**4. Discussion**

The results presented above validate a potential new antimalarial strategy where heparin binding to ookinetes will prevent the interaction of this *Plasmodium* stage with the mosquito midgut and consequently its development into an oocyst. It has been suggested that chondroitin sulfate is a ligand for the circumsporozoite- and thrombospondin-related anonymous protein-related protein (CTRP) [9], a key molecule for ookinete mobility and parasite development [21]. Characterizing the sGAG ookinete binding domain and sulfation pattern will be important regarding the development of future antimalarials acting on this stage of the parasite's cycle. To start unraveling the relevance of the sulfation pattern in blocking ookinete progression, hypersulfated heparin has been tested here, and has shown interesting potential since the lowest concentration used resulted in a significantly larger inhibition of ookinete development than the same concentration of native heparin. Chemical modifications of heparin or its binding to nanocarriers are strategies that could contribute to increase activity and midgut residence time in view of the potential development of sGAG-based antimalarials as disruptors of the life cycle of *Plasmodium* in the mosquito.

So far, transmission-blocking approaches have focused on the concept of treating humans with vaccines or drugs that will target mosquito stages [22]. A largely unexplored avenue, however, is targeting *Plasmodium* in the insect vector directly [23]. The implementation of antimalarial medicines designed to be delivered directly to mosquitoes might reduce treatment and development costs because the clinical trials otherwise required for therapies to be administered to people could be significantly simplified. Strategies that control malaria using direct action against *Anopheles* are not new but mostly focus on eliminating the vector, either by killing it with pesticides [24] or through the release of sterile males [25]. The administration of drugs to mosquitoes during their blood feed is being used to deliver ivermectin, an endectocide that, at concentrations found in human blood after treatment, is toxic to all *Anopheles* species examined [24]. When *P. falciparum*–infected female *Anopheles gambiae* mosquitoes were exposed to surfaces treated with the antimalarial drug atovaquone, the development of the parasite was completely arrested [26]. Although this strategy is unlikely to work for large hydrophilic molecules like heparin, several other approaches are available for direct drug delivery to mosquitoes, some of which have been used to deliver to dipterans lipid-based [27] and chitosan nanoparticles [28].

The failure of heparin in inhibiting parasite development when delivered in a sugar meal prior to infection of the mosquito indicates that heparin must be present in the midgut simultaneously with ookinetes. This poses a significant obstacle regarding the practical implementation of future antimalarial strategies based on the observations reported here. Heparin is normally present in human plasma in values ranging from 1 mg/L to 2.4 mg/L [29], whereas heparin in the mosquito midgut is active at concentrations >100 times higher. The anticoagulant activity of heparin prevents its administration to people in the amounts required to block ookinete development, although sGAG mimetics [9] or modified heparins having low anticoagulant capacity [30] offer promising perspectives. The use of limited heparin amounts in infected patients for transmission-blocking might actually be beneficial given the described pro-coagulant effects of *Plasmodium*-infected red blood cells [31]. However, to be present in the circulation at the moment of a mosquito bite, the blood residence time of these molecules should be extremely long. An alternative approach could be provided by offering heparin to mosquitoes in an artificial diet simulating vertebrate blood. The available technology is capable of manufacturing artificial blood for mosquito feeding from hemoglobin obtained from citrated rabbit blood [32], outdated bovine blood [33], and other blood-free artificial liquid diets [34–36]. These substitutes mimic in the mosquito the physiological effects of a fresh vertebrate blood meal, supporting ovarian and egg maturation and normal development of offspring into functional adults. Such artificial feedings can substitute direct feeding on mammals and often have prolonged shelf life and do not require refrigeration. For their delivery to mosquitoes, a number of artificial blood feeders are currently under study [37]. This approach would require the presence of attractants in the artificial diet to lure mosquitoes that have already taken a human blood meal and thus potentially carrying ookinetes in their midguts.

The economic landscape of malaria calls for new strategies that take into account the costs of bringing a medicine into the market, which due to expensive clinical trials often prevent promising new drugs from a fast entry into the production pipeline. As a possible approach to solving this problem, the administration of heparin to mosquitoes offers two advantages: first, blocking the life cycle of *Plasmodium* in the mosquito vector through direct drug delivery to the insect, can dramatically shorten product development due to the avoidance of large-scale human tests. Second, applying the three Rs of drug development (rescue, repurpose, reposition) to previously discarded compounds is an interesting strategy to return value to potential treatments in decline or on hold. Heparin is a natural polysaccharide that can be abundantly obtained in large amounts from the intestinal mammalian mucosa and which has a widespread medical use. The results presented here can inspire researchers and entrepreneurs, especially those in malaria endemic regions, to pursue the development of an efficient and economically affordable antimalarial strategy. A chain of heparin production could easily start with the usually discarded mucosae of pigs, goats, sheep, or cows that are consumed for food. In addition, potential strategies to deliver heparin to mosquitoes might involve the use of small containers filled with mosquito attractants, which can boost the economy of many developing regions through either the fabrication of such dispensers made of plastic, glass or aluminum, or the recycling of bottles and cans.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/10/8/1136/s1, Figure S1. Gating strategy for Figure 1a; Figure S2. Photomicrograph gallery of different time points after heparin-Cy5 administration in sugar feed; Figure S3. Photomicrograph gallery of different time points after heparin-Cy5 administration in MFA; Figure S4. Gating strategy for the data analysis presented in Figure 4b; Table S1. GFP-positive events (n) and corresponding percentages presented in Figure 4b.

**Author Contributions:** Conceptualization, X.F.-B. and E.L.; methodology, E.L., F.N., H.S., and X.F.-B.; formal analysis, E.L.; investigation, E.L., J.F., J.G., and F.N.; resources, C.R.A.-V., F.N., H.S., and X.F.-B.; writing—original draft preparation, X.F.-B. and E.L.; writing—review and editing, X.F.-B., E.L., H.S., and F.N.; visualization E.L.; supervision, F.N., H.S., and X.F.-B.; project administration, X.F.-B.; funding acquisition, F.N., H.S. and X.F.-B. All authors have read and agreed to the published version of the manuscript.

**Funding:** X.F.-B. received funding support from (i) Spanish Ministry of Science, Innovation and Universities (http: //www.ciencia.gob.es/), grant numbers PCIN-2017-100 and RTI2018-094579-B-I00 (which included FEDER funds), (ii) BIOIBERICA, and (iii) ERA-NET Cofund EURONANOMED (http://euronanomed.net/), grant number 2017-178 (NANOpheles). H.S. and F.N. received funding support from Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, GHTM–UID/04413/2020 (https://ghtm.ihmt.unl.pt/).

**Acknowledgments:** ISGlobal and IBEC are members of the CERCA Programme, *Generalitat de Catalunya*. We acknowledge support from the Spanish Ministry of Science, Innovation and Universities through the *"Centro de Excelencia Severo Ochoa 2019-2023"* Program (CEX2018-000806-S). This research is part of ISGlobal's Program on the Molecular Mechanisms of Malaria, which is partially supported by the *Fundación Ramón Areces*. We are indebted to the Cytometry and Cell Sorting Facility of the *Institut d'Investigacions Biomèdiques August Pi i Sunyer* (IDIBAPS) for technical help. E.L. would like to thank Rafael Oliveira and Helio Rocha for their support and feedback.

**Conflicts of Interest:** X.F.-B. has received funding from BIOIBERICA and C.R.A.-V. is employed by BIOIBERICA. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
