(T1530-05-25) Decorated Drug Nanocrystals as Highly-Loaded Trojan Horses for Nose-to-Brain Delivery in Cerebral Malaria

Purpose: Malaria is one of the most threatening parasitic diseases, killing 608,000 people only in 2022.¹ Cerebral malaria (CM) is a severe neurological complication of the infection with Plasmodium falciparum malaria where the redox environment is created by highly concentrated glutathione in brain parasite reservoirs.¹ The World Health Organization (WHO) recommends a combination of artemether (ART) and lumefantrine (LUM) as the gold standard treatment for malaria, including CM.¹ Crucially, this drug combination is only available in oral tablets which lead to erratic bioavailability and limited concentration of drugs reaching the central nervous system (CNS).² Nose-to-Brain (N2B) drug delivery is a potential strategy to enable drugs to bypass the blood-brain barrier, which can effectively increase drug accumulation in the CNS.² As an outstanding subfamily of nanoparticles, nanocrystals (NCs) work as highly loaded drug vectors, opposite to other conventional nanoparticles, only loading 5-15% of drug.³ This work focuses on the surface modification of ART or LUM NCs via coating chitosan (CS) and conducting a two-step synthesis reaction with N-acetyl cysteine (NAC) and PEG-thiol in order to obtain mucopenetrative drug NCs for N2B administration.

Methods: ART or LUM NCs were fabricated by a media milling method previously reported by our group⁴ and underwent purification using dialysis membranes for 3 days.² Then the purified drug NCs were incubated with 0.5% w/v CS at room temperature overnight. Subsequently, NAC and PEG-thiol were added to this system, where the CS-NAC-PEG drug NCs were formed as illustrated in Fig. 1. Particle properties of drug NCs were characterised in each step using a Zetasizer, including particle size and zeta potential. Changes on the NC surface were monitored by Fourier transformed infrared spectroscopy (FTIR), proton nuclear magnetic resonance (¹H NMR), carbon-13 nuclear magnetic resonance (¹³C NMR), and scanning electron microscopy (SEM) as well.

Results: The particle size of both drug NCs increased significantly (p < 0.05) after each reaction step. In terms of ART NCs, particle size, PDI, and zeta potential reached 281.70 ±1.27< nm, 0.39 ± 0.06, and -5.10 ± 1.00 mV respectively, as illustrated in Fig. 2a. As for LUM NCs, particle properties changed to 192.45 ± 0.35> nm, 0.28 ± 0.01, and -2.57 ± 0.24 mV respectively, as demonstrated in Fig. 2b. Further characterisation of FTIR spectra revealed that the absorption peaks of CS-NAC ART NCs at 1651.2 cm⁻¹ (amide I band), 1493.6 cm⁻¹ (amide II band), and 1286.8 cm⁻¹ (amide III band), and the absorption peaks of CS-NAC LUM NCs at 1647.4 cm⁻¹ (amide I band), 1486.4 cm⁻¹ (amide II band), and 1291.1 cm⁻¹ (amide III band) were stronger than those of CS, which can be attributed to the additional amide group formed from the reactions between NAC and CS, as shown in Fig. 2c & d. Afterward, ¹H NMR (Fig. 2e & f) and ¹³C NMR (Fig. 2g & h) further confirmed that PEG-thiol connected to CS-NAC drug NCs and disulfide bonds formed successfully. Moreover, SEM micrographs of both drug NCs (Fig. 2i & j) displayed spherical particle shapes with a smooth surface, dense and uniform arrangement, without obvious aggregation.

Conclusion: This study reveals that CS-NAC-PEG drug NCs with desirable particle properties were successfully obtained. These NCs have the potential to work as highly loaded Trojan horses for direct N2B drug delivery in CM. Future work will include ex vivo and in vivo studies in malaria models to evaluate the treatment efficacy.

References: 1. World malaria report 2023. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023.
2. McGuckin, M. B. et al. Nanocrystals as a master key to deliver hydrophobic drugs via multiple administration routes. Journal of Controlled Release 345, 334–353 (2022).
3. Agrawal, M. et al. Recent strategies and advances in the fabrication of nano lipid carriers and their application towards brain targeting. J Control Release 321, 372–415 (2020).
4. Fuster, M. G., Wang, J., Fandiño, O., Víllora, G. & Paredes, A. J. Folic Acid-Decorated Nanocrystals as Highly Loaded Trojan Horses to Target Cancer Cells. Mol Pharm (2024).

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).


Fig. 1. Schematic representation of NCs manufacture and surface modification process of ART or LUM NCs.


Fig. 2. Comparison of particle size, PDI, and zeta potential from the ART NCs (A) and LUM NCs (B) following coating with chitosan, NAC, and PEG-thiol (means + SD, n=3). FTIR spectra (C & D), 1H NMR spectra (E & F), 13C NMR spectra (G & H), and SEM micrographs of the formulated ART NCs and LUM NCs.