Enhancing malaria control using Lagenaria siceraria and its mediated zinc oxide nanoparticles against the vector Anopheles stephensi and its parasite Plasmodium falciparum

In many developing countries, there are certain health problems faced by the public, one among them is Malaria. This tropical disease is mainly caused by Plasmodium falciparum. It is categorized as a disaster to public health, which increases both mortality and morbidity. Numerous drugs are in practice to control this disease and their vectors. Eco-friendly control tools are required to battle against vector of this significant disease. Nanotechnology plays a major role in fighting against malaria. The present paper synthesized Zinc oxide nanoparticles (ZnO NPs) using zinc nitrate via simple green routes with the help of aqueous peel extract of Lagenaria siceraria (L. siceraria). The synthesized ZnO NPs were characterized by various biophysical methods. Moreover, the extract of L. siceraria and their mediated ZnO NPs was experimented against III instar larvae of An. stephensi. The impact of the treatment based on ZnO NPs concerning histology and morphology of mosquito larval was further observed. In the normal laboratory environment, the efficiency of predation of Poeciliareticulata (P. reticulata) against An. Stephensi larvae was found to be 44%, whereas in aqueous L. siceraria extract and its mediated ZnO NPs contaminated environment, P. reticulate showed predation efficiency of about 45.8% and 61.13% against An. Stephensi larva. L. siceraria synthesized ZnO NPs were examined against the Plasmodium falciparum CQ-sensitive strains. The L. siceraria extract and its mediated ZnO NPs showed the cytotoxic effects against HeLa cell lines with an IC50 value of 62.5 µg/mL. This study concludes that L. siceraria peel extract and L. siceraria synthesized ZnO NPs represent a valuable green option to fight against malarial vectors and parasites.


Materials and methods
Chemicals and materials. Zinc nitrate, Xylene, Ethanol and all the chemicals required for carrying out the experiment was procured from Sigma-Aldrich. Fresh L. siceraria was obtained from Vellore local market, Tamil Nadu, India. Zinc nitrate was obtained from Sigma Aldrich, India. All aqueous solutions were prepared using deionized water. All glass wares were cleaned with chromic acid followed by thorough washing with deionized water and then with acetone for prior use.
Preparation of L. siceraria aqueous extract. The impurities like dust, scum and other kinds of stuff were removed from the gathered L. siceraria by first washing it using tap water and then using distilled water. Later, they were peeled cautiously to isolate the epicarp and instantly dried in the shade. The peels were dried to obtain a fine powder. The 10 g of L. siceraria powder was measured and fetched in a beaker that already comprises of 100 mL of distilled water. The mixture was boiled for 10 min 49,50 . Whatmann No.1 filter paper was used to filter the obtained extract and a separate flask was used to accumulate the filtrate and stored in the refrigerator for further use.
Bio-synthesis and characterization of ZnO NPs using L. siceraria peel. Zinc nitrate and L. siceraria aqueous peel extract were used to amalgamate ZnO NPs. The detailed synthesis and characterization have been reported in our earlier work 50 . An. stephensi rearing. An. stephensi larvae were collected from rice fields and stagnant waters from the nearby areas of Melvisharam (12°56′23″ N, 79°14′23″ E) and identified in Zonal Entomological Research Centre, Vellore (12°55′48″ N, 79°7′48″ E), Tamil Nadu. To start the colony, larvae were kept in plastic trays containing tap water. All the experiments were carried out at 27 ± 2 °C and 75-85% relative humidity under 14:10 light and dark cycles. Larvae were nourished under a diet of dog biscuits, algae and brewer's yeast in 3:1:1 proportion, respectively 34 . Larvicidal activity. In this assay, An. StephensiIII instar larvae were left in a glass beaker for 24 h containing 250 mL of dechlorinated water along with aqueous L. siceraria extract (80, 160, 240, 320 and 400 ppm) and its mediated ZnO NPs (30,60,90, 120 and 150 ppm). Using distilled water, the control set-up was made. As a result, a number of larval deaths were noticed after exposure of 24 h. The experiment was repeated five times against the An. Stephensi III instar larvae 51,52 . The death rate can be calculated by using the following formula.
Histopathological and stereomicroscopic analysis. In order to conduct a histopathological study,

An.
Stephensi larvae with the aqueous extract of L. siceraria and ZnO NPs were treated for 24 h with 10% buffered formaldehyde and then dehydrated through the solutions of xylene and ethanol (70-100%) and at last they were mounted in paraffin blocks. With the help of glass knives, larval tissues were segmented in the rotary Mortality (% ) = (number of dead individuals/number of treated individuals) × 100 Scientific Reports | (2020) 10:21568 | https://doi.org/10.1038/s41598-020-77854-w www.nature.com/scientificreports/ microtome for a thickness of 8 µm. Each cut sections were mounted on the glass slides and stained by eosin and haematoxylin. After this process, under the microscopic light, each section was examined for the histopathological test. Through the stereomicroscope, the collected larval tissues and its damages were observed 53,54 . Predation efficiency assays. In this experiment, the predation efficiency of Poeciliareticulata (P. reticulata) (National Institute of Health Guidelines) against III instar An. Stephensi larvae was examined. In each and every single trail, with one P. reticulate nearly 150 larvae were introduced in glass beakers containing 250 mL of dechlorinated water treatment. Aqueous L. siceraria extracts and its mediated ZnO NPs (i.e. for plant extract and NPs, nearly 1/3 of LC 50 ) were calculated against III instar larvae. The experiment was also performed under standard laboratory conditions (especially with no treatment of plant extract and NPs). Control was dechlorinated water and mosquito larvae without P. reticulata. Chandramohan et al. 55 and Murugan et al. 56  Drug dilutions. Chloroquine (CQ) is a stock solution which is formulated in water (milli-Q grade). Accordingly, dimethyl sulfoxide (DMSO) was used for the preparation of the ZnO NPs. To attain the necessary concentrations, the entire stock solutions were later diluted with culture medium.
In vitro anti-plasmodial assay. The aqueous peel extract of L. siceraria and its mediated ZnO NPs (1.56, 3.12, 6.25, 12.5, 25, 50, 100 µg/mL) was evaluated against the Chloroquine sensitive (3D7) strains of P. falciparum for conducting the antiplasmodial activity. For conducting the drug screening, SYBR green I-based fluorescence test was set up. The positive control should be maintained at a culture of parasitized blood cells which must be later treated with chloroquine. With fresh red blood cells, and 2% parasitized P. falciparum diluted to 2% hematocrit, the negative control was maintained. With fresh red blood cells, 100 µl of P. falciparum diluted to 2% of hematocrit was incorporated in the 96 well tissue culture plates. In an atmosphere filled with 5% of air and CO 2 mixture, the plates were kept in a CO 2 incubator at 37 °C. After 72 h, a 100 µl of lysis buffer containing 2 × concentration of SYBR Green-I (Invitrogen) was added to it and incubated at 37 °C for 60 min. The plate was analyzed at 530 ± 20 nm of emission and 485 ± 20 nm of excitation for relative fluorescence units using fluorescence plate reader (BIOTEK, FLX800). The fluorescence counts were plotted against the concentration of the drug in dose-response curves 57,58 . With the help of a microscope, the results were validated after 48 h with Giemsa stain and the average percentage of suppressed parasitemia can be calculated using this formula: Data analysis. The antiplasmodial activity of aqueous L. siceraria extract and its mediated ZnO NPs was expressed by the percentage growth inhibition. The concentrations causing 90% inhibition of parasite growth (IC 90 ) and 50% inhibition of parasite growth (IC 50 ) were calculated using the drug concentration-response curves.
β-Hematin formation assay. The potential L. siceraria extract's antimalarial activity and its mediated ZnO NPs were estimated using Afshar et al. 59 technique with slight alterations. In short, L. siceraria extract and its mediated ZnO NPs with different concentrations (0-2 mg/mL in DMSO) were incubated with 10 mM oleic acid, 1 M HCl, and 3 mM of hematin. The end volume was finely-tuned to 1 mL by mixing sodium acetate buffer (pH5). Later, the samples were protected at 37 °C with constant shaking during the night. During this process, chloroquine diphosphate was applied as a positive control. After that, the samples were centrifuged at 21 °C for 10 min at 14,000 rpm, and the samples were frequently added with 2.5% (w/v) SDS existing in buffered saline in order to purify thehemozoin pellets (usually 3-8 washes). After this process, it was washed with 0.1 M sodium bicarbonate till the removal of supernatant. In the end, clean pellets were dissolved with 1 mL of NaOH, and a UV spectrophotometer was used to measure the absorbance at 400 nm. DMSO was used as a negative control. The outcomes were noted since the heme crystallization/polymerization's percentage inhibition (I%) was compared towards the positive control (chloroquine) with the help of the given formula: Predatory efficiency The cell line HeLa was seeded and propagated in a 96-well plate as 1 × 10 5 cells approximately in every well and incubated for 24 h. Once the cell reached the confluence, the different concentrations of ZnO NPs were added and kept for incubation for 24 h at 37 °C with a 5% CO 2 condition. Then the sample was taken out from the well and washed with phosphate-buffered saline maintained at pH 7.4. 100 µl/well (5 mg/mL) of 0.5% 3-(4, 5-diphenyl-tetrazolium bromide (MTT) 5-dimethyl-2-thiazolyl)-2, was added and incubated for next 4 h. 1 mL of DMSO was added in every well after the incubating process. The measurement of the absorbance at 570 nm was done with UV-spectrophotometer while DMSO was kept as a blank set-up. Measurements were performed and the concentration required for a 50% inhibition (IC 50 ) was determined graphically. By using the below formula, viability % of the cell can be determined.
Graphs were plotted using the concentration of the sample in X-axis and cell viability % at Y-axis. Sample control and cell control were included in all assays to fully compare the assessment of cell viability 43 . Ethics declaration. Use of experimental animals, and human participants. This is to confirm that all methods were carried out in accordance with WHO guidelines and Regulations. The fish (P. reticulata) was handled according to the National Institute of Health Guidelines for the handling and care of experimental animals and the animal utilization protocol was approved by the Institutional Animal Care, VIT, Vellore.

Results and discussions
Larvicidal activity. The 62 documented that Ag NPs synthesized from the aqueous leaf extract of Mimusopselengi were highly effective against larvae and pupae of the malaria vector An. Stephensi (LC 50 ranged from 12.53 to 23.55 ppm) and the arbovirus vector Aedesalbopictus (LC 50 ranged from 11.72 to 21.46 ppm). It is imperative to know their functioning during the consideration of ZnO NPs larvicidal activity. The impact of ZnO NPs and that of the biochemical components of the An. stephensi III instar larvae was regulated. Overall, it was revealed that there were changes caused by the tested samples in the normal biochemical components with a decrease or increase in the action in comparison to the control.
Histopathological and stereomicroscopic analysis. The study of histopathology regarding An. Stephensi III instar larvae, where it was made to treat with L. siceraria aqueous extract and mediated ZnO NPs, shows the decomposed layer of the epithelial' s outer cuticle along with entire decomposition of the abdominal area, special caeca, and midgut. This out-turned in the depletion of caudal and lateral hairs (Figs. 1, 2). In the aspect of stereomicroscopic analysis, An. Stephensi III instar larvae treated with L. siceraria aqueous peel extract      63 reported that green synthesized NPs will not possess any toxicity towards mosquito natural enemies and predatory fishes. Murugan et al. 56 stated that P. reticulata's predation towards larvae of C. quinquefasciatus had a remarkable increase of predation and there was no notable impact of toxicity accused on guppies as they come into the contact of Ag NPs infected ecosystem. Benelli 64 found very less toxicity level in non-target organism, P. reticulata using green synthesized Ag NPs.
In vitro antiplasmodial assays. The antiplasmodial activity of aqueous L. siceraria extract and its mediated ZnO NPs was tested at a different concentrations ranging from 100, 50, 25, 12.5, 6.25, 3.12, 1.56 µg/mL and chloroquine diphosphate was used as a positive control. The IC 50 values of aqueous L. siceraria extract and its mediated ZnO NPs against P. falciparum strains at 48 h of parasitemia suppression are listed in Table 3 and Fig. 4. The microscopic observation involved in anti-plasmodial activity of aqueous extract of L. siceraria and its mediated ZnO NPs against P. falciparum strains is shown in Fig. 5. In the same way, Mishra and Sharma 39 noticed that the aqueous extract of leaves of Neem and Ashoka has the property of antiplasmodial at IC 50 value which is 30 µg/mL and 8 µg/mL. The two medicinally significant plants, namely, Thalictrumfoliolosum and Aris-    34 discovered that these medicinal plants are powerful against the resistant and sensitive strains of Chloroquine. The antiplasmodial activity of green synthesized metal oxide and metal NPs were fully studied by Ishwarya et al. 51 .   The mechanism of ZnO NPs and L. siceraria on P. falciparum strains. The plasmodium parasite outbreaks the host erythrocyte in order to utilize hemoglobin for synthesizing the essential requirements to develop and proliferate. During this process, a massive amount of heme is generated as a toxic undesirable byproduct which is pernicious for malaria parasite. Therefore, to protect itself, the parasite neutralizes large amounts of heme to hemozoin or water-insoluble malaria pigment via the biocrystallization process. Hence, inhibition of hemozoin formation by means of peel extract of L. siceraria aqueous mediated ZnO NPs is regarded as an incomparable target to combat the malaria (Fig. 6).
Cytotoxicity activity on HeLa cells using MTT assay. The in vitro cytotoxicity of L. siceraria mediated ZnO NPs was evaluated against human cervical (HeLa) cancer cell lines at different concentrations (1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8 µg/mL). The L. siceraria mediated ZnO NPs exhibited potent cytotoxicity/anticancer activity in the tested cell lines ( Table 5). The effect was compared with normal HeLa cell lines. Results showed that at higher concentrations there is a significant mortality (Fig. 7). The inhibitory effect was observed after 24 h of incubation. Figure 8 shows the changes in the percentage of inhibition in NPs treated HeLa cells. The results also showed that HeLa cells were inhibited by L. siceraria mediated ZnO NPs with an IC 50 value of 62.5 µg/mL (

Conclusion
Overall this paper reviews the use of nanomaterials for controlling malaria and mainly examines the malaria life cycle, epidemiology and prevalence in global and India perspective. From this review, it is clear that female anopheles mosquito play a significant role in transmits malarial disease and hence in recent days, many researchers applied various nanotechnology methods to control it specifically nanomimics strategy. However, the application of nanotechnology for controlling malaria has both positive and negative effects but it provides various adverse effects to humans therefore it is recommended to apply for medicinal purpose in future. Nonetheless, further field studies are required to investigate the effective method of behavior and side effects.  License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.