Tiny Green Army: Fighting Malaria with Plants and Nanotechnology

Malaria poses a global threat to human health, with millions of cases and thousands of deaths each year, mainly affecting developing countries in tropical and subtropical regions. Malaria’s causative agent is Plasmodium species, generally transmitted in the hematophagous act of female Anopheles sp. mosquitoes. The main approaches to fighting malaria are eliminating the parasite through drug treatments and preventing transmission with vector control. However, vector and parasite resistance to current strategies set a challenge. In response to the loss of drug efficacy and the environmental impact of pesticides, the focus shifted to the search for biocompatible products that could be antimalarial. Plant derivatives have a millennial application in traditional medicine, including the treatment of malaria, and show toxic effects towards the parasite and the mosquito, aside from being accessible and affordable. Its disadvantage lies in the type of administration because green chemical compounds rapidly degrade. The nanoformulation of these compounds can improve bioavailability, solubility, and efficacy. Thus, the nanotechnology-based development of plant products represents a relevant tool in the fight against malaria. We aim to review the effects of nanoparticles synthesized with plant extracts on Anopheles and Plasmodium while outlining the nanotechnology green synthesis and current malaria prevention strategies.


Introduction
In 2021, there were a total of 247 million cases of malaria, causing 619 thousand deaths, with a mortality rate of 14.8 per 100,000 population at risk.Compared to the previous year, there was an increase of 2 million malaria cases globally [1].Changes in malaria epidemiology have been attributed to increased risk of transmission, parasite genetic variability, growing drug resistance, and the COVID-19 pandemic [2].In the first year of the pandemic, malaria combat was compromised because of the reduced distribution of Insecticide-Treated Nets (ITNs) and the disruption of treatment and diagnosis of malaria cases, especially in the World Health Organization (WHO) African Region [1].Due to the pandemic, the lockdown limited the action of malaria prevention programs, causing a reduction in the distribution of ITNs.Also, health systems were overwhelmed with a shortage of health workers due to a lack of protection equipment, the closure of clinics due to quarantines, and all efforts being directed to control the pandemic, directly impacting the management and prevention of new malaria cases [3].The WHO reported that in 2021, there was an increase in malaria cases, mainly in the African and Asian continents, where substances as reducing agents, such as sodium borohydride, hydrazine, formaldehyde, and hydrogen [22].So, to overcome toxicity, several nanocarriers are being evaluated for developing these particles, such as plant extracts, for a biogenic synthesis approach.Nanotechnology has numerous biomedical applications in wound healing [23], cancer treatment [24][25][26], dentistry [27], and the cosmetic industry [28].Also, a clinical trial evaluated the effects of thyme and carvacrol NPs on Aspergillus fumigatus isolated from patients in intensive care (found in clicaltrial.govwith the ID NCT04431804).
Because of economic benefits, easy accessibility, and variety, plant extracts are a relevant alternative [29].NPs synthesized with plant extracts have appropriate size and shape, are consistent with average NPs, and are efficient because of high yield in less synthesis time [30].Also, using plant extracts requires the bottom-up method of NPs synthesis, characterized by the self-assembly of atoms and molecules from chemical interactions, which is simpler and more efficient [20].A very commonly used metal in the synthesis of NPs, silver (Ag), has antimicrobial and anti-inflammatory effects [29].Thus, using plant extracts to synthesize Ag NPs can enhance these activities because polyphenols also show beneficial properties [31].Moreover, Ag NPs are harmful to aquatic organisms and are considered a pollutant.However, the green synthesis of these NPs can reduce Ag toxicity by increasing molecule stability and reducing the agglomeration of particles [30].Although these advantages are significant, some drawbacks must be considered, such as possible toxicity, the lack of biocompatibility of the materials used, and the high cost of obtaining nano systems compared to conventional pharmaceutical formulations to treat a neglected disease [12].
The green synthesis of NPs refers to using biological materials as the capping agents to improve the form, functionality, and stability of NPs [32].The process of green-synthesizing NPs is illustrated in Figure 1.
Pharmaceutics 2023, 15, x FOR PEER REVIEW3 of 25 in the pharmaceutical industry, or inorganic, which are metal-based.Nano systems differ in terms of composition, the method of preparation, and structural organization [21].The most common methods for synthesizing nanoparticles (NPs) utilize expensive and toxic substances as reducing agents, such as sodium borohydride, hydrazine, formaldehyde, and hydrogen [22].So, to overcome toxicity, several nanocarriers are being evaluated for developing these particles, such as plant extracts, for a biogenic synthesis approach.Nanotechnology has numerous biomedical applications in wound healing [23], cancer treatment [24][25][26], dentistry [27], and the cosmetic industry [28].Also, a clinical trial evaluated the effects of thyme and carvacrol NPs on Aspergillus fumigatus isolated from patients in intensive care (found in clicaltrial.govwith the ID NCT04431804).
Because of economic benefits, easy accessibility, and variety, plant extracts are a relevant alternative [29].NPs synthesized with plant extracts have appropriate size and shape, are consistent with average NPs, and are efficient because of high yield in less synthesis time [30].Also, using plant extracts requires the bottom-up method of NPs synthesis, characterized by the self-assembly of atoms and molecules from chemical interactions, which is simpler and more efficient [20].A very commonly used metal in the synthesis of NPs, silver (Ag), has antimicrobial and anti-inflammatory effects [29].Thus, using plant extracts to synthesize Ag NPs can enhance these activities because polyphenols also show beneficial properties [31].Moreover, Ag NPs are harmful to aquatic organisms and are considered a pollutant.However, the green synthesis of these NPs can reduce Ag toxicity by increasing molecule stability and reducing the agglomeration of particles [30].Although these advantages are significant, some drawbacks must be considered, such as possible toxicity, the lack of biocompatibility of the materials used, and the high cost of obtaining nano systems compared to conventional pharmaceutical formulations to treat a neglected disease [12].
The green synthesis of NPs refers to using biological materials as the capping agents to improve the form, functionality, and stability of NPs [32].The process of greensynthesizing NPs is illustrated in Figure 1.
Figure 1.Schematic diagram for the biosynthesis of nanoparticles (NPs) via a green route using plant extract.A plant extract is a rich mixture of various metabolites and reductive biomolecules, each playing a pivotal role in the intricate process of reducing metal ions.Biomolecules stand out for their ability to transfer electrons to metallic ions, acting as potent reducing agents that instigate the ions' reduction.The consequence of this reduction process is the development of structured patterns in the form of a crystalline formation known as a nucleus.The initiation of nucleation involves the aggregation of monomers in supersaturated systems.This crucial step establishes the foundation for the subsequent growth of these nuclei, leading to the formation of metal nanoparticles.As these atomic nuclei form, they provide a foundational platform for the reduced ions, facilitating their gradual deposition onto the surface.This deposition process significantly contributes to the enlargement of the particles.Biomolecules, in addition to their role as reducing agents, also serve as capping agents.They bind to the particle surface, acting as molecular guardians that effectively stabilize the size of the particles.
Capping agents refer to molecules with an amphiphilic character, featuring a polar head and a non-polar tail region.The polar head group forms coordination bonds with the metal atoms within nanocrystals formed during agglomeration, while the tail region interacts with the surrounding medium.The right reducing and capping agents used during the synthesis of NPs are necessary to ensure optimal activity.The bioactive compounds of plant-based products are currently underestimated due to degradation and volatilization in field conditions.One option to avoid these drawbacks is to formulate bioactive plant products using polymers, plasticizers, stabilizers, and biodegradable antioxidants [33].A plant extract is composed of a variety of metabolites and reductive biomolecules that play a crucial role in reducing metal ions.These components include terpenoids, flavones, ketones, aldehydes, amides, carboxylic acids, carbohydrates, proteins, and vitamins [34].During the process of green synthesis, bio-compounds form a coating on the surface of NPs [32].This supplementary layer consequently amplifies the biological attributes of green NPs in contrast to those generated through other chemical reduction methods.The conventional green synthesis reaction involves a straightforward combination of a natural extract and a metal salt solution, serving as the precursor for NPs [35].This strategy has garnered attention over the past decade, especially for Ag and Au NPs, which are considered safer than other types of metallic NPs [36].Other metals, like nickel (Ni), manganese (Mn), Titanium (Ti), titanium trichloride (TiO 2 ), palladium (Pd), cerium (Ce), platinum (Pt), and ZnO, have been utilized in the creation of plant-based NPs and green NPs synthesis [35,36].
Biomolecules exhibit the capacity to transfer electrons to metallic ions, functioning as potent reducing agents that initiate the ions' reduction.Consequently, the reduced ions develop structured patterns that resemble a crystalline formation called a nucleus.The nucleation process begins with the monomer aggregation in supersaturated systems.The formation of an atomic nuclei is followed by the expansion of these nuclei into metal nanoparticles [37].These nuclei offer a foundation for the reduced ions, facilitating their gradual deposition onto the surface, thereby contributing to the enlargement of the particles.The expansion process is halted by biomolecules that can also function as capping agents, binding to the particle surface and effectively stabilizing their size [37,38].
Besides metallic NPs, the discussion extends to nano emulsions and nanogels.Nanogels are hydrogel particles composed of crosslinked polymers that can swell in an aqueous environment in nanoscale size.Notably, nanogels can accommodate a substantial drug load and achieve efficient encapsulation.Also, they excel at encapsulating larger quantities of substances and sustaining the release of entrapped molecules [39].On the other hand, nano emulsions are nanostructures formed by the dispersion of two immiscible liquids, such as oil in water or water in oil, that are stabilized by a surfactant.It has a variety of dosage forms, including liquids, creams, sprays, gels, aerosols, and foams.The pesticide industry uses it as an aqueous base for organic deliverables [40].The characteristics of NPs directly influence their interaction with the environment.Hence, they should be thoroughly investigated to prevent any adverse effects.The small size of NPs and the negative charge on their surface were associated with the potential of Ag NPs to cause hemolysis [41].
The application of nanotechnology for both vector control and patient therapy represents the most promising solution to this challenge.The main objective of malaria treatment is to provide the ideal drug concentration for the intracellular parasite [42].Nanotechnology protects drugs from chemical degradation, increases the drug's circulation time in the blood, improves cellular interaction with the parasite, and promotes the maintenance of plasma levels at a constant concentration.Also, it increases therapeutic efficacy, the progressive and controlled release of the drug, decreases toxicity by reducing plasma peaks at maximum concentration, and adds to the possibility of directly affecting specific targets [43].Nanoparticles can be functionalized with ligands that specifically bind to receptors on the surfaces of malaria parasites.This targeted drug delivery ensures that anti-malarial drugs are delivered directly to the parasites, increasing treatment efficacy while minimizing side effects on healthy cells.Also, nanoparticles can serve as carriers for vaccine antigens, enhancing their stability and immunogenicity [44].

Plants and Nano Formulations
In their natural habitat, plants already show defense mechanisms against plagues, predators, and other competing species [33,45].Thus, to use those against a naturally occurring mosquito is to take full advantage of the biological power displayed by these plants in favor of human health whilst causing minimal environmental impact.
Especially in the malaria scenario, plants that grow in tropical and subtropical regions can facilitate green insecticide production because of easy access, rare toxicity against humans, and low-dose effectiveness [46].For example, extracts of various seaweed species, especially Caulerpa acemose, are toxic to mosquito larvae, including A. stephensi, at the halflethal concentration (LC50) of 0.06 µg/mL [47].Zingiber cernuum essential oil is an effective larvicide and oviposition deterrent against malaria vectors A. stephensi and A. subpictus and does not affect non-target mosquito predators [48].Heracleum sprengelianum essential oil is also effective against A. subpictus larvae with LC50 33.4 µg/mL [49].
Notably, diverse plant species are commonly used in traditional medicine practices to treat malaria and alleviate Campo's symptoms [50] because these plants show antiparasitic properties, as seen in [51][52][53][54][55]. Historically, plants have originated the active substances used in malaria drug treatments, such as quinolines derived from Cinchona trees, which originate chloroquine [56].Also, the current drug of choice to treat malaria, artemisinin, is derived from the plant Artemisia annua L. Its discovery can be traced back to the sixties when it was used as an herbal medicine in China [57].
The challenge in using crude plant extracts is the degradation and evaporation of the chemical compounds upon field conditions [33].In addition, most isolated plant-derived substances have a short half-life [58], emphasizing the need to develop a better form of administration.Phytochemical nanoformulation (i.e., non-nutrients derived from plants) can improve bioavailability, water solubility, and protection from enzymatic degradation [59].Botanical-based NPs can be an eco-friendly alternative to current chemical pesticides, which are highly toxic to the environment [60].
Apart from plants, many other biological sources have been used to synthesize NPs.Nano-pesticide synthesized using Metarhizium robertsii showed larvicidal activity against A. stephensi fourth instar larvae in low doses and was not toxic to non-target organisms, such as earthworms and crustacea [61].Sardine-fish-scale Ag NPs showed potent larvicidal, pupicidal, and ovicidal activity against A. stephensi.Interestingly, the study showed increased predatory activity against larvae from the mosquitofish Gambusia affinis [62].Ag NPs synthesized by Bacillus marisflavi showed great ovicidal, larvicidal, and pupicidal activity against A. stephensi, the mortality rates of eggs and pupae being the highest compared to other mosquito species [63].Chitosan Ag NPs showed toxicity against A. stephensi larvae (fourth instar LC50 5.51 ppm) and pupae (LC50 6.54 ppm) [64].Mycosynthesized Ag NPs using Cochliobolus lunatus were toxic to second-fourth instar larvae of A. stephensi at concentrations of 5 and 10 ppm while presenting no danger to non-target Poecilia reticulata [65].Earthworms' coelomic fluids have been extensively used due to their therapeutic properties, such as cytotoxic, proteolytic, and antibacterial.Researchers used the Eudrilus eugeniae earthworms as reducing and stabilizing agents in synthesizing Ag NPs tested against A. stephensi.E. eugeniae Ag NPs were effective against larvae and pupae, and the predation efficiency of the mosquitofish G. affinis was boosted from 68.50% to 89.25% in second instar larvae [66].G. affinis and P. reticulata, among other species, are larvivorous fish commonly used in the biological control of Anopheles mosquito by malaria prevention programs [67] and demonstrated to be highly effective in a malaria-free country [68].
Additionally, nanotechnology can be applied to other strategies to fight malaria, such as better drug delivery in malaria cases, as reviewed by our group [12], and the nanoformulation of repellents to control disease transmission [85].

Malaria Prevention Strategies
Whereas targeting the mosquito vector, the main combat strategies are Indoor Residual Spraying (IRS) and ITNs [9].In sub-Saharan Africa, ITNs are the primary prevention strategy, and even though there has been an increase in ownership and use of ITNs over the years, population-level access has decreased since 2017.In addition, the levels of people protected by IRS in endemic countries have decreased, reaching 2.4% in 2021 compared to 5.5% in 2010 [1].Although IRS and ITNs have effectively reduced the malaria burden, these strategies have faced challenges because of household accessibility [86] and vector resistance to insecticides [87].Pyrethroid-only long-lasting insecticidal nets (LLINs) are the current main WHO recommendation, but mosquito resistance to pyrethroids has compromised its efficacy.Pyrethroids are the synthetic version of pyrethrins derived from the flowers of the Pyrethrum genus, which have insecticidal effects.The development of pyrethroids was incited due to the molecular instability of pyrethrins [88].Pyrethroids increase the opening of insects' sodium channels, disrupting nervous impulse propagation and the overactivation of nerve cells [89], causing mosquito paralysis, the same effect observed in nanoformulated plant extracts and further discussed in this review.Other synthetic insecticides are proposed with conditional recommendation because costeffectiveness is still uncertain and lacks evidence [9].Pesticides are known to be hazardous to the environment [90].They are lethal to aquatic organisms [91,92] and potentially affect human development due to prenatal exposure [93] and hormone signaling dysfunction [94].
Currently, the WHO does not recommend insecticide space-spraying due to a lack of evidence about its impact on malaria and the short life of the used chemicals [9].This review summarizes the effects of numerous eco-friendly alternatives to synthetic pesticides, emphasizing nanotechnology's use to improve efficacy (Table 1).Senna occidentalis and Ocimum basilicum coils showed significant toxicity against A. stephensi, causing mortality rates of 38% and 52%, respectively, similar to the pyrethrin-based control coil, which caused a 42% mortality rate [95].Pteridium aquilinum Ag NPs coils synthesized with different plant parts caused mortality rates comparable to pyrethrin-based coils [96].Also, in both studies, the plant-containing smoke could reduce parasite transmission because it caused a reduction in the percentage of fed mosquitoes.Coils from Ulva lactuca were effective against A. stephensi, causing a mortality rate of 66%, higher than that of the pyrethrin-based coil, which was 41% [97].
In addition, the green nanotechnology strategy can promote a biological form of vector control because most plant extracts can increase the predatory activity of nontarget organisms against the early developmental stages of the mosquito.For instance, chitosan-synthesized Ag NPs increased the predation efficacy of Danio rerio against larvae of A. stephensi at a concentration of 1 ppm [64].Citrus limon gold-palladium (Au-Pd) NPs had no negative impact on the predatory habit of flying insects, which increased over time [98].Likewise, Lagenaria siceraria zinc oxide (ZnO) NPs increased P. reticulata predation efficacy from 45.8% in the aqueous extract to 61.13% [99].Also, Cymbopogon citratus Au NPs boosted Mesocyclops aspericornis predation against A. stephensi from 26.8 to 45.6% [100].The predation efficacy improvement could be due to the decreased mobility of larvae, which are typically fast-paced, after exposure to the green-synthesized NPs [98].Additionally, Ag NPs synthesized with Ichnocarpus frutescensi [101], Rubus ellipticus [102], Naregamia alata [103], and Hugonia mystax [104] had a negligible toxic effect on non-target aquatic organisms, such as G. affinis, Diplonychus indicus, and Anisops bouvieri.Solanum xanthocarpum-synthesized Ag NPs [105] and Momordica charantia leaf extract TiO 2 NPs [106] had no toxicity against P. reticulata at the same concentrations lethal to mosquitos.It is crucial to outline the choice of material used to synthesize NPs because CdS showed toxicity against non-target aquatic organisms [107].These are predators of mosquito breeding sites, hence the importance of developing insecticides harmful only to the intended species to maintain the ecologic niche balance.Malva sylvestris Ag NPs were not toxic to D. indicus and G. affinis, as the longevity and swimming habits did not change after exposure to NPs, and the suitability index showed that treatment with NPs was more toxic to the mosquito larvae [108].
The studies above follow the recently defined WHO preferred product characteristics (PPCs) for vector-control products.PPCs were determined to incentivize the search and development of new strategies to control Anopheles mosquitoes while addressing multiple parameters, including indoor and outdoor transmission control, complementarity to existing methods, and safety for humans and the environment [87].Green-synthesized nanotechnology fulfills most of the demanded PCCs, thereby being a suitable candidate to fight malaria.

Plant Extracts and Green NPs Action Mechanisms
The NPs' action mechanisms are still unclear, and the current knowledge is limited to mosquitoes' morphological, molecular, biochemical, and physiological levels [133].Table 1 summarizes the effects of plant extracts in these numerous levels with the nanoparticle's corresponding type and size.The high mortality rates exerted by NPs on mosquito larvae may be explained by the small size of the particles, which enables the passage through the respiratory tubes and/or insect cuticle and into cells where they interfere with molting and other physiological processes [66,122].After penetrating the exoskeleton, the Ag NPs exert their effects in the intracellular space, interacting directly with proteins or DNA and causing genotoxic effects.The depletion of antioxidants induces oxidative stress, the oxidative dissolution of Ag NPs, and mitochondria perturbation [134].It also can induce apoptosis through the activation of procaspase-3, the downregulation of the pro-survival protein Bcl-2, the enhanced expression of pro-apoptotic gene products, and the release of cytochrome c into the cytosol [135,136].
The toxicity of NPs is intricately linked to their propensity to accumulate within various organs.Upon entering the bloodstream, NPs exhibit the capacity to disperse throughout the body, accumulating within organs such as the liver, spleen, lungs, and kidneys.This distribution of NPs within the body is primarily influenced by their surface area-to-size ratio, a factor that affects their tendency to accumulate in distinct tissues and organs [137].The pathways through which NPs exert toxicity within the organs go through several critical factors, including producing reactive oxygen species (ROS), DNA damage, the alteration of protein structures and functions, and compromising membrane integrity.Notably, NP characteristics that seem to enhance these mechanisms involve their expansive surface areas, which promote molecular interactions within the specific target sites [138].Toxicity is not limited to the target species but also to the environment, especially in the management of the vector in-field conditions.NPs released in water affect aquatic organisms, consequently resulting in reports of blood acidosis, causing circulatory collapse, bioaccumulation, hepatotoxic effects, oxidative stress, and embryonic development.However, such effects were observed at elevated concentrations [29], in opposition to the low concentrations used in nanoparticles.
The neurotoxic effects of plant-derived compounds are linked to acetylcholinesterase (AChE) inhibition, the modulation of gamma-aminobutyric acid (GABA), and octopamine (OA) receptors (GABAr and OAr, respectively).Essential oils are a complex mixture of green chemicals, mainly terpenoids, that act through these mechanisms, resulting in the overstimulation or paralysis of the insect's nervous system [139].Studies using Ag NPs evaluate the expression of glutathione S-transferase (GST) in midges, pointing to the importance of GST genes protecting against oxidative stress induced after exposure to Ag NPs [140].
In field or semi-field experiments, it is hypothesized that the lethal effect of plant extracts may be due to the formation of an oil layer in the aqueous medium that blocks proper water oxygenation and allows for the active penetration of phytochemicals in larvae [110].The water quality and compounds also implicate mosquito habits, such as egg-laying, because female gravid mosquitoes are attracted to breeding sites according to olfactory cues, such as pheromones and environmental odors, with species-dependent preferences [145].A. coluzzii were more attracted to sites with early stages larvae [146] and A. gambiae had a preference to water sites treated with nonane, a pheromone produced by larvae [147].Plant extracts may prevent oviposition due to their larvicidal effects, thus eliminating a mosquito attraction factor.Conversely, volatile plant compounds from natural habitats can attract mosquitoes and favor egg-laying, such as limonene, β-pinene, β-elemene, and β-caryophyllene detected in preferred A. gambiae breeding sites [148].In this case, we hypothesize that the oviposition-deterrent activity by nanoformulations is due to the stability of these compounds in the water and the higher capacity for penetrating the insect's body, causing toxicity.
The disadvantage of inorganic nanoparticles is the metal ion's toxicity to the environment, highlighting the demand for an eco-friendly and modern intervention.For that reason, NPs synthesized from biomolecules is a relevant tool in fighting malaria whilst causing minimal impact on the environment.To summarize, the effects and possible action mechanisms are depicted in Figure 2, and we hypothesize that the toxicity of green NPs may be caused by the known effects of phytochemicals, which play a part in the functionality of NPs (as seen in Figure 1).In the left panel, we summarize the phytochemicals' effects.These molecules are recognized for their larvicidal and mosquitocidal properties, modulating the mosquito nervous system, by inducing either overstimulation or inhibition.This neuro-modulation results in observable behavioral changes, such as the reduced swimming and restless movement of larvae.In the right panel, effects of green nanoparticles (NPs) are illustrated.NPs with their optimal size effortlessly penetrate the membranes of mosquitos.Their mechanism of action involves interactions with DNA, contributing to enhanced apoptosis and oxidative stress within the target organisms.Notably, when deployed in field or semifield conditions, these nanoparticles form an oily layer, a phenomenon correlated with a reduction in water oxygenation.Furthermore, both phytochemicals and green NPs influence factors such as the attraction of female mosquitoes to breeding sites, which are guided by olfactory cues susceptible Figure 2. Action mechanisms of plant extracts and plant-derived nanoparticles.In the left panel, we summarize the phytochemicals' effects.These molecules are recognized for their larvicidal and mosquitocidal properties, modulating the mosquito nervous system, by inducing either overstimulation or inhibition.This neuro-modulation results in observable behavioral changes, such as the reduced swimming and restless movement of larvae.In the right panel, effects of green nanoparticles (NPs) are illustrated.NPs with their optimal size effortlessly penetrate the membranes of mosquitos.Their mechanism of action involves interactions with DNA, contributing to enhanced apoptosis and oxidative stress within the target organisms.Notably, when deployed in field or semi-field conditions, these nanoparticles form an oily layer, a phenomenon correlated with a reduction in water oxygenation.Furthermore, both phytochemicals and green NPs influence factors such as the attraction of female mosquitoes to breeding sites, which are guided by olfactory cues susceptible to water quality.The effects of green-synthesized NPs may be due to the known effects of biomolecules present in plant extracts.

Antimalarial Effects of Green NPs
Multiple studies have evaluated the plant extracts potential to fight malaria at its many fronts, parasite elimination, or vector control.Regarding the vector population control, the main parameters assessed were the induction of larvae, pupae, and adult mortality, the impediment of oviposition, and egg hatchability.The main effects of green-synthesized NPs against mosquitoes and parasites are illustrated in Figure 3.  Effects of green-synthesized NPs on malaria prevention.NPs synthesized with plant extracts are toxic to early developmental stages of the malaria mosquito vector, Anopheles, acting against all four larvae stages (instar) and pupae, preventing the emergence of adult mosquitos capable of disease transmission.These nanoformulations also show adulticidal effects, impacting mosquito behavior and causing morphological damage.Furthermore, some studies showed that plant-extract NPs reduced the feeding of mosquitoes by preventing the biting habit.Oviposition is hindered in water treated with the nanoformulated plant extracts, thus reducing breeding site suitability.Green-synthesized NPs also improve the feeding activity of mosquito predators, lowering the survival chances of mosquito larvae and pupae.These formulations are eco-friendly because predators are not harmed.In addition, nanotechnology emphasizes the well-known antiparasitic activity of plant extracts.The antiplasmodial effects of plant-derived nanoformulations are parasitemia suppression by increased parasite mortality and metabolic impairment.Therefore, plant-derived nanoformulations are a potential tool to fight malaria.
Among the studied vector species are A. stephensi and A. subpictus.A. stephensi is an essential vector of both P. falciparum and P. vivax, and has been documented to spread to other geographic areas, especially urban environments, where it can thrive, posing a challenge to vector control [1].Thus, the use of natural products in these settings would be beneficial.Using plant extracts to synthesize NPs increases the mortality rate of mosquitoes in all developmental stages, and this effect is obtained using lower concentrations of the extract.For example, Ag NPs synthesized with Euphorbia hirta [124], Nerium oleander [125], U. lactuca [97], Codium tomentosum [114], M. sylvestris [108], Figure 3. Effects of green-synthesized NPs on malaria prevention.NPs synthesized with plant extracts are toxic to early developmental stages of the malaria mosquito vector, Anopheles, acting against all four larvae stages (instar) and pupae, preventing the emergence of adult mosquitos capable of disease transmission.These nanoformulations also show adulticidal effects, impacting mosquito behavior and causing morphological damage.Furthermore, some studies showed that plant-extract NPs reduced the feeding of mosquitoes by preventing the biting habit.Oviposition is hindered in water treated with the nanoformulated plant extracts, thus reducing breeding site suitability.Greensynthesized NPs also improve the feeding activity of mosquito predators, lowering the survival chances of mosquito larvae and pupae.These formulations are eco-friendly because predators are not harmed.In addition, nanotechnology emphasizes the well-known antiparasitic activity of plant extracts.The antiplasmodial effects of plant-derived nanoformulations are parasitemia suppression by increased parasite mortality and metabolic impairment.Therefore, plant-derived nanoformulations are a potential tool to fight malaria.
Among the studied vector species are A. stephensi and A. subpictus.A. stephensi is an essential vector of both P. falciparum and P. vivax, and has been documented to spread to other geographic areas, especially urban environments, where it can thrive, posing a challenge to vector control [1].Thus, the use of natural products in these settings would be beneficial.Using plant extracts to synthesize NPs increases the mortality rate of mosquitoes in all developmental stages, and this effect is obtained using lower concentrations of the extract.For example, Ag NPs synthesized with Euphorbia hirta [124], Nerium oleander [125], U. lactuca [97], Codium tomentosum [114], M. sylvestris [108], Holostemma adakodien [81], H. mystax [104], Quisqualis indica [130], Nicandra physalode [131], Gmelina asiatica [132], Musa paradisiaca [149], Drypetes roxburghii [150], Vinca rosea [151], Cocos nucifera [112], Pergularia daemia [152], L. siceraria ZnO NPs [99], and C. citratus Au NPs [100] had the highest larvicidal and pupicidal effect against A. stephensi, in comparison to the crude plant extract.S. xanthocarpumsynthesized Ag NPs showed significant larvicidal effects up to 72 h after A. stephensi larvae exposure [105].Also, against larvae of A. subpictus, Ag NPs synthesized with extracts of diverse plant species, such as Zornia diphylla [127], Mussaenda glabra [128], Anisomeles indica [129], Barleria cristata [119], Ammannia baccifera [120], Nelumbo nucifera [153], Tinospora cordifolia [154], and Mimosa pudica [155], and Vitex negundo ZnO NPs [117] were more toxic than the crude leaf extract, presenting lower lethal concentrations.The LC50 of green NPs and the affected mosquito species are summarized in Table 2.A eucalyptus nanoemulsion had larvicidal activity against A. stephensi in laboratory and semi-field conditions.The larvae population had low density for up to 6 days in breeding sites sprayed with the nanoemulsion.In contrast to the breeding sites treated with regular eucalyptus oil, which had increased larvae after only two days [118].In laboratory conditions, Aristolochia indica leaf extract-synthesized Ag NPs were toxic against A. stephensi larvae and pupae, with an LC50 ranging from 3.94 to 15.65 ppm, respectively.In field experiments, the crude extract and NPs had similar larvicidal activity, reaching about 50% larvae mortality in 24 and 100% in 72 h [110].Artemisia dracunculus essential oil nanoemulsion and nano gel were toxic to A. stephensi larvae.The nanogel preparation was more lethal than the nanoemulsion, with an LC50 of 6.6 and 13.5 µg/mL, respectively [82].Nanoliposomes and pupae of A. stephensi [161].The peanut (Arachis hypogaea) peel extracts were utilized to synthesize Ag NP and evaluated against fourth instar larvae of A. stephensi.When exposed to a concentration of 15 mg/L, a 100% mortality rate was observed among the larvae within 24 h, accompanied by significant morphological changes in the cuticular membrane [116].A similar outcome was observed with the Ag from the aqueous leaf extracts of Leucas aspera and Hyptis suaveolens, where a mortality rate of 100% was achieved at a concentration of 10 mg/L [162].Biosynthesized Au NPs of Couroupita guianensis flower extract were used in water storage reservoirs on larval populations of A. stephensi.The Au NP flower extract had larvicidal and pupicidal effects, and the adulticidal activity showed 95% mortality.The predation efficiency of Aplocheilus lineatus after treatment with ultra-low dosages of C. guianensis Au NP was enhanced (96.04%) [113].
A few reports indicated that green-synthesized NPs may also have toxic effects against malaria parasites in cellular and animal models.The most studied Plasmodium strains are the P. falciparum chloroquine-sensitive (CQs) strain 3D7, and chloroquine-resistant (CQr) strains RKL9 and INDO.An early in vivo report assessed the potential of orally administered palladium (Pd) NPs synthesized from Eclipta prostrata in mice infected with the P. berghei CQs strain NK 65.Pd NPs were more effective in reducing parasitemia than the aqueous extract [84]. A. cordifolia Ag NPs were more toxic to 3D7 and RKL9 strains than crude extract, with IC50 values 8.05 and 20.27 µg/mL for 3D7, 10.31 and 32.55 µg/mL for RKL9, respectively [109].Ag NPs synthesized with Azadirachta indica inhibited parasite growth.The IC50 (µg/mL) values for A. indica bark Ag NPs against 3D7 and RKL9 strains were 8 and 7.8, respectively.A. indica leaf Ag NPs had an IC50 of 9.2 for 3D7 and 11.1 for RKL9.Both NPs were effective at lower concentrations than the aqueous extract [41].Antiplasmodial effects were also seen for L. siceraria ZnO NPs, with parasitemia suppression at IC50 4.32 µg/mL, comparable to chloroquine with IC50 2.5 µg/mL [99].Compared to the crude leaf extract, M. charantia-synthesized TiO 2 NPs were more effective in the antiplasmodial assay against 3D7 and INDO strains [106].C. tomentosum Ag NPs inhibited the parasitemia of 3D7 and INDO strains, with an IC50 of 72.45 and 76.08 µg/mL, respectively.The treatment was similar to chloroquine, with an IC50 of 80 (CQs strain) and 85 µg/mL (CQr strain) [114].Interestingly, Ag NPs and crude extract from U. lactuca [97] and P. aquilinum [96] showed higher toxicity to 3D7 and INDO parasite strains than chloroquine.The C. guianensis extract Au NPs showed a higher growth inhibition than chloroquine against 3D7 and INDO strains [113].Also, the L. siceraria ZnO NPs exhibited the potent inhibition of hematin formation [99].E. eugeniae Ag NPs showed higher toxic activity in antiplasmodial assays than chloroquine against P. falciparum [66].After the infection of red blood cells, P. falciparum digests hemoglobin, generating a free heme group, which is toxic to the parasite.Within its digestive vacuole, Plasmodium converts the heme group into hemozoin, also called β-hematin, a non-toxic crystallized molecule [163].So, the inhibition of hemozoin formation is crucial in the research for novel antimalarials.Thus, pilling evidence shows the potential use of green NPs to fight malaria.

Conclusions
While it is well-documented in the literature, there needs to be more research discussions regarding the relevance of green-synthesized NPs in malaria prevention and its impact on the vector.Many studies have indeed explored its potential in treatment, but there needs to be more exploration of its role in vector prevention.In this paper, our primary objective was to conduct a comprehensive review of previously synthesized nanoparticles that are potentially used in controlling malaria transmission.By addressing this gap in research, we aim to contribute valuable insights into malaria prevention.Nanotechnology is leading with the most recent research for better drug delivery and the development of novel tools.The nanoformulation of compounds enables low concentrations compared to the bulk chemicals, thereby reducing toxicity.Indeed, nanotechnology characteristics and materials are an essential topic in discussion since they can influence the biological impact and behavior of the NPs.There are disadvantages to inorganic NPs, so the biogenic synthesis of NPs has been extensively reported in the literature, especially regarding the use of plant extracts in the synthesis of inorganic NPs with potent activity against the malaria vector and parasite.This green synthesis of NPs allows the bio compounds plant polyphenols to enhance the effects of metallic NPs, causing an overall toxic impact on all developmental stages of the mosquito vector, reflected in larval mortality, reduced oviposition, impaired feeding habit, and morphological and behavioral changes.In addition, NPs synthesized with plant extracts show antiplasmodial effects by inhibiting parasite growth.Furthermore, choosing interventions against malaria should take into account equal access, sustainability, and human health impact.

Future Perspectives
The lack of evidence of the plant extracts' mechanism of action becomes a significant issue in this review.The complex chemical composition of plants can hinder the emergence of resistance because the toxic compounds can vary between plant species.Although plant-derived nanoformulations are not currently used, we advocate for robust research regarding the long-term bioavailability of green nanoparticles, potential toxicity, production scaling, and administration feasibility.

Figure 2 .
Figure 2. Action mechanisms of plant extracts and plant-derived nanoparticles.In the left panel, we summarize the phytochemicals' effects.These molecules are recognized for their larvicidal and mosquitocidal properties, modulating the mosquito nervous system, by inducing either overstimulation or inhibition.This neuro-modulation results in observable behavioral changes, such as the reduced swimming and restless movement of larvae.In the right panel, effects of green nanoparticles (NPs) are illustrated.NPs with their optimal size effortlessly penetrate the membranes of mosquitos.Their mechanism of action involves interactions with DNA, contributing to enhanced apoptosis and oxidative stress within the target organisms.Notably, when deployed in field or semifield conditions, these nanoparticles form an oily layer, a phenomenon correlated with a reduction in water oxygenation.Furthermore, both phytochemicals and green NPs influence factors such as the attraction of female mosquitoes to breeding sites, which are guided by olfactory cues susceptible Pharmaceutics 2023, 15, x FOR PEER REVIEW13 of 25

Figure 3 .
Figure3.Effects of green-synthesized NPs on malaria prevention.NPs synthesized with plant extracts are toxic to early developmental stages of the malaria mosquito vector, Anopheles, acting against all four larvae stages (instar) and pupae, preventing the emergence of adult mosquitos capable of disease transmission.These nanoformulations also show adulticidal effects, impacting mosquito behavior and causing morphological damage.Furthermore, some studies showed that plant-extract NPs reduced the feeding of mosquitoes by preventing the biting habit.Oviposition is hindered in water treated with the nanoformulated plant extracts, thus reducing breeding site suitability.Green-synthesized NPs also improve the feeding activity of mosquito predators, lowering the survival chances of mosquito larvae and pupae.These formulations are eco-friendly because predators are not harmed.In addition, nanotechnology emphasizes the well-known antiparasitic activity of plant extracts.The antiplasmodial effects of plant-derived nanoformulations are parasitemia suppression by increased parasite mortality and metabolic impairment.Therefore, plant-derived nanoformulations are a potential tool to fight malaria.

Table 1 .
Characteristics of NPs synthesized with plant extracts that have beneficial use and its effects on malaria vector and parasite.The nanoparticle sizes are according to transmission electron microscopy (TEM) or scanning electron microscopy (SEM) analysis.

Table 2 .
Half-lethal concentration (LC50) of nanoformulated plant products and bulk formulations in late developmental stages (third-fourth instar and pupae) of Anopheles species after 24 h of exposure.