(M1330-01-05) Is a Minimally Invasive Amphotericin B Treatment Feasible? Nanocrystals-Loaded Microneedles for Cutaneous Leishmaniasis

Purpose: Six hundred million people are threatened by leishmaniasis globally, with 50% of new cases occurring in children.¹An alternative to toxic pentavalent antimonial drugs is intravenous liposomal amphotericin B (L-AmB). Despite promising clinical outcomes, the use of L-AmB in low- and middle-income countries where the disease is more prevalent is complex since it often requires inexistent dedicated infrastructure and personnel.² Moreover, patients need to attend multiple hospital appointments, often travelling far distances and losing days of work. Here, we report the development and characterisation of AmB nanocrystals (AmB-NCs) loaded in dissolving microneedle array patches (MAP) as a patient-centric alternative to painful and expensive intravenous L-AmB.

Methods: AmB-NCs were produced by media milling using 0.5% w/v poloxamer 188 as stabiliser.³ Afterwards, the AmB-NCs was obtained by a freeze-drying process of the liquid nanosuspensions and particle size analysed by dynamic lights scattering. The in vitro cell viability study of leishmania promastigotes and raw macrophages were tested in comparisons of AmB and AmB-NCs. The ex vivo viability of intramacrophage amastigotes from murine splenic explants and axenic amastigotes from infected bone marrow of Balb/c mice were investigated in comparisons of AmB and AmB-NCs. The two-layered dissolving MNs were prepared. The moulds for MNs manufacture composed of 16 x 16 pyramidal-cubical shaped needles with the height of 850 μm. The first layer of the MAP formulation contained the mixture of AmB-NCs, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and deionised water. The second layer was composed of PVP and glycerol as support.⁴ The in vitro dissolution of the AmB-NCs was tested and compared with the dissolution kinetics of AmB by using MN tips with the release medium PBS and 1% w/v SDS in a 37 ± 0.5 °C, 40 rpm incubator. The Franz diffusion cell was used for the ex vivo full-thickness porcine skin deposition study. The in vivo plasma pharmacokinetic, lymphatic pharmacokinetic and biodistribution study were investigated in 12-week age female Sprague‒Dawley^® in comparison of intradermal dissolving AmB-NCs MAPs and intravenous AmBisome^®.

Results: The resultant AmB-NCs mean particle size was 92.63 ± 0.26 nm with PDI value of 0.252 ± 0.004 after milling (n =3). Dissolving MAPs loaded with AmB-NCs were successfully produced (Figure 1a). The average height of MN tips was 812 ± 21 μm. AmB-NCs dissolved quicker than AmB in vitro, with 100% cumulative release achieved at 5h (Figure 1b). The AmB-NC MNs group from the drug in-skin deposition study achieved 247.86 ± 25.23 μg drug deposition at 24 h which is higher than the crude AmB MNs as shown in Figure 1d. The novel MAPs led to an enhanced ex vivo deposition when compared to AmB MAPs (Figure 1d). Upon administration to rats, AmB-NCs MAPs led to selective accumulation of AmB in lymph nodes, with a higher AUC than that of L-AmB (Figure 1e&f). This is a promising finding for potential application in cutaneous leishmaniasis. Ex vivo efficacy and biocompatibility studies demonstrated that AmB in solution and AmB-NCs had similar antileishmanial effects (Figure 2), with AmB-NCs showing less toxicity in healthy macrophages. The dose response curves were investigated to obtain the EC50 values in promastigotes, macrophages, intra-macrophagic amastigotes and axenic amastigotes of AmB and AmB-NCs. The emitted infrared fluorescence was plotted against different concentrations of AmB and AmB-NCs. As shown in Figure 2, AmB and AmB-NCs showed similar effect on promastigotes, intra-macrophagic amastigotes and axenic amastigotes. The concentration of AmB-NCs required to produce the same toxic effect in healthy macrophages was higher than AmB, which means AmB NCs has less toxic effect than AmB on macrophages.

Conclusion: This patient-centric alternative could revolutionise the management of leishmaniasis, allowing for at-home, minimally invasive administration of AmB, replacing expensive and difficult-to-deliver L-AmB. Translation of this formulation to patients would allow for healthier populations in countries where disease is more prevalent.

References: 1- Visceral leishmaniasis, DNDi (2024). 2- Stone NR, Bicanic T, Salim R, Hope W. Liposomal amphotericin B: a review of the pharmacokinetics. Clinical Experience and Future Directions. Drugs. 76(4):485-500 (2016). 3- Zhang C. et al. Curcumin nanocrystals-in-nanofibres as a promising platform for the management of periodontal disease. Int J of Pharm. 648, 123585 (2023). 4- Larrañeta E. et al. Microneedle arrays as transdermal and intradermal drug delivery systems: Materials science, manufacture and commercial development. Materials Science and Engineering. 104, 1-32 (2016).

Acknowledgements: This work was partially supported by the Royal Society (grant RGS\R1\231342) and the Engineering and Physical Sciences Research Council (grant EP/Y001486/1).


Figure 1. (a) Optical microscope graphs of bilayer NCs MAP, (b) In vitro dissolution profile, (c) Light microscope image of the skin after 24 h Franz cell study, (d) Ex vivo drug deposition, (e) In vivo lymphatic distribution (Means ± SD, n = 4).


Figure 2. In vitro antileishmanial effect on (a) Leishmania promastigotes, biocompatibility in (b) THP-1 healthy macrophages, Ex vivo antileishmanial efficacy on (c) intra-macrophagic amastigotes and (d) axenic amastigotes (Means ± SD, n = 3), (e) Confocal images of infected macrophages treated with AmB-NCs, Scale bar: 5 μm.