Application of DHA@ZIF-8 in treating cerebral malaria in mice

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to an application of DHA@ZIF-8 in treating mouse cerebral malaria. The present invention first establishes a mouse cerebral malaria model, then uses ZIF-8@DHA on the model for treatment, and observes the therapeutic effect of the nano drug on mouse cerebral malaria by behavioral, pathological, molecular biological and other technical means; the present invention uses ZIF-8 as a carrier to achieve the effect of sustained release of the drug, thereby extending its action time in the body to more effectively kill malarial parasites. Compared with natural administration, the action time of DHA is extended.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of DHA@ZIF-8 in treating cerebral malaria in mice. Background Art

[0002] Cerebral malaria (CM) is a neurological term published in 2020. It is a parasitic disease of the brain caused by direct invasion of malarial parasites, especially Plasmodium falciparum. It is a serious disease with a high mortality rate and is one of the most serious types of malaria. About 1% to 2% of falciparum malaria will eventually develop into cerebral malaria, which mostly occurs in children under 5 years old in Africa, with a mortality rate of about 15% to 20%. Clinically, cerebral malaria may present with mild hemiparesis, convulsions, ataxia, coma and impaired consciousness, meningeal irritation signs and even death. Even for patients who receive early standard antimalarial chemotherapy drugs, there is still a very high mortality rate after the malarial parasites are cleared from the body. In addition, about 25% of survivors will have sequelae of neurological complications and cognitive dysfunction. 10% to 20% of surviving children have persistent neurological deficits, cognitive dysfunction, behavioral disorders, and motor impairment.

[0003] Currently, quinine and artemisinin are recommended drugs for the treatment of CM. Although artemisinin drugs have a highly effective antimalarial effect, the dead parasites after killing the malarial parasites still block the local microvessels. The local ischemic changes and microvascular damage caused by the blockage cannot be effectively repaired, making the mortality rate of cerebral malaria in children and adults still as high as 18% and 30%. Studies have also shown that artemisinin-based combination therapies (ACTs) often fail to protect against cell death, neural damage and cognitive deficits. Therefore, artemisinin drugs, especially dihydroartemisinin (DHA), alone are not enough to prevent death or neurological dysfunction in all patients with cerebral malaria. This suggests that we need to continue to explore new artemisinin-based drug combinations, treatment plans and auxiliary treatments based on existing treatment strategies.

[0004] Zeolitic imidazolate frameworks (ZIFs) are composed of metallic zinc. 2+It is a tetrahedral structure, and the four zinc nodes are coordinated with the nitrogen atoms of 2-methylimidazole (Hmim) to form ZIF-8. Zinc is the second most abundant metal in the human body, and the imidazole group exists in human histidine, and the cytotoxicity is negligible, so ZIF-8 has good biocompatibility and safety. ZIF-8 has good thermal stability and chemical stability, and dissolves under acidic conditions, which is conducive to the release of drugs on demand under the control of pH. In addition, ZIF-8 has a high specific surface area and porosity, a flexible structure, and adjustable pore size, which can control the release of drugs. Premature degradation of drugs often makes it difficult to play a therapeutic role. Adsorbing them on ZIF-8 can prevent premature release of drugs. On the one hand, the ZIF-8 shell has a protective effect on the drug. Under the wrapping of ZIF, the sensitivity of the drug to the external environment is reduced; on the other hand, the metal ions of ZIF-8 interact with the drug, causing the drug to transform into a stable isomer and enhance stability. Drug release is carried out in stages, that is, the drug on the surface of ZIF-8 is dissolved and released first; then the drug inside the material gradually diffuses outward and dissolves along the concentration gradient; as the skeleton collapses, the drug wrapped in the ZIF-8 cavity is released, and these stages ultimately achieve sustained and slow drug release. Pore size adjustment or structural modification of ZIF-8 can change the affinity between the drug and the nanocarrier and control the release rate of the drug. Modification of targeting ligands can deliver drugs to the lesion site and achieve precise treatment of the disease. Integrating multiple drugs for combined treatment of CM into the same nanocarrier is an important research direction for the treatment of CM in the future. Therefore, we believe that ZIF-8 is a very potential nano drug carrier for the treatment of CM.

[0005] To this end, the present invention constructs a nano drug delivery system ZIF-8@DHA, establishes a mouse cerebral malaria model, uses the nano drug for intervention, and evaluates its effect in treating mouse cerebral malaria from the perspectives of behavior and pathology, in order to provide an effective solution for the treatment of human cerebral malaria.

[0006] Studies have shown that ZIF-8@DHA has a therapeutic effect on diseases such as tumors. However, whether and how it works in infectious diseases, especially cerebral malaria, has not been reported. The reason for the above-mentioned lack of research may be that the use of this nano drug belongs to the interdisciplinary subject involving materials science, pharmacology, and parasitology. Experts and scholars in the field of malaria are more concerned about the molecular biological basis of Plasmodium pathogenicity. In terms of treatment, more attention is focused on developing new drugs to replace artemisinin, or on studying vaccines for the prevention or treatment of malaria through various technical means. At present, there are some progress in the above, but there is still a certain distance from actual clinical treatment. The present invention is based on the perspective of interdisciplinary studies, and is prompted by the use of nano drugs to treat major diseases such as tumors, and research on nano drugs to treat malaria has been carried out earlier in China.

[0007] To this end, the present invention attempts to reveal the role of ZIF-8@DHA in the treatment of mouse cerebral malaria and its possible mechanism of action. Specifically, a mouse cerebral malaria model was first established, and then ZIF-8@DHA was used for treatment on the model. The therapeutic effect of the nanomedicine on mouse cerebral malaria was observed through behavioral, pathological, molecular biological and other technical means. Summary of the invention

[0008] The present invention first provides an application of DHA@ZIF-8 in treating cerebral malaria, wherein the application is in vivo in mice.

[0009] In certain embodiments, the cells acted upon in vivo are mouse erythrocytes infected with Plasmodium berghei.

[0010] In certain embodiments, the cerebral malaria is mouse cerebral malaria.

[0011] In certain embodiments, the administration concentration of DHA@ZIF-8 is 0.5-5 mg / kg of DHA as the final concentration.

[0012] In certain embodiments, the administration concentration of DHA@ZIF-8 is 3 mg / kg of DHA as the final concentration.

[0013] The present invention also provides an application of DHA@ZIF-8 in preparing a medicine for treating cerebral malaria.

[0014] In certain embodiments, the in vitro is a cell.

[0015] In certain embodiments, the cerebral malaria is mouse cerebral malaria.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] In view of the short half-life of dihydroartemisinin in vivo, the present invention uses ZIF-8 as a carrier to achieve the effect of sustained release of the drug, thereby prolonging its action time in vivo to more effectively kill malarial parasites. Compared with natural administration, the action time of DHA is prolonged. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Effects of different drug treatments on behavioral indicators of ECM mice. A. Body weight; B. RMCBS; C. Protozoaemia; D. Survival rate.

[0019] Figure 2 Detection of blood-brain barrier permeability in ECM mice treated with different drugs. A. Representative images of brain tissue; B. Quantitative evaluation of blood-brain barrier permeability.

[0020] Figure 3Histopathological observation of mouse HE staining. A. Brain tissue (1000×); B. Liver (400×); C. Spleen (400×).

[0021] Figure 4 Quantitative analysis of hemochromatin and red pulp area in liver and spleen tissues of mice. A. Hepatic hemochromatin ratio; B. Spleen hemochromatin ratio; C. Spleen red pulp area (400×). *, P<0.05; **, P<0.01; ***, P<0.001. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 Materials and Methods

[0024] Experimental animals, Plasmodium strains and strains:

[0025] Female C57BL / 6 mice (18-22 g, 8-10 weeks old) were purchased from HNSJA Co., Ltd., Changsha, China. They were housed under specific pathogen-free conditions and fed with UV-irradiated feed and purified water to maintain appropriate living and feeding standard experimental conditions (25 ± 3 °C). All mice were acclimatized to the environment 1 week before the experiment.

[0026] The Plasmodium berghei strain ANKA was kindly donated by Professor Xu Wenyue of the Army Medical University and was cryopreserved in liquid nitrogen for long-term storage in our laboratory.

[0027] The experimental instruments are shown in Table 1;

[0028] Table 1 Experimental instruments

[0029] name model Origin and Company Scales 76175 China, Bei Xi Man OLYMMPUS Microscope BX53 Olympus Corporation, Japan Centrifuge 5424R Eppendorf, Germany Constant temperature metal bath MK-20 China, Aosheng Tissue Slice Image Imaging System BX53 Olympus Corporation, Japan Micropipette 2-volume pipette us imaging system Eppendorf, Germany Multifunctional ELISA reader SYNERGY-HT BioTek, USA Mouse tail vein injection instrument KW-XXY Calvin, China refrigerator -20℃，-80℃ Haier, China

[0030] The experimental reagents and consumables are shown in Table 2;

[0031] Table 2 Experimental reagents and kits

[0032]

[0033]

[0034] Example 2

[0035] Resuscitation and passage: Place the PbA malarial parasite strain frozen in liquid nitrogen in a 37°C water bath, and draw blood with a 1mL syringe after it is thawed. Immediately inoculate it intraperitoneally into C57BL / 6 mice at a dose of 0.2mL / mouse. This is blood seed resuscitation. When the parasitemia level of the blood seed mice reaches 15%-30%, remove the eyeballs and draw blood in a heparin anticoagulant tube. 6 The iRBCs-infected erythrocytes were passaged into offspring C57BL / 6 mice at a dose of 0.2 mL / mouse.

[0036] (1) Constructing ECM model

[0037] The P.bergheiANKA malarial parasite strain (PbA) frozen in liquid nitrogen was taken out and placed in a 37°C water bath. After thawing, blood was drawn with a 1 mL syringe and immediately injected intraperitoneally into C57BL / 6 mice at a dose of 0.2 mL / mouse. This was blood seed recovery. When the parasitemia level of the blood seed mice reached 15%-30%, the eyeballs were removed and blood was collected in heparin anticoagulation tubes. 1×10 6 The blood of infected red blood cells (iRBCs) was passaged to the offspring C57BL / 6 mice at a dose of 0.2 mL / mouse. When the protozoanemia level of the offspring mice reached 15%-30%, the eyeballs were removed and blood was collected in heparin anticoagulant tubes. 6 iRBCs were inoculated into experimental mice at a dose of 0.2 ml / mouse, which was recorded as the inoculation day 0, which was the establishment of ECM.

[0038] (2) Drug synthesis

[0039] Zinc nitrate hexahydrate 45 mg was dissolved in 1.5 mL of deionized water. 2-Methylimidazole 99 mg was dissolved in 2.7 mL of methanol. DHA 5 mg was dissolved in 0.25 mL of DMF. At room temperature, 2-methylimidazole and DHA solution were added to the aqueous solution, stirred continuously, centrifuged at 12,000 rpm for 8 min, and DHA@ZIF-8 nanoparticles were obtained, which were washed with methanol three times. DHA@ZIF-8 was synthesized and kept at 4 °C for subsequent use. The morphological characteristics of DHA@ZIF-8 were examined by scanning electron microscopy and transmission electron microscopy. In addition, the size and zeta potential of DHA@ZIF-8 were evaluated using a Malvern laser particle size analyzer. Transmission electron microscopy and scanning electron microscopy showed that DHA@ZIF-8 had a typical crystalline structure with a physical size of 250 nm. The zeta potential of DHA@ZIF-8 verified by the Malvern zeta potentiometer showed a positive value. It is worth noting that the value increased slightly with the loading of DHA. Overall, these results indicated that DHA@ZIF-8 had been successfully synthesized.

[0040] (3) Experimental groups and drug treatment plans

[0041] According to different dosing strategies, after the experimental mice were adaptively fed, the C57BL / 6 mice were weighed and randomly divided into 4 groups, with 10 mice in each group. DHA, DHA@MOF and DHA@ZIF-8 were dissolved in 5% dimethylsulfoxide (DMSO) in normal saline (0.9% NaCl) with a final concentration of 3 mg / kg DHA. The infected untreated group (PbA) mice were injected with 5% DMSO on the third day after infection. The DHA group was given 3 mg / kg DHA on the third day after infection. The MOF group was given 3 mg / kg DHA@MOF on the third day after infection. The ZIF group was given 3 mg / kg DHA@ZIF-8 on the third day after infection. Each drug was administered for 5 consecutive days, with a one-time administration of 200 μL.

[0042] (4) Basic indicator testing

[0043] Protozoaemia observation: Blood was collected from the tail vein to prepare thin blood smears, which were then stained with Giemsa. Protozoaemia was evaluated by Giemsa-stained thin blood smears under a 100x optical microscope. Protozoaemia was examined and quantified by counting the number of iRBCs in at least 1,000 red blood cells.

[0044] Detection of basic behavioral indicators: Starting from day 0 after infection, the weight, neurological characteristics, parasitemia and survival rate of mice were monitored daily. The rapid murine coma and behavioral scale (RMCBS) was used to evaluate the neurological characteristics, and 10 parameters (hair, limb strength, defense ability, auricular reflex, toe reflex, contact reflex, gait, balance, body posture and exploratory activity) were used for evaluation. If the mice gradually develop characteristics such as unsteady walking, ataxia, messy hair, no stretching, loss of toe reflex, loss of auricular reflex, convulsion, coma, and even death, ECM is successfully established.

[0045] Blood-brain barrier integrity assessment: The protection of the brain was assessed by blood-brain barrier integrity (BBB). 1% EB dye was diluted in 0.9% NaCl. On day 8 after infection, 200 μl of 1% EB dye was injected into the tail vein of mice, the dye was allowed to circulate for 30 min, and then the mice were anesthetized and 0.9% NaCl was perfused into the right atrium through the heart to allow the liquid to flow out. The brain was quickly separated, photographed, weighed, and then placed in a 1.5 mL centrifuge tube for grinding. 1 mL of formamide was added to each brain sample and then incubated in a constant temperature metal bath at 37°C for 48 h. These samples were centrifuged at 1000 rpm for 10 min, and the absorbance was measured at 630 nm using an ELISA reader to quantify EB according to the standard curve.

[0046] Histopathological observation: After the end of administration, i.e., on the 8th day after infection, tissue samples were collected. After euthanasia, brain, liver, spleen, and small intestine tissues were immediately collected and washed three times in cold PBS to remove blood. Then, they were fixed with a universal tissue fixative for 24 hours and embedded in paraffin. Continuous 4 μm thick sections were cut and HE stained to observe microvascular obstruction and leakage. The sections were observed using an optical microscope, and Olympus cellSens standard 1.13 software was used for image acquisition.

[0047] result:

[0048] ① Basic behavioral indicators: PbA-infected mice were treated with DHA, DHA@MOF and DHA@ZIF-8, respectively. The results of phenotypic behavioral experiments showed that on the 8th day after infection, the average body weight of the DHA@MOF group was significantly higher than that of the PbA group (P=0.003). On the 18th day after infection, the body weight of the DHA@ZIF-8 group was the highest, larger than that of the DHA group (P=0.047) ( Figure 1 A). The RMCBS scores of all mouse groups showed a downward trend. The RMCBS scores of the DHA and DHA@ZIF-8 groups decreased slightly but remained stable. At 8 dpi, the RMCBS scores of mice in the DHA@MOF group were significantly higher than those in the PbA group (P = 0.007). The DHA@ZIF-8 group had the highest RMCBS score during the same period. Compared with the DHA group, the DHA@ZIF-8 group had a higher score (P = 0.019) ( Figure 1 B). On the 9th day after infection, the DHA@ZIF-8 group had the highest body weight, RMCBS score, and survival rate, and the lowest parasitemia, while DHA@MOF showed no significant insecticidal effect ( Figure 1 C). All mice in the PbA group died within 12 days, while mice in the DHA@ZIF-8 and DHA groups did not die within 18 days, and the survival rate of the DHA@ZIF-8 group was significantly higher than that of the DHA group ( Figure 1 D). In summary, DHA@ZIF-8 can be considered as a candidate nanodrug delivery system for DHA treatment of CM.

[0049] ② Blood-brain barrier permeability assessment: Visual assessment and EB quantification showed that the EB in the PbA group had obvious leakage, the DHA group had less leakage, and the DHA@ZIF-8 group had no visible leakage ( Figure 2 A). In the therapeutic application of DHA combined with ZIF-8, there was no statistical difference in EB leakage between the DHA group and the DHA@ZIF-8 group (P=0.21) ( Figure 2 B). In addition, the EB level in the DHA@MOF group was higher than that in the DHA@ZIF-8 group (P<0.0001).

[0050] ③ Histopathological indicators: The results of brain histopathological analysis showed that the DHA@ZIF-8 group was the most effective in reducing the sequestration of iRBCs in brain capillaries and the adhesion and aggregation of inflammatory cells, suggesting that DHA@ZIF-8 can significantly improve cerebrovascular injury and has a significant protective effect on ECM-induced brain injury ( Figure 3 A). Liver tissue pathological analysis showed that the DHA@ZIF-8 group had the most complete liver lobule structure, the most effective reduction of iRBCs isolation in the hepatic vein, almost no malarial pigment deposition and leukocyte adhesion and aggregation, revealing that the DHA@ZIF-8 group had the best ability to clear malarial pigment ( Figure 3 B). Quantitative analysis of hemochromatin in the liver was performed ( Figure 4 A). The DHA@ZIF-8 group had the lowest hemozoin content. The efficacy of DHA combined with ZIF-8 on hemozoin metabolism exceeded that of DHA alone (Р=0.023). A significant difference in hemozoin was observed between the DHA@MOF group and the PbA group (Ρ=0.016). Splenic histopathological analysis showed that the RePu area of ​​DHA@ZIF-8 was the smallest, indicating that anemia was improved and extramedullary hematopoiesis was reduced, confirming that DHA@ZIF-8 had a significant therapeutic effect on the spleen ( Figure 3 C) Quantitative evaluation of spleen hemozoin, PbA group had the largest RePu area. The RePu area of ​​DHA@MOF group was significantly reduced compared with PbA group (P<0.0001). Although DHA@MOF reduced the RePu area, extramedullary hematopoiesis still persisted. DHA@ZIF-8 group had the lowest hemozoin content ( Figure 4 B). There was a significant difference between the DHA@ZIF-8 group and the DHA group (P=0.013). Compared with the DHA group, the RePu area in the DHA@ZIF-8 group was significantly reduced (P=0.006), indicating a decrease in extramedullary hematopoiesis ( Figure 4 C).

[0051] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of DHA@ZIF-8 in the preparation of a drug for treating cerebral malaria, characterized in that: The DHA is dihydroartemisinin.

2. The use according to claim 1, characterized in that: The cerebral malaria is mouse cerebral malaria.

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