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# Institution and analysis of glucose-modified nanocomposite liposomes for the remedy of cerebral malaria | Journal of Nanobiotechnology

### Screening goal supplies

Within the examine of molecular dynamics simulation, the phospholipid bilayer construction, the realm per lipid, deuterium order and the electron density within the simulation system have been chosen as parameters to investigate the reliability of the bilayer construction fashioned by numerous supplies and phospholipid. Primarily based on the outcomes, the popular focused materials was used for preparation of brain-targeted liposomes.

#### The formation strategy of lipid bilayer

Within the examine, the molecular dynamic simulation trajectory footage of phospholipid and supplies have been taken at 0 ns, 50 ns, 100 ns, 300 ns, 550 ns, 700 ns, 850 ns and 1000 ns. And the morphologies of phospholipid bilayer fashioned by supplies and phospholipid have been evaluated intuitively. The outcomes confirmed that in contrast with different supplies, the cholesterol-undecanoate-glucose conjugate was like ldl cholesterol and will type a steady bilayer construction with phospholipid (Fig. 3A–D and Extra file 1: Figs. S2–S4).

The soybean lecithin and cholesterol-undecanoate-glucose conjugate initially fashioned a phospholipid bilayer inside 50 ns however didn’t utterly type a phospholipid bilayer till 700 ns, and the construction fragments of glucose within the conjugates (crimson) confronted exterior the phospholipid bilayer, which was conducive to the popularity of mind focused liposomes by GLUT1 (Fig. 3E). As talked about above, the cholesterol-undecanoate-glucose conjugate was chosen as goal materials for additional examine.

#### The world per lipid at 1000 ns

The world per lipid (AL) at 1000 ns was calculated utilizing gridMAT-MD [54]. The AL of the highest and backside layers was 0.65 nm2 and 0.69 nm2 within the PLPC/ldl cholesterol simulation, respectively. The AL of prime and backside layers within the PLPC/cholesterol-undecanoate-glucose conjugate, PLPC/arbutin and PLPC/undecane-glucose conjugate was 0.43 nm2 and 0.42 nm2, 0.42nm2 and 0.52nm2 and 0.52 nm2 and 0.48 nm2, respectively. It could possibly be discovered that conjugates (cholesterol-undecanoate-glucose conjugate and undecane-glucose conjugate) and arbutin decreased the AL. Nevertheless, the AL is an insufficient parameter to estimate the standard of a drive area in a simulation. It’s extremely delicate not solely to the drive area itself but in addition to a variety of secondary simulation parameters. An alternative choice to AL as a measure of the standard of a simulation is the amount per lipid (VL). VL converges quicker than AL and exhibits much less fluctuation than AL [46]. VL might be calculated from the next equation [44, 46]:

$${V}_{L}=frac{V-{n}_{w}{V}_{w}}{{n}_{L}}$$

the place V is the amount of the simulation field and nL and nw are the variety of phospholipids (128) and water molecules (2000), respectively. Vw is the amount per water molecule. Vw = 3.15 × 10−2 nm3 was discovered at 323 Okay and a stress of 1 bar [46]. The VL was 1.72 nm3, 1.77 nm3, 1.23 nm3 and 1.24 nm3 within the PLPC/ldl cholesterol, PLPC/cholesterol-undecanoate-glucose conjugate, PLPC/arbutin and PLPC/undecane-glucose conjugate at 1000 ns, respectively. So, cholesterol-undecanoate-glucose conjugate is not going to be considerably affected the VL of phospholipid bilayers in contrast with ldl cholesterol.

#### Deuterium order

Deuterium order (SCD) is a parameter generally used to characterize the diploma of order of fatty chains in phospholipid bilayers. SCD might be calculated from the next equation [46, 55]:

$${S}_{CD}=frac{1}{2}(3{cos}^{2}{theta }_{i}-1)$$

the place θi is the angle between the ith molecular axis and the bilayer regular (z-axis). The deuterium order of the PLPC/ldl cholesterol and PLPC/cholesterol-undecanoate-glucose conjugate, PLPC/undecane-glucose conjugate and PLPC/arbutin are proven in Fig. 4A–D, respectively. The SCD of sn-1 and sn-2 in each simulations exhibited related adjustments, however the hydrocarbon chains within the PLPC/conjugate had decrease SCD. This discovering signifies that the SCD of the phospholipids within the conjugate was decrease than that of ldl cholesterol. However SCD in cholesterol-undecanoate-glucose conjugate is increased than each arbutin and undecane-glucose conjugate, which implies, examine with arbutin and undecane-glucose conjugate, the cholesterol-undecanoate-glucose conjugate could make phospholipid bilayers extra order and compact [56, 57]. It might conclude that PLPC/cholesterol-undecanoate-glucose conjugate liposomes might encapsulate extra medicine and stably in vivo ship medicine to focus on the mind.

#### The electron density within the simulation system

The electron density within the simulation system was analyzed, and the electrons within the hydrocarbon chain in PLPC/ldl cholesterol have been primarily gathered within the heart of the simulation field. The fees of the water and phospholipid heads have been primarily distributed on the sting of the field (Fig. 4E). Within the PLPC/ cholesterol-undecanoate-glucose conjugate, PLPC/undecane-glucose conjugate and PLPC/arbutin, the cost distribution was comparatively uniform (Fig. 4F–H). Within the PLPC/cholesterol-undecanoate-glucose conjugate, the costs of the phospholipid hydrocarbon chains have been primarily distributed at − 0.5 Å within the heart of the field. The fees of the cholesterol-undecanoate-glucose conjugates have been primarily concentrated at 1.8 Å within the heart of the field.

In abstract, by means of the evaluation of dynamic simulation knowledge, the outcomes confirmed that the cholesterol-undecanoate-glucose conjugate might higher type a steady and ordered phospholipid bilayer construction with phospholipid than different focused supplies. So, the cholesterol-undecanoate-glucose conjugate was chosen as GLUT1 focused materials for additional examine.

### Synthesis of cholesterol-undecanoate-glucose conjugate and its characterization

The synthesis of the cholesterol-undecanoate-glucose conjugate is printed in Fig. 5A. Glucose is a polyhydroxy compound, and there are a number of hydroxyl teams concerned in its synthesis, which will increase the problem of its purification. Due to this fact, an arbutin-containing fragment of glucose was reacted with bromine to generate cholesterol-undecanoate-glucose conjugate.

Ldl cholesterol-undecanoate-glucose conjugate was a white amorphous powder, soluble in ethanol and tetrahydrofuran. ESI–MS confirmed m/z: 847.6 [M + Na]+ (Extra file 1: Fig. S13), suggesting its molecular formulation is C50H80O9; infrared spectrum confirmed that the compound had a hydroxyl characterization absorption of three,395 cm−1, a saturated alkane attribute absorption of 2932 cm−1 and the attribute absorption of ester carbonyl at 1735 cm−1 (Fig. 5F). 1H NMR spectrum confirmed δH 0.69 (3H, s, H-18), 1.03 (3H, s, H-19), 0.99 (3H, d, J = 6.4 Hz, H-21), 0.91 (6H, d, J = 6.6 Hz, H-26, 27), 4.88 (1H, m, H-3) have been the attribute proton sign of the 4 nook methyl teams of ldl cholesterol and H-3. The 13C NMR knowledge of ldl cholesterol structural fragments have been as follows: δC 37.8 (C-1), 29.0 (C-2), 74.4 (C-3), 39.1 (C-4), 140.5 (C-5), 123.3 (C-6), 32.5 (C-7), 32.7 (C-8), 50.8 (C-9), 37.3 (C-10), 21.8 (C-11), 40.2 (C-12), 43.0 (C-13), 57.3 (C-14), 25.0 (C-15), 28.7 (C-16), 56.9 (C-17), 12.5 (C-18), 19.9(C-19), 36.5 (C-20), 19.5 (C-21), 37.0 (C-22), 24.7 (C-23), 40.4 (C-24), 28.7 (C-25), 23.5 (C-26), 23.2 (C-27), the above-mentioned spectral knowledge recommended that the cholesterol-undecanoate-glucose conjugate molecule had ldl cholesterol structural fragments. Within the 1H NMR spectrum, δH 2.43 (2H, t, J = 7.4 Hz, H-2′), 1.72 (2H, m, H-3′), 1.74 (2H, m, H-4′), 1.25–1.28 (8H, m, H-5′-8′), 1.43 (2H, m, H-9′), 1.75 (2H, m, H-10′), 3.89 (2H, t, J = 6.5 Hz, H -11′) have been the attribute sign of undecanoate proton. The corresponding 13C NMR knowledge have been as follows: δC 173.5 (C-1′), 35.3 (C-2′), 25.9 (C-3′), 29.9 (C-4′), 28.7 (C-5′), 30.0 (C-6′), 30.2 (C-7′), 30.1 (C-8′), 26.8 (C-9′), 30.2 (C-10′), 69.1 (C-11′); The above spectral knowledge recommended that the cholesterol-undecanoate-glucose conjugate molecule had undecanoate structural fragments. 1H NMR spectrum confirmed δH 7.38 (2H, d, J = 9.0 Hz, H-2”, H-6”) and 6.99 (2H, d, J = 9.0 Hz, H-3”, H-5′ ‘) have been the para-substituted fragrant ring proton sign, δH 5.53 (1H, d, J = 7.5 Hz, H-G-1), 4.29 (1H, m, H-G-2), 4.08 (1H, m, H-G-3), 4.33 (1H, m, H-G-4), 4.34 (1H, m, H-G-5), 4.54 (1H, d, J = 11.6 Hz, H-G-6a), 4.39 (1H, d, J = 11.6 Hz, H-G-6b) have been a set of proton alerts of glucose fragments. The corresponding 13C NMR knowledge have been as follows: δC 153.2 (C-1”), 118.8 (C-2”, 6”), 116.2 (C-3”, 5”), 155.3 (C- 4”), 103.8 (G-1), 75.5 (G-2), 79.3 (G-3), 71.8 (G-4), 79.0 (G-5), 62.9 (G-6). The above spectral knowledge recommended that the molecule of cholesterol-undecanoate-glucose conjugate had arbutin structural fragment.

Via the evaluation of the above-mentioned spectral knowledge, it could possibly be decided that the structural fragment of the cholesterol-undecanoate-glucose conjugate had a sequence of structural options of the goal product. With a purpose to make clear the connection mode of every structural fragment and the attribution of the hydrocarbon sign, two-dimensional nuclear magnetic spectroscopy HSQC, HMBC and 1H-1H COSY assessments have been additional carried out. Within the HMBC spectrum, the δH 4.88 (1H, m, H-3) of the ldl cholesterol construction fragment was remotely associated to the δC 173.5 (C-1′) of the undecanoate construction fragment, indicating that the eleventh alkanoate structural fragments have been related within the type of ester bonds by means of C-1′ and C-3 of ldl cholesterol. As well as, the HMBC spectrum confirmed that the δH 3.89 (2H, t, J = 6.5 Hz, H-11′) within the undecanoate construction fragment and the δC 155.3 (C -4”). There was a long-distance correlation, indicating that the undecanoate structural fragment was related within the type of an ether bond by means of C-1′ and the C-4″ of the arbutin structural fragment (Fig. 5B–E). Mixed with the evaluation of the HMBC spectrum, it may be decided that the cholesterol-undecanoate-glucose conjugate was the goal product, particularly: cholesterol-undecanoate-glucose conjugate.

### Characterizations of liposomes

The sizes of na-ATS/TMP@lipoBX, iv-ATS/TMP@lipoBX and na-ATS/TMP@lipo have been 86.2 ± 0.9 nm, 96.2 ± 2.0 nm and 96.7 ± 1.1 nm, respectively (Fig. 3A). Via the commentary of TEM, the liposomes confirmed a clean spherical form (Fig. 6E, H and Extra file 1: Fig. S15). The looks of liposomes was translucent (Fig. 6G, H and Extra file 1: Fig. S16). The DSC spectrum of iv-ATS/TMP@lipoBX was proven in Extra file 1: Fig. S17.

In comparison with na-ATS/TMP@lipoBX and na-ATS/TMP@lipo, the liposomes (na-ATS/TMP@lipoBX) modified by the conjugate haven’t any vital adjustments of their characterizations. The end result might not directly confirm the cholesterol-undecanoate-glucose conjugate can type a steady bilayer construction with phospholipid, which is a conclusion of molecular dynamics simulation.

### The encapsulation effectivity of liposomes

The encapsulation effectivity of ATS and TMP within the na-ATS/TMP@lipoBX and iv-ATS/TMP@lipoBX have been measured by high-performance liquid chromatography (HPLC). The encapsulation effectivity of ATS within the three sorts of liposomes was increased than 90%, and the encapsulation effectivity of TMP was near 30% (Fig. 6B).

### In vitro drug launch

The in vitro launch fee is an important parameter for management of drug high quality. The cumulative launch charges of ATS from iv-ATS/TMP@lipoBX and na-ATS/TMP@lipoBX have been about 80% and 50% within the first 8 h, respectively. The cumulative launch charges of TMP from iv-ATS/TMP@lipoBX and na-ATS/TMP@lipoBX have been about 65% and 55% within the first 1 h, respectively (Fig. 6C, D). The discharge of ATS and TMP from the above two liposomes could possibly be categorised into two phases: a speedy launch section throughout which unencapsulated ATS and TMP coordinated on the floor of the lipid bilayer could be launched . This preliminary section was adopted by a gentle launch, often called the sluggish launch section [58].

ATS was launched from iv-ATS/TMP@lipoBX and na-ATS/TMP@lipoBX at totally different charges through the speedy launch section, and this end result could be defined by the upper the phospholipid content material within the na-ATS/TMP@lipoBX, which encapsulated ATS tighter. This launch characterization of na-ATS/TMP@lipoBXg is helpful to inhibit the recurrence of malaria.

### Stability examine of liposomes

#### Placement stability of liposomes

The RSDs of particle measurement and encapsulation effectivity of na-ATS/TMP@lipo at weeks 0,1,2,3, and 4 have been as follows: 0.98% and 0.59% (ATS), 1.50% (TMP). The RSDs of particle measurement and encapsulation effectivity of iv-ATS/TMP@lipoBX at weeks 0,1,2,3 and 4 have been as follows: 0.82% and 1.82% (ATS), 5.61% (TMP) (Fig. 7A–C).

#### Stability of liposomes in serum

The particle measurement RSDs of na-ATS/TMP@lipo inside 24 h and 48 h in serum have been as follows:1.72% and a pair of.63%; the particle measurement RSDs of iv-ATS/TMP@lipoBX inside 24 h and 48 h in serum have been as follows:7.11% and 15.54% (Fig. 7B).

### In vivo brain-targetability analysis

The distribution of the liposomes within the mice was evaluated dynamically utilizing a DiR fluorescent probe. The outcomes confirmed that na-DiR@ATS/TMP@lipoBX (brain-targeted liposomes) had an extended retention time within the mind in comparison with na-DiR@ATS/TMP@lipo (non-targeted liposomes) (Fig. 8B). At 48 h after administration, a sure depth of fluorescence sign might nonetheless be detected within the remoted mind of mice within the focused administration group, whereas the fluorescence depth within the mind of the non-targeted administration group decreased quickly at 4 h after administration (Fig. 8D).

The consequences of in vivo mind fluorescence imaging and ex vivo mind fluorescence imaging was in contrast within the examine. Within the in vivo fluorescence imaging outcomes, apparent fluorescence alerts could possibly be seen within the mouse head 1 h after administration, however the mind ex vivo fluorescence imaging outcomes confirmed that the focused liposomes entered the mind with a sure delay. The fluorescence depth within the mind reached the utmost worth 4 h after administration (Fig. 8C).

In mind, fluorescence imaging confirmed that liposomes first reached the olfactory bulb after nasal administration. Some research have proven that cerebral malaria may cause hemorrhage and irritation within the olfactory bulb [59,60,61], so brain-targeted liposomes can ship medicine to the olfactory bulb to attain focused remedy. The efficacy analysis outcomes on this paper verified some great benefits of na-DiR@ATS/TMP@lipoBX within the remedy of cerebral malaria.

After iv-ATS/TMP@lipoBX injection, the fluorescence sign could possibly be detected quickly within the remoted mind, however the fluorescence depth decreased considerably over time, and there was no apparent fluorescence sign within the remoted mind after 8 h.

In contrast with intranasal administration of DiR@ATS/TMP@lipoBX, the injection of iv-ATS/TMP@lipoBX had a shorter retention time within the mind, which affected the antimalarial efficacy (Fig. 8A).

### Modeling cerebral malaria

Smears of crimson blood cells obtained from wholesome mice and mice contaminated with P. falciparum are proven in Fig. 9A. The crimson blood cells within the wholesome mice have been uniform in measurement, formed like a double concave disc, and harbored no irregular constructions within the cytoplasm. The crimson blood cells contaminated with P. falciparum had the form of rings, trophozoites and schizonts (crimson arrow), which indicated that the modeling was profitable.

### Impact of medication on speedy murine coma and habits scale (RMCBS) of mice

When observing the habits of mice throughout train and exploration, their nervousness, despair, and different signs can replicate the impact of medication. Mice within the mannequin and drug-free lipo group started to indicate unsteady strolling, decreased limb power, and a lack of fur gloss by the fifth day of the experiment, whereas their RMCBS confirmed a downward pattern all through the experiment. The RMCBS of the opposite teams confirmed a slight downward pattern through the commentary interval. In contrast with the mannequin and drug-free lipo teams, there was a big distinction (P < 0.05). The na-ATS/TMP group confirmed a big lower in RMCBS on the ninth day, presumably as a result of the small nasal space of the mice absorbed an inadequate quantity of the drug, and the answer lacked a liposome-like sustained-release impact, which allowed the drug to clear from the physique quickly. On the twelfth day, the RMCBS of the na-ATS/TMP@lipoBX group was increased than that within the different administration teams (Fig. 9B).

### Impact of medication on weight of mice

The physique weights of mice within the mannequin, drug-free lipo and na-ATS/TMP (intranasal administration of ATS and TMP options) teams through the commentary interval first elevated after which decreased. The physique weights of the mice within the na-ATS/TMP@lipo teams confirmed an rising pattern that reached a most on the ninth day; through the commentary interval, the physique weights of mice within the iv-ATS/TMP (intravenous administration of ATS and TMP options) and iv-ATS/TMP@lipoBX teams confirmed a pattern of first rising, then lowering after which rising. The physique weights of mice within the na-ATS/TMP@lipo and the iv-ATS/TMP teams have been increased than these of the mannequin group (P < 0.05) and considerably increased than these within the drug-free lipo group (P < 0.01) (Fig. 9C).

### Impact of medication on an infection fee of mice

The an infection fee is a crucial indicator for evaluating drug efficacy, and on this case, the an infection fee is set by the variety of crimson blood cells contaminated by P. falciparum divided by the full variety of crimson blood cells (both 1000 or greater than 1000 blood crimson cells). On this examine, the therapeutic results of the answer teams and the liposome teams on the cerebral malaria mannequin mice after injection and intranasal administration have been evaluated. Desk 1 exhibits that the an infection fee of the na-ATS/TMP@lipoBX (brain-targeted liposomes) group was considerably decrease than that of the opposite administration teams, and the an infection fee on the ninth day of commentary was solely 3.34%. This was considerably decrease than the mannequin, drug-free lipo and na-ATS/TMP teams (P < 0.01) and decrease than the na-ATS/TMP@lipo, iv-ATS/TMP@lipoBX and iv-ATS/TMP teams (P < 0.05). There was no vital distinction between the an infection fee of the mannequin and drug-free lipo teams through the commentary interval (P > 0.05), indicating that drug-free lipo had no pharmacological results (Fig. 9D). These outcomes confirmed that brain-targeted based mostly on GLUT1 might considerably improve the therapeutic impact towards cerebral malaria.

### Results of medication on destructive conversion fee of mice

The destructive conversion fee was indicated by no P. falciparum or an an infection fee < 0.5% for every mouse after stopping administration for twenty-four h, and the destructive mice have been categorised by the full mice in every group. The destructive conversion fee of the iv-ATS/TMP group was 70%, that of the na-ATS/TMP group was 90%. And the destructive conversion fee of the iv-ATS/TMP@lipoBX group was 80%, the destructive conversion charges of na-ATS/TMP@lipoBX and na-ATS/TMP@lipo teams have been each 90% (Fig. 9E). In response to the above, the destructive conversion charges of the intranasal administration teams have been higher than that of the injection teams.

### Impact of medication on recurrence fee of malaria

The recurrence fee of the destructive mice after stopping administration was evaluated. The na-ATS/TMP@lipoBX (brain-targeted liposomes) confirmed vital benefits in stopping the recurrence of cerebral malaria. Cerebral malaria recurred in all mice of the injection (iv-ATS/TMP@lipoBX and iv-ATS/TMP) teams on the sixth day after stopping administration. Cerebral malaria recurred in all mice of the na-ATS/TMP@lipo teams on the seventh day after stopping administration. Nevertheless, the mice within the na-ATS/TMP@lipoBX group had a decrease recurrence fee, solely 50% of mice have a resurgence of malaria on the ninth day after stopping administration (Fig. 9F).

The outcomes of this examine are per the outcomes of in vivo brain-targetability analysis. After nasal administration, brain-targeted liposomes (na-ATS/TMP@lipoBX) could possibly be successfully enriched within the mind and extend the retention time of the drug within the mind with the assistance of cholesterol-undecanoate-glucose conjugate. Because of this, brain-targeted liposomes might successfully lower the resurgence fee of cerebral malaria. It’s of nice significance to the remedy of cerebral malaria.

### Pharmacokinetic and mind biodistribution research

The focus curves and pharmacokinetic parameters of ATS, TMP and dihydroartemisinin (artesunate metabolites [62]) in plasma or mind after intranasal (i.n.) administration of na-ATS/TMP@lipoBX and intravenous (i.v.) administration of na-ATS/TMP@lipoBX in mice are proven in Fig. 10 and Desk 2. The outcomes confirmed that na-ATS/TMP@lipoBX after i.n. administration might enter the systemic circulation in massive portions. The relative bioavailabilities of ATS and TMP in plasma by i.n. administration was roughly was 91% and 97%, respectively, in contrast with that of i.v. administration. This means that the focused liposomes might ship medicine to the mind throughout the blood–mind barrier through this pathway.

After i.n. administration of na-ATS/TMP@lipoBX, the focus of the drug within the mind was considerably increased than that within the i.v. administration group. The relative bioavailabilities of ATS and TMP within the mind by i.n. administration was roughly 415% and 183%, respectively, in contrast with that of i.v. administration. Primarily based on this end result, it could possibly be speculated that focused liposomes might additionally ship medicine on to the mind by means of the nasal-brain pathway [63]. The in vivo imaging outcomes (In vivo brain-targetability analysis) of na-ATS/TMP@lipoBX verified this speculation, and the examine confirmed that na-ATS/TMP@lipoBX might combination on the olfactory bulb to attain direct brain-targeted supply.

As well as, ATS could possibly be metabolized within the physique into one other antimalarial energetic drug, dihydroartemisinin (DHA) [62]. The focus of DHA within the mind was quantitatively analyzed, the focus curve and pharmacokinetic parameters of DHA in mind after i.n. administration of na-ATS/TMP@lipoBX or i.v. administration of iv-ATS/TMP@lipoBX was proven in Fig. 10C and Desk 2. After i.n. administration, the Cmax and AUC (0-t) of DHA have been 10.4 and 4.5 instances these of i.v. administration, respectively. The relative bioavailability of DHA in mind by i.n. administration was roughly 449% in contrast with that of i.v. administration. A better focus of DHA delivered to the mind might kill the malaria parasite in mind extra successfully.

The above outcomes confirmed that na-ATS/TMP@lipoBX might enter the mind in two methods (systemic circulation and nasal-brain pathways) to supply antimalarial results after i.n. administration and the liposomes had an excellent brain-targeted drug supply capability by i.n. administration.

### Histological analysis.

The pathological outcomes of every administration group are proven in Fig. 11. No apparent histological abnormalities have been brought on by the nano-preparations in any of the principle organs of the mice after intranasal administration of na-ATS/TMP@lipoBX (brain-targeted liposomes). This end result exhibits that na-ATS/TMP@lipoBX has good biosafety.

As well as, in contrast with the intranasal administration group, the alveolar partitions of the mice within the tail vein injection teams (iv-ATS/TMP@lipoBX group and drug-free lipo group) and mannequin group have been considerably thicker; the mannequin and drug-free lipo teams have been thought to indicate pathological adjustments in response to P. falciparum invasion. Within the intranasal administration of na-ATS/TMP@lipoBX group, the nuclei of liver cells stained extra evenly, and the liver cells have been organized radially across the central vein (Fig. 11). The above-mentioned pathological outcomes additionally confirmed that na-ATS/TMP@lipoBX had sure benefits within the remedy of cerebral malaria in mice.

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