Effect of nanostructure lipid carrier of methylene blue and monoterpenes as enzymes inhibitor for Culex pipiens

Solid lipid nanoparticles second generation, nanostructure lipid carrier (NLC), is one of the most important biodegradable nanoparticles. Nanostructure Lipid carrier (NLC) was used to encapsulate methylene blue (MB) dye, carvacrol and citronellal and their efficacy as insecticidal against Culex pipiens (Cx. pipiens) were distinguished. The prepared nanoformulation revealed very good physicochemical properties, especially the homogeneity of the particle size. Transmission electron microscope showed spherical shaped nanoparticles within range less than 200 nm. The prepared NLC-MB-MT system showed a very competitive insecticidal activity and high virulence against the mosquito larvae with higher mortality rate of LC50 of 0.141 µl/mL, in addition to high level of Oxidative stress parameters obtained through all the tested enzymes including hydrogen peroxide (4.8 ppm), protein carbonyl amount (0.12 OD/mg protein), ascorbic acid (0.15 mg) and Superoxide dismutase (SOD) showed strong increasing (0.09 OD/mg protein/min) at 6 µg/mL, respectively. Whereas paradoxical results of the oxidative stress enzymes were obtained from different concentration of nanoformulation that introduce a convenient reason for their potential insecticidal effect. The cytotoxic effect of NLC-MB-MT was evaluated using WI38 human lung cell lines, the LC50 was 6.4 mg/mL. The low cytotoxic reactivity towards the tested cell line makes the NLC-MB-MT nanoformulation has its promising insecticidal efficacy. Molecular docking study for each component were done against acetylcholine esterase protein and accepted binding modes achieved by the three compounds.

www.nature.com/scientificreports/ creation, steroid synthesis, detoxification reactions, phagostimulation, or neuromodulation in addition to its potential general role in detoxifying superoxide (O 2 ) and hydrogen peroxide (H 2 O 2 ) 48,49 . Therefore, the current study aimed to improve and understand the use of NLC incorporated with MB carvacrol and citronellal mono Terpenes (NLC-MB-MT) as insecticide via studying to oxidative stress parameters as key of protective indicators towards Cx. pipiens larvae and to evaluate: (i) ROS concentration (H 2 O 2 ) (ii) determines enzymatic antioxidants activity (Ascorbic acid concentration& SOD activity) (iii) protein carbonyl amount after treatment by NLC-MB-MT with different concentrations on the fourth instars of Cx. pipiens with respect to control insects.
Synthesis of encapsulated nanostructure lipid carrier. The preparation of lipid carrier nanoparticles encapsulated MB, carvacrol and citronellal monoterpenes (NLC-MB-MT) was done by the assistance of the homogenization method according to Radwan et al. 50 . It was prepared by using three different beaker (B1), (B2) and (B3). Firstly, the aqueous solution prepared (B1), 4 mL de-ionized water, 3 mL Tween 20 and 0.2 mL butanol as co-surfactant were mixed to a well-stirred and pre-warmed mixture of 0.25 g sodium glycocholate and 0.25 g sodium taurocholate solubilized in 10 mL water then kept warmed at 45 °C.
On another beaker (B2), the lipid contents with the drugs need to be encapsulated, were placed with the quantities of 0.9 g of stearic acid, and 0.018 g of MB. A stock mixture of methanol and chloroform 3:1 (v/v) was prepared and 2 mL withdrawn from the solvent stock solution and used to dissolve MB, the temperature raised gradually up to 85 °C, consequently the stearic acid became molten and the MB dissolved, keeping the heating for 7 min further until all the solvent evaporated (use digital balance to make sure that all the weighted solvent evaporated and no effervescence due to solvent still present). Allow the beaker (B2) to cool up to 70 °C using infra-red thermometer to monitor the temperature change and keep heating to this temperature, the molten should be clear blue solution if there was any non-dissolved MB the dissolution step should be repeated with increasing the quantity of stearic acid.
Using 5 mL beaker (B3), exactly 1.2 g of oleic acid and 0.02 g of both carvacrol and citronellal (each one 0.01 g) were added. The mono terpenes (MT) was injected beneath the oleic acid layer using micropipette to reduce volatile ability, then the mixture stirred at 150 rpm for 2 min. The content of beaker (B3) was added to beaker (B2) and mixed together mechanically at 250 rpm at the same temperature for one min (the overheat avoided) quickly, the beaker (B1) was added to the mixture with stirring at 600 rpm for 2 min and the final mixture was quenched by adding 20 mL ice cold water and sonication applied using probe sonicator (VCX 750 sonicator & 13 mm probe) for 15 min then the final emulsion was kept in 50 mL falcon tube at a temperature 10 °C.
Characterization of nanostructure lipid carrier. The average particle size (DLS), polydispearsity index (PDI) and zeta potential. The radius and polydispersity index (PDI) were measured by using the dynamic light scattering technique (DLS). The measuring conditions were set to be at 25 °C (the room temperature) with an angel of 173°. Zeta potential (z.p) was investigated by estimating the frequency shift change of the scattered light at a scattering angle of 12° due to laser beam irradiation. The average size, PDI and zeta potential measurements were done using (Zeta sizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) in the Egyptian petroleum research institute (EPRI). The sample preparation by dissolving or dispersing about 5-10 mg of the solid NLC (if lyophilized) or the solution under investigation in 20 mL de-ionized water at the room temperature (25 °C) with homogenization (preferred to used probe sonication) for 5 min before investigating the sample, the sample investigated at zero day and 7-day preparation.
Internal morphology by transmission electron microscope (TEM). The shape of the NLC prepared and the internal structure visualization was carried out using Field Transmission Microscopy (HR-TEM, JSM-7100F) in the central labs of National Research center institute (NRC), Giza, Egypt. The images were recorded with JEOL JEM-2100-115 high-resolution transmission electron microscopes system the electron-accelerating voltage varied from 100 to 200 kV. The NLC sample prepared as follow, 1 µL of NLC was highly diluted with de-ionized (1:200 v/v) and placed and fixed on a 200 mesh carbon-coated grid, after 2 min the excess of liquid NLC was removed by cellulose filter. Phosphotungstic acid (PTA) was dropped (2-3 drops) to the grid for 10 s to allow the negative staining to occur then the excess PTA was removed via filter paper by absorption.
In-vitro cytotoxicity effect of the NLC-MB-MT nanoformulation on normal human cell lines (MTT assay). The fibroblast human lung cell lines WI38 (Siga Aldrich, Merck, Darmstadt, Germany) was used to perform the cytotoxicity assay of the NLC-MB-MT nanoformulation (American type culture collection, CCl-75). The cell line cultured RPMI media containing 10% fetal bovine serum (FBS). Antibiotics 100 unit/ mL were added, penicillin and streptomycin (each one 100 µg/ml to prevent any bacterial growth) and then incubated using CO 2 incubator at 37 °C with humidity rate 5%. The cell lines were transported and seeded in 96-well plates at a density 1.0 × 10 5  Docking procedure. The reference drug or co-crystallized drug (imidacloprid) was labeled in green color to be distinguished easily, binding sites was recognized automatically form the surface sand maps options to recall the co-crystallized ligand binding site directly and the docking completed after ligands and protein preparation, using default setting were used via Rotate Bonds" choice to permit flexible ligand-rigid receptor docking. The score function was changed to be the London G with triangle matcher replacement were set, 30 conformer was adjusted instead of the automatic option conformer of the best score ligand and low energetically were retained. The top five conformers scoring of ligand-receptor docking was then showed by two-and three-dimensional ligand-receptor interactions 51,52 . Insecticidal evaluation. Insecticidal effect and enzyme assessment. A colony of Cx. pipiens larvae were supplied from Department of Entomology, Faculty of Science, Cairo University. The larvae were kept in a plastic plate 20 * 20 * 10 cm 3 for 200 larvae in each plate. Larvae were supplied daily with fish food. The NLC-MB-MT was prepared by dissolving the different concentrations in 5 mL distilled water (1, 2, 4, 6 µL/mL) then adding larvae in each concentration. Control insects were treated with distilled water only. The experiment was stand along 24 h. Then, the mosquito larvae were collected using mesh and then were homogenized in 5 mL of an ice-cold phosphate buffer with additions from (60 mL of 50 mM phosphate buffer, 10 mL of 0.1% Triton 100, 5 mL of 0.05 mM CaCl 2 ; the mixture was filled with distilled water to a volume of 100 mL after correcting pH to 7.0 with HCl or NaOH). The samples were centrifuged at 2000×g for 10 min at 4 °C after homogenization (10 strokes/30 s using a pestle). Three replicates of the measurements were made (each replicate was a pool of ten insects). The Levine et al. 53 method was used to calculate the amounts of protein carbonyls. The bovine serum albumin (BSA) fraction V (Sigma-Aldrich, Missouri, United States) was used as the protein standard to measure the total protein concentration of the samples using the Bradford 54 technique by spectrophotometry.
The Misra and Fridovich 55 technique was used to measure SOD activity. The reaction mixture looked like this: 87 mL of the appropriate tissue's supernatant, 402 mL of a sodium carbonate buffer (200 mM; pH 10.0), 35 mL of EDTA (10 mM), and 2835 mL of newly produced epinephrine (15 mM). In order to quantify the absorbance, a UV/Vis Jenway-7305 spectrophotometer was used (Bibby Scientific Limited, Staffordshire, UK). OD/g protein/ min was used to express SOD activity.
For Ascorbic acid determination, 10 g of the sample was blended in the blender, the sample extract is created. A 250 mL conical flask was then filled with the sample and 50 mL of a 5% metaphosphoric acid acetic acid solution. The flask was filled with the remaining 50 mL of phosphoric acid solution. After using Whatman filter paper to filter the solution, the filtrate was collected to be tested for vitamin C. A small amount of bromine solution was added and blended with the filtered sample solution. To eliminate the bromine solution, a few drops of thiourea solution were then added to the sample solution. The sample solution then mixed with 1 mL of a 2,4 DNPH solution. 2,4 DNPH solution causes the coupling process. All of the standards and sample solution were held at 37 °C for 3 h to allow the reaction to finish. 5 mL of H 2 SO 4 was added after the solutions had cooled on an ice bath for three hours. Thus, colored solutions were produced, and their absorbance was measured at a certain wavelength 56 .
The method of Junglee et al. 57 was used to determine the concentration of H 2 O 2 spectrophotometrically. Simply put, a one-step extraction-colorimetric procedure was used, consisting of a homogenization step using PBS, pH = 7.0 mixed with 0.25 mL Trichloroacetic acid (TCA) (0.1% (w:v)), 0.5 mL KI (1 M), and then centrifuging the 1 mL samples at 12,000 xg for 15 min at 4 °C to measure the absorbance at 240 nm.  11.0%, and moisture 6.0%). To ensure best rearing conditions, the water lost due to evaporation was compensated with fresh distilled water and the dead larva were removed day by day. Using a collecting sieve, the pupae removed from the breeding pans into plastic pots (7 cm × 6 cm) half filled with de-ionized water then the pupae were transported to the adult breeding cages for adult emergence. Emerging adult Cx. pipiens mosquitoes were placed into a mesh-screened wooden cage of dimensions (35 × 35 × 35 cm) with a hole in the front for pupae insertion and the other daily-routine work of food insertion, removal of laid eggs, and cage cleaning. The cages were supplied with a small petri dish with a cotton pad soaked in a 10% sucrose sugar solution as a source of carbohydrates for both males and females. The sucrose solution was changed every day to prevent fungus contamination. To confirm blood feeding which was provided 3-4 days following the emergence of the female, a domestic pigeon was tied to the top of the rearing cage at the night shift, noticing that before the first blood meal the sugar was withheld for 12 h. A 250 mL plastic cups partly filled with dechlorinated tap water were used to allow the gravid females to oviposit while also increasing the relative humidity in the cage. Daily collections of oviposited egg rafts were made, and these were delicately transferred with a tiny paintbrush to white enamel pans (25 cm in diameter and 9 cm in depth), which were half filled with dechlorinated tap water. These pans were preserved until the eggs hatched and are covered to prevent the oviposition of other species.

Results
Polydispersity index and homogeneity. According to Fig. 1S and Table1 the polydispersity index of the NLC-MB-MT equal to 0.096 and 0.092 for zero-day and 7-day preparation, respectively. This result indicated PDI value < 0.1 by the mean the size distribution was highly homogeneous and that is very close to be monomodal particle size distribution.
Zeta potential and charge. After 7 days preparation the value of zeta potential of the prepared NLC-MB-MT nanoformulation exhibited negative sign as net charge density as clarified in Fig. 2S and Table 1 with high numerical value of − 27.4 mV and − 25 mV, respectively at zero-day and 7-day preparation indicating very good physical stability behavior and low tendency to aggregation.
Internal structure and particle morphology. The prepared NLC-MB-MT depicted regular spherical and semispherical particle shape in rage of size less than 200 nm as showed Fig. 1a and with the presence of negligible aggregation (marked with red cycle). Qualitatively, the fields in Fig. 1a and b presented particles in comparable and narrow size distribution also most of the nanoparticles in range of 66 nm. Furthermore, the encapsulation of lipid layer as outer layer and target compounds (MB, carvacrol and citronellal) on the inner layer that was clearly showed in Fig. 1c and d.

In-vitro cytotoxicity effect of the NLC-MB-MT nanoformulation on normal human cell lines (MTT assay).
The fibroblast human lung cell lines WI38 was chosen randomly as preliminary test to evaluate the cytotoxicity effect of the NLC-MB-MT nanoformulation. The lower IC 50 concentration indicated the more potent drug activity and that is not favorable in the cytotoxic activity evaluation using WI38 cell lines as it is indicating more sensitivity towards the cell lines and consequently the human tissue. After 48 h incubation the half maximal inhibitory concentration that inhibits 50% of the cells IC 50 equal to 6.4 mg/mL as showed in Table 2 and Fig. 2. Such result is very good relative to chemical pesticides as 2,4-phenoxy acetic acid (IC 50 after 72 h = 115 ± 4.39 µg/mL) 59 .

Molecular docking of acetylcholine esterase enzyme. Molecular docking study was performed
within the active site pocket of 2ZJU protein crystalized with imidacloprid insecticide Fig. 3. Comparing to the co-crystallized ligand (imidacloprid) binding interactions showed in Fig. 4, docking results revealed good binding affinity towards all the tested compounds (tested ligands) with accepted binding energies and RMSD values as shown in Table 3 and Fig. 5.

Effect of NLC-MB-MT on the hydrogen peroxide level (H 2 O 2 ). The larval tissue homogenates from
the control groups contained the least amount of H 2 O 2 . The fluctuation increasing pattern of the concentration  was found to be maximum at 1 µL/mL, then it decreased to 2 µL/mL, fluctuated by increasing at 4 µL/mL, and eventually decreased to 6 µL/mL, till it virtually approached the control groups at 12 h post injection, Fig. 6.

Effect of NLC-MB-MT on the protein carbonyl amounts.
The protein carbonyl content of the Cx.
pipiens larvae was significantly affected by the NLC-MB-MT nanoformulation treatment at 12 h after injection, particularly at 1 µL/mL where it reached the highest level (0.15 OD/mg), followed by 0.12 OD/mg at 6 µL/mL, before total protein abruptly decreased to 0.02 OD/mg at 4 µL/mL, and continued until reaching the control at 2 µL/mL recording 0.01 OD/mg. The total protein was dramatically reduced after reaching a level that was higher than the controls 12 h after injection as shown in Fig. 7.

Effect of NLC-MB-MT on the SOD activity and Ascorbic acid concentration.
There were nonsignificant differences (P > 0.05) in both SOD activity and ascorbic acid concentration of mosquito larvae after treatment with NLC-MB-MT nanoformulation concentrations (1, 2, 4, 6 µL/mL) after 12 h post injection com-         Table 5. Subsequent to stressor application compared to relevant controls, experimental larvae exhibited significant physiological changes in activities of the tested parameters in response to stressor challenge, as can be seen.

Discussion
The quality of any nanoparticles mainly determined by measuring their size and morphology, the particle size could be measured using DLS technique which measures the correlations (automatic correlation function) of the time-dependent fluctuations of the light scattered made by the nanoparticle's suspension which is governed by their Brownian motion 60 . By fitting the autocorrelation function, the diffusion coefficient determined and then the Stocks-Einstein is used to calculate the radius and consequently the hydrodynamic diameter. The present results is one of the smallest size of nanostructured lipid carrier with average size diameter of 69.38 nm, as usual NLC have particle size within range from 200 to 400 nm 61 and some other NLC have extended size up to 1000 nm 62 . Preparation of smaller size than 200 nm required excessive increasing in the surfactant concentration however, producing NLC smaller than 100 nm is attracting particular interest as a result of their superior to penetrate cellular barrier especially, in biological system 63 . PDI is very important parameter indicating homogeneity and stability of the synthesized nanoparticles. PDI for width sizePDI values for wide-size distribution is ranging from 0 to 1, smaller values of PDI = 0 indicates complete monodispears size distribution (monomodal), while PDI > 0.5 referees to polydisprese or bread size distribution 50,64 . The analysis of DLS in addition polydispersity index assumes the measured nanoparticles objects are of ideal spherical shapes. As a result of the fact of the particle size is exponential function-dependent to the scattering intensity, that means very small number of large molecules often masks the small ones, that will introduce misleading results and makes the device will not differentiate between small aggregated particles and larger ones 65 . Decreased of PDI values in case of NLC-MB-MT nanoformulation confirmed that the particle size distribution was best to be described with "narrow-size", that means all the particle size have comparable sizes and consequently wide size variation is limited.
The preparation of narrow size distributed nanoparticle is a magnificent challenging but really the stability factor should be taken into consideration, zeta potential is a physical property exhibited by any particle in a suspension comes from the idea of most colloidal system in aqueous medium carry an electric charge. Knowing the value of zeta potential could reduce the number of trials needed to design stable nanoformulations. Zeta potential of the prepared NLC-MB-MT nanoformulation exhibited negative sign not only due to the acidic lipid contents (oleic and stearic acid) but also to the negatively charged particles of the phenolic carvacrol and the polar aldehyde citronellal mono terpenes in addition the slightly acid MB dye. As a reason of the accumulation of the negative charges generates sufficient high repulsion force between the particles that makes the dispersion resist the flocculation or coagulation and the colloidal system will be stable. As increasing magnitude of the z.p values indicated more repulsion force and enhanced stability 66 . The best value of zeta potential are > + 30 and < − 30 mV 67 . Zeta potential of the nanoformulation NLC-MB-MT was found to be − 25 mV after 7 days preparation, this value is not the best zeta potential ever but it is indicating very good physical stability 68 and high repulsive forces between the colloidal particles.
The internal morphology of the nanoparticles is best to be described by the transmission electron microscope as it is support us with in-situ vision of the particle shapes and homogeneity 69 . The prepared NLC-MB-MT presented spherical and semispherical particle varied in their regularity in comparable rage of size less than 200 nm. Some of negligible aggregation presented, in-consistent with the outcomes obtained from zeta potential results. Qualitatively, Most of the synthesized nps presented comparable particle sizes with narrow size variation in addition most of the nanoparticles in range of 66 nm. The DLS, Zeta potential inconsistence with TEM analysis, confirmed the synthesis of stable narrow size-distributed nanostructure lipid carrier.
To determine how the co-delivered compounds could bind to the protein active site, docking study was done using chain A. From Table 1, all one of the tested compounds could bind to the active site with two different bonds varied from hydrogen bonding to hydrophobic bonds (aromatic), for example both carvacrol and MB could bind with one hydrogen bond and another aromatic interaction, whereas citronellal bind with two aromatic interactions. Comparing the docking results of the three ligands under investigation to the imidacloprid as cocrystalized ligand, the imidacloprid showed one aromatic interaction with the aminoacid (Thr185), meanwhile citronellal and carvacrol have interactions with the amino acids (Trp143) and (Thr185) with Root Mean Square Deviation (RMSD) values 0.901263118and 1.08430552, respectively. Such accepted RMSD values 52 values with low binding energy (− 4.06693888and − 3.93443322, respectively) confirmed that all the three compounds have successive binding modes more effective than imidacloprid to the vicinity of 2ZJU protein active site with acceptable binding energy. The use of bioinformatics is crucial to reduce the probabilities, in this study and according to the results of the molecular docking study which confirmed that using carvacrol, citronellal and MB will be effective and each one of them could replace the Imidacloprid with some types of accepted interaction and accepted binding modes. AChE inhibition assay were done for each separate compound in addition to the nanoformulation which contains merging the three active ingredients. The synthesized nps showed very  71 . The lower LC 50 concentration of the NLC-MB-MT nanoformulation indicated the more potent drug activity and that is not favorable in the cytotoxic activity evaluation using WI38 cell lines. Those results are very good relative to chemical pesticides as 2,4-phenoxy acetic acid (LC 50 after 72 h = 115 ± 4.39 µg/mL) 59 .
There is limited data on the effects of NLC-MB-MT nanoformulation on the parameters of oxidative stress, namely total antioxidant ability and reducing power. This information was taken into consideration while interpreting the findings of this experiment. Elevated quantities of detoxifying and antioxidant enzymes are produced by insects as a result of their defense mechanisms against certain insecticides [72][73][74] . By focusing on the conversion of superoxide anion radicals (O 2 ⋅−) into oxygen and H 2 O 2 , these findings provide an explanation of how SOD enzymes function 75 . The highly created H 2 O 2 concentration is converted by CAT enzymes to oxygen and water 76,77 . Hydroxyl radical production causes protein oxidation and denaturation when the rate of O 2 anions or H 2 O 2 breakdown is insufficient.
Oxidative stress parameters as a biomonitoring of pesticides applications were evaluated in various studies 78,79 . Due to a number of foreign and internal causes, (including the treatment of NLC-MB-MT nanoformulation) increases the formation of H 2 O 2 in living things, the levels of ROS generation are raised. Similar to previous findings, the current findings demonstrated a notable raise in H 2 O 2 generation rate nearly in each concentration of NLC-MB-MT. After 12 h post injection, the treatment of Cx. pipiens larvae causes an increase in ROS generation, particularly H 2 O 2 . The findings supported by Fridovich 80 theory that endogenous toxicant emit ROS that reveal elevated amounts of H 2 O 2 when they enter an insect's body 81 . H 2 O 2 damages cell membranes by oxidizing the proteins that make up the membranes (protein oxidation) 39,82 . The observed drop in total protein may be the result of protein breakdown into free amino acids brought on by the stress of the treatment. This may indicate that the insect was attempting to physiologically counteract the effects of the H 2 O 2 by producing defense proteins 83 .
The ROS accumulate as a result of oxidative stress when there is an imbalance between their synthesis and elimination 84,85 . As a result of oxidative stress, substances like DNA single strand breaks are all examples of damage to macromolecules 44,86 , protein carbonylation 2,87 and lipids suffer oxidative damage 73,88 . The cell macromolecular processes that produce oxidative damage can be minimized by an antioxidant defense system that includes both enzymatic and non-enzymatic mechanisms. Differences in biotic and abiotic circumstances, as well as harsh environmental variables including bacterial, fungal, and viral infections, insecticide, low or high temperature, among others, govern oxidative damage among insect species. Super oxide dismutases (SODs) are frequent enzymes in living things that become active when there is oxygen present. According to Łukasik et al. 42 , increasing oxidative stress caused a considerable decrease in ascorbate, the main non-enzymatic antioxidant that, along with ascorbate peroxidase which helps aphids get rid of harmful H 2 O 2 . These enzymes help O 2 be transformed into oxygen and H 2 O 2 . The ROS levels are regulated by SOD enzymes. However, in addition to the various concentrations, there was a reduction in Ascorbic acid and SOD activity in the mosquito larvae. Likewise, compared to the control, as shown in Spodoptera exigua larvae exposed to high humidity had significantly decreased SOD, CAT, and peroxidase activity 89 . These findings shed light on the various ways by which high insect mortality results in depleted antioxidant systems. The potential for using the antioxidant response of mosquito larvae or other oxidative stress indicators as a marker of NLC-MB-MT nanoformulationwas also evaluated. Additionally, a new approach of possible application of NLC-MB-MT nanoformulation as a disruptor of the mosquito larval tissues was presented.

Conclusion
Finally, our results demonstrate that these toxic effects of NLC-MB-MT were evidenced by significant changes in the activity of defensive enzymes. The present study addressed the use of natural monoterpenes and MB as co-delivery system incorporated into NLC-MB-MT as insect control. The reactive oxidative stress led to some biochemical changes in the mosquito larval tissues dependent on model of NLC-MB-MT nanoformulation application. The cytotoxic evaluation against WI38 cell line give initiative cytotoxic results so, it may help in a further investigation of the NLC-MB-MT nanoformulation toxicity in field application. Further studies on the pharmacological characterization of insect NLC-MB-MT nanoformulation may help us to develop new specific insecticides for pest management.

Data availability
This article presents all data created or analysed throughout the investigation.