Extracellular Vesicles Derived from Plasmodium -infected and Non-infected Red Blood Cells as Targeted Drug Delivery Vehicles

1 The mainAmong several factors behind drug resistance evolution in malaria isare the difficulty 2 challengeto of administering appropriate overall doses that are not toxic for the patient but that, 3 locally, are sufficiently high to rapidly kill the parasites. Thus, a crucial antimalarial strategy is 4 the development of drug delivery systems capable of targeting antimalarial compounds to 5 Plasmodium with high specificity. as a result of the complex life cycle of Plasmodium , the short 6 half-lives of most antimalarial drugs, and the blood fluidic conditions affecting the interaction of 7 molecules with target cells. Extracellular vesicles (EVs) have been widely explored as delivery 8 vectors of nucleic acids and proteins. In the present study, extracellular vesicles (EVs) were 9 have been evaluated as a drug delivery system for the treatment of malaria. EVs derived from 10 naive red blood cells (RBCs) and from Plasmodium falciparum -infected RBCs (pRBCs) were 11 isolated by ultrafiltration followed by size exclusion chromatography. Lipidomic characterization 12 showed that there weare no significant qualitative differences between the lipidomic profiles of 13 pRBC-derived EVs (pRBC-EVs) and RBC-derived EVs (RBC-EVs). Both EVs were taken up by 14 RBCs and pRBCs, although pRBC-EVs were more efficiently internalized than RBC-EVs, which 15 suggested their potential use as drug delivery vehicles for these cells. When loaded into pRBC- 16 EVs, the antimalarial drugs atovaquone, lumefantrine and tafenoquine inhibited in vitro P. 17 falciparum growth more efficiently than their free drug counterparts, indicating that pRBC-EVs 18 can potentially increase the efficacy of several small hydrophobic drugs used for the treatment 19 of malaria.


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In this scenario, the development of more efficient treatments for malaria is urgent.

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The unbound label was finally removed by SEC as described above. Fractions 8 and 9 were 219 concentrated in Amicon Ultra-4 cCentrifugal fFilters (100 kDa cut off).

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The acquisition was configured to stop after recording 50,000 events within the RBC population.

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Growth inhibition was determined by comparison of the parasite growth between treated and 258 non-treated cultures.

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A drug-free EV concentration range from 3 to 200 µg protein/mL was also evaluated for 260 potential in vitro antimalarial activity. Cell-free RPMI-AG was processed identically as pRBC and

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RBC cultures for EV isolation, and the resulting EV-free fractions were used as control.

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Differences between two data groups were analyzed using t-tests. Comparisons between more 265 than two groups were made using one-way ANOVA with a Tukey post hoc test. Statistical

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Lipidomic analysis did not reveal significant qualitative differences between the lipid composition 313 of EVs derived from RBCs and pRBCs (Fig. 32)

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Notably, the mechanism of EV interaction with cells, which depends in part on the recipient cell

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To investigate EV targeting to RBCs and pRBCs, rRhodamine-labeled EVs were

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At the lower drug doses tested, both tafenoquine and atovaquone encapsulated in the 440 lower pRBC-EV concentration assayed (6.10 µg protein/mL) were more efficient than equal free 441 drug amounts in decreasing parasite growth in an in vitro P. falciparum culture (Fig. 5A4A-C).

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Nevertheless, a dose effect was not observed upon coincubation of lower drug doses with 443 higher EV numbers, which suggests a likely saturation of pRBC-EV drug intake. At higher 444 concentrations of EVs, however, these could still accommodate more drug in higher drug 445 concentration assays. Lumefantrine offered the worst results at all drug and EV concentrations 446 tested (Fig. 5B4B). It is conceivable that its high lipophilicity (log P = 8.7) confers it a strong 447 affinity for lipid bilayers, which might stabilize its permanence in the pRBC plasma membrane,

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where its antimalarial activity would be minor.
It is also worth noting that tafenoquine is more potent inhibiting parasite growth at the 450 lower drug dose encapsulated into pRBC-EVs than at the higher drug dose (Fig. 5C4C).

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Although there is no obvious explanation for this result, one can speculate a destabilizing effect

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The same amounts of drug-free pRBC-EVs as those used in drug-containing samples 458 had no effect on parasite growth (Fig. 5A4A-D). Drug-free RBC-EVs and pRBC-EVs started

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The current consensus is that a combination of different treatment strategies is required 467 to eradicate malaria. In the drug therapy approach, it is important to have a drug delivery 468 system that will efficiently encapsulate the drug and deliver it to the intended cell type. This will 469 enable the use of overall reduced drug doses, with decreased adverse effects, but at the same 470 time it will provide the capacity to deliver sufficiently high local amounts of drug to rapidly kill the