(T0930-06-35) Feasibility and Application of Anti-Malarial Endoquine-Like Quinolone (ELQ) on Mosquito Bed Nettings and HDPE Films for Anti-Malarial Properties using Solution Coatings and Hot Melt Extrusion Methods

Purpose: Malaria transmission occurs through the bite of Plasmodium-infected mosquitoes of the genus Anopheles, and in much of sub-Saharan Africa, transmission of the deadliest Plasmodium falciparum parasites is mediated by the sibling mosquito species Anopheles gambiae and An. coluzzii.  Insecticide-based mosquito control represents the cornerstone of malaria prevention and elimination efforts,ⁱ however, mass distribution of long-lasting insecticide impregnated mosquito bed nets (LLINs) has driven widespread resistance to pyrethroid insecticides in Anopheles populations.ⁱⁱ Therefore, the development of new tools to prevent malaria transmission are needed.  It was recently demonstrated by our collaborators that antimalarials applied to solid surfaces can kill the mosquito stages of P. falciparum parasites when Anopheles females absorb these compounds via their legs.ⁱⁱⁱ From a preliminary in vivo efficacy screen, they further identified structurally similar endoquine-like quinolone (ELQ) compounds to efficiently target different sites (Qo and Qi, which importantly do not induce cross-resistance) on the P. falciparum cytochrome bc1 complex.^iv The Work described here is primarily related to 1) the task of infusing ELQ onto polyester netting coupons and 2) the fabrication of blended ELQ-polyethylene films.

Methods: To achieve the ultimate objective of obtaining efficacious, full sized bed nets infused with ELQ compounds two methods were envisioned.  Method 1) involved solution coating of ELQ compounds onto the surface of polyester netting and Method 2) involved the extrusion blending of molten ELQ into polyethylene. Method 1: Polyester (Apex Mills, RC47) coupons were prepared by cutting to a 25cm x 25cm size, washing in a 0.2 wt% aqueous Alconox solution, rinsed with DI-water, and then dried. In an effort to preserve supply of ELQ-453, Sudan Blue II (SBII) was employed as a simulant due to its similar melting point and solubility (See Figure 1A). Netting coupons were soaked in jars containing coating solutions of SBII with and without polycaprolactone (PCL) in DCM, then removed to air dry. Coatings with ELQ/PCL were performed in a similar fashion. Coated coupons were prepared with and without annealing to observe and compare its effects on SBII or ELQ release. To meet the 2013 “Guidelines for laboratory and field-testing of long-lasting insecticidal nets” by the World health Organization (WHO), coatings need to withstand 10 washing cycles using a soap solution. Thus, coated coupons were prepared with and without this extensive washing with a 0.2% Alconox solution to later observe and compare the washing effects. After annealing and washing trials, coupons were extracted with ethanol for 2min and for 24hr to remove available SBII or ELQ for quantification, then using UV-vis analysis for content determination. Method 2: High density polyethylene (HDPE) pellets were cryogenically milled to 1mm. Blends of 0.1%, 1%, and 3% ELQ-453 in milled HDPE were prepared, then extruded at 150°C. Resulting filament formulations were pelletized and heat-pressed into thin films (10cm x 10cm) and ethanol extraction testing.  Alconox wash cycles were not performed on HDPE thin films.

Results: Method 1: The SBII-coated coupons provided excellent formulation and process information. The data shows that a fair amount of SBII remained after the 10x Alconox wash. Additionally, thermally annealed samples exhibited a reduced amount of total measured SBII extracted, thus annealing would not be beneficial for ELQ-coated coupons. The addition of PCL improved the total amount of available SBII, but did not significantly change in the Alconox wash performance, while excess (2x) PCL did not significantly increase the amount of available SBII. The data garnered from the SBII trials led to proper targeting of ELQ amounts onto coupons, even after the extensive Alconox washing steps. The ELQ extraction data also shows that we can vary the ELQ content fairly easily. Here we achieved up to ~16mg of ELQ-453 per coupon (25cm x25cm) which equates to ~256mg per square meter of cut fabric netting, surpassing our preliminary target of 100mg per square meter of cut fabric netting. br>Method 2: Uniform ELQ-HDPE films were obtained with occasional entrapped air bubbles in portions of the films. UV-vis analysis of 24 hr ethanol extraction showed active ELQ on the surface for each of the 3 different drug loadings. The highest drug loaded film sample, 3 wt% ELQ-HDPE, exhibited an extraction value of 4.47 mg/film (10cm x 10cm), which would equate to a surface coverage of 221.5 mg/m², or 447mg per square meter of cut fabric netting.

Conclusion: Polyester mosquito netting was successfully coated with ELQ-453, surpassing our preliminary efficacious target of 100mg per square meter of cut fabric netting, and satisfying the wash guidelines of the WHO. SBII proved to be reasonable simulant in narrowing our coating processes to include PCL in coating formulations and to avoid annealing steps. Thin films of ELQ-loaded HDPE were also successfully prepared with ample extractable ELQ-453, also surpassing the efficacious target, albeit without the Alconox washing. Both extrusion and solution-coating methods show promise for industrial manufacturing of ELQ-coated mosquito bed nettings.

References: i. Bhatt S, Weiss DJ, Cameron E, et al. “The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015”. Nature. 2015;526(7572):207-210E.
ii. World malaria report 2023. Geneva: World Health Organization. Published online 2023.
iii. Burrows J, Fidock DA, Miller RS, Rees S. “Blocking Plasmodium Development in Mosquitoes: A Powerful New Approach for Expanding Malaria Control Efforts”. Am J Trop Med Hyg. 2019;101(4):734-735.

iv. Paton DG, Childs LM, Itoe MA, Holmdahl IE, Buckee CO, Catteruccia F. “Exposing Anopheles mosquitoes to antimalarials blocks Plasmodium parasite transmission”. Nature. 2019;567(7747):239-243.

Acknowledgements: A special thank you to our partners: Harvard T.H. Chan School of Public Health and The Oregon Health Science University/Portland VA Research Foundation. Funding provided by: Open Philanthropy and The National Institutes of Health (R01)


Figure 1. Chemical structures of A) Sudan Blue II (SBII) and B) ELQ-453.


Figure 2. Amounts of SBII from Alconox washes and ethanol extraction.


Figure 3. Amounts of ELQ-453 from Alconox washes and ethanol extraction.