing on allicin and its practical applications due to its numerous benefits.

Controlled in-situ synthesis of allicin near a target site may be the feasible way to (re)utilize its full potential. For that, it is essential to physically separate and stabilize allicin's precursors (enzyme and substrate) and introduce a controlling mechanism to harness the beneficial properties of allicin. Driven by the urgency for novel approaches to prevent pandemic scenarios caused by multi-drug resistant bacteria, we propose spray-dried carriers suitable for a topical or pulmonary application, in-situ formation, and controlled release of antibacterial thiosulfinates. This approach can significantly extend the limited palette of currently used antibacterial compounds for thiosulfinates, overcoming the lack of antibiotics in the near future and issues associated with their gradual accumulation in the environment. Moreover, the fact that allicin (in contrast to other antibiotics) is volatile means that the physical presence of the carrier in the vicinity of the target site, e.g., bacterial infection, is preferable but not critical. Therefore, volatile thiosulfinates can reach even regions not accessible for the typical drug carriers commonly utilized in topical and pulmonary delivery.

In this work, we investigated various carrier materials and optimized the process parameters of spray drying, e.g., the inlet temperature of the drying air, use of external cooling, and type of atomizer, with the goal of maintaining a high specific activity of encapsulated alliinase in the final product. Using the innovative non-contact diffusion method, we demonstrated that the spray-dried powders activated by air moisture or manually pre-wetted powder could be applied for in-situ generation of therapeutically effective doses of allicin. The knowledge about the relationship between carrier material and enzyme activity allows for prolonged allicin formation and avoids its high spatiotemporal concentrations or burst release, leading to undesired side effects such as skin irritation or severe chemical burns. These findings may contribute to the development of sustainable nature-inspired products based on the controlled in-situ synthesis of highly reactive antibiotics from their stabilized precursors.

1. Stokes, J.M., et al., A Deep Learning Approach to Antibiotic Discovery. Cell, 2020. 180(4): p. 688-702.e13.
2. Gupta, K. and R. Viswanathan, Combined Action of Streptomycin and Chloramphenicol with Plant Antibiotics against Tubercle Bacilli. Part I: Streptomycin and Chloramphenicol with Cepharanthine. Part II: Streptomycin and Allicin. Antibiotics & Chemotherapy, 1955. 5(1): p. 24-7.
3. Leontiev, R., et al., A comparison of the antibacterial and antifungal activities of thiosulfinate analogues of allicin. Scientific reports, 2018. 8(1): p. 1-19.
4. Bhattacharya, S., et al., Kinetics of bactericidal potency with synergistic combination of allicin and selected antibiotics. Journal of bioscience and bioengineering, 2022. 133(6): p. 567-578.