Nano preparation acting on drug-resistant plasmodium in targeting manner and preparation method of nano preparation
Through targeted nanocarriers, dihydroartemisinin and PI3K inhibitor idealisib were packaged, and using the PH-GSH dual response release mechanism, the problem of difficult reversal of artemisinin drugs to drug-resistant Plasmodium in the prior art was solved, and efficient antimalarial treatment effect was achieved.
Patent Information
- Application Number
- CN202411321729.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art is difficult to effectively reverse the resistance of artemisinin drugs to drug-resistant Plasmodium, resulting in poor malaria treatment.
The targeted nanocarrier was used to co-pack the dihydroartemisinin and the PI3K inhibitor idealisib. Through glucosamine modification and polydopamine coating, the double response release of PH-GSH was achieved, and targeted the red blood cells infected by Plasmodium.
It improves the targeting and bioavailability of drugs, enhances the killing effect of drug-resistant Plasmodium, reverses drug resistance, and improves the antimalarial efficacy.
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Figure CN119925632A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug preparation, and in particular to a nano preparation which acts on drug-resistant malarial parasites in a targeted manner to reverse drug resistance, and a preparation method thereof. Background Art
[0002] In recent years, the spread of artemisinin-resistant malaria has been increasing, and artemisinin resistance is a bottleneck problem in the current treatment of malaria.
[0003] The development of artemisinin resistance is the result of multiple factors. Recent studies on the mechanism of artemisinin resistance have shown that the main reason is that the sequence of the malarial parasite K13 protein changes and its content decreases, which leads to an increase in the downstream malarial parasite PI3K kinase and its phosphorylated product PI3P, causing the malarial parasite to reduce hemoglobin endocytosis and stagnate in the ring stage, which reduces the sensitivity of the malarial parasite to artemisinin; at the same time, the reduction of hemoglobin causes a decrease in the content of ferrous heme, which blocks the activation of ARTs and weakens the efficacy of antimalarial drugs. Therefore, it can be seen that the change of the malarial parasite K13 protein is the main reason for the development of resistance.
[0004] PI3P is a key mediator of artemisinin resistance, and Plasmodium PI3K is an important target for reversing resistance. The PI3P level is linearly correlated with the degree of resistance, and PI3P is considered to be an important signaling molecule that mediates and reflects resistance. Plasmodium PI3K-mediated phosphatidylinositol phosphorylation is the only source of ring-phase PI3P, and the PI3K inhibitor idealisib can effectively inhibit Plasmodium PI3K-mediated phosphatidylinositol phosphorylation, reduce the expression level of PI3P, and increase the sensitivity of Plasmodium to artemisinin drugs. The present invention targets and co-delivers the Plasmodium PI3K inhibitor idealisib and dihydroartemisinin into the parasite, which can enhance the efficacy of the drug and reverse the resistance from the mechanism, thereby improving the antimalarial efficacy.
[0005] Glucose transporter (GLUT1) and hexose transporter (HT) are highly expressed on the host erythrocyte membrane and malarial parasite plasma membrane, respectively. These two transporters transport glucose into malarial parasites, providing them with the energy necessary for development and reproduction. This preparation uses glucosamine as a target head to modify nanoparticles, which can enter erythrocytes through the GLUT1 transporter on the surface of the host cell membrane, and then enter malarial parasites through the specific HT on the malarial parasite plasma membrane. It improves the efficiency of nanoparticles in targeting infection of erythrocytes, and delivers drugs to intracellular malarial parasites through HT, which has a synergistic effect on ARTs antimalarial.
[0006] When malarial parasites invade red blood cells (RBCs), the host cell membrane expresses new permeability channels (NPPs) to obtain nutrients. NPPs allow particles and proteins smaller than 80 nm to enter infected red blood cells (iRBCs) to improve their growth and reproduction. At the same time, iRBCs contain a large amount of glutathione (GSH), which helps protect malarial parasites from damage by ARTs oxidative stress. The pH value in the body of malarial parasites is slightly acidic (5.2-5.8), which is conducive to the action of proteases, allowing malarial parasites to use the hemoglobin of red blood cells to synthesize the nutrients they need.
[0007] The nanoparticles are coated with polydopamine (PDA) material. When the nanoparticles are in the slightly acidic environment and high level of GSH in the body of malarial parasites, PDA cleaves, thereby promoting the dual-response release of the drug. When dihydroartemisinin reaches the body of malarial parasites, it will lead to an increase in reactive oxygen species (ROS) and a decrease in GSH levels. At the same time, the PDA coating will further reduce the GSH level. When the PI3K inhibitor idealisib reaches the body of malarial parasites, it will reduce the expression level of PI3P, achieving the effect of reversing drug resistance. Summary of the invention
[0008] In view of the deficiencies in the prior art, the present invention provides a nanocarrier, a preparation and a preparation method thereof that are targeted at malarial parasites, and can co-encapsulate two drugs with different properties therein. The preparation has a PH-GSH dual response, and glucosamine enables the drug coated by the nanocarrier to target red blood cells infected with malarial parasites and reverse the red blood cells' resistance to artemisinin-based drugs.
[0009] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention designs a nanoparticle that can encapsulate two drugs with different physical properties, an antimalarial drug and Pf PI3K inhibitors are delivered to infected red blood cells. In vivo, the polydopamine coating allows the nanoparticles to remain stable in the body and delay the release of the drug, improving the short-term effect of traditional antimalarial drugs until they enter the infected red blood cells and rapidly lyse and release the drug under high GHS and slightly acidic conditions. At the same time, the glucose derivatives on the surface of the carrier enable the nanoparticles to better target infected red blood cells, improve the bioavailability of the drug, and develop a safe and efficient method for treating malaria.
[0010] (2) The two drugs provided by the present invention can act together on red blood cells infected with malarial parasites: dihydroartemisinin destroys the structure of malarial parasites by interfering with the surface membrane and mitochondria of malarial parasites, thereby killing malarial parasites, while PfThe PI3K inhibitor idealisib can effectively inhibit PfPI3K-mediated phosphatidylinositol phosphorylation, reduce the expression level of PI3P, and increase the sensitivity of Plasmodium to dihydroartemisinin. In in vitro experiments, the two drugs have a synergistic effect, and the combination of the two drugs has the effect of killing Plasmodium against both artemisinin-sensitive and -resistant strains. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 This is a transmission electron microscopy image of the glucosamine-modified dihydroartemisinin and idelalisib co-loaded dual-responsive nanoparticles in an example of the present invention.
[0012] Figure 2 The release results of dihydroartemisinin and idelalisib in the nanoparticles of the embodiment of the present invention are shown in FIG.
[0013] Figure 3 This is a schematic diagram of the co-localization results of coumarin 6-modified nanoparticles and infected red blood cells in an example of the present invention.
[0014] Figure 4 The pharmacodynamic results of the nanoparticles in the examples of the present invention in mice infected with artemisinin-sensitive and artemisinin-resistant Plasmodium burgdorferi K173. DETAILED DESCRIPTION
[0015] In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. The purpose of these embodiments is only to explain and not to limit the present invention in any way.
[0016] For the convenience of description, some drugs in this embodiment and the drawings of the specification are abbreviated in common English, as follows: Reduced glutathione: GSH; Polydopamine: PDA; Dihydroartemisinin: DHA; Poly(lactic-co-glycolic acid): PLGA; Phosphate buffered saline: PBS; Coumarin 6: C6; Infected red blood cells: iRBCs; Glucose transporter 1: GLUT1; Hexose transporter: HT; New permeability channels and macromolecular transport pathways: NPPs; Dihydroartemisinin and idelalisib mixed solution: DHA+idelalisib-sol; DHA+idelalisib co-loaded nanoparticles: DI-NPs; DHA+idelalisib co-loaded dual-responsive nanoparticles: DI@PDA-NPs; Glucosamine-modified DHA+idelalisib co-loaded dual-responsive nanoparticles: DI@PDA-Glu-NPs; Coumarin 6 labeled glucosamine modified dual-responsive nanoparticles: C6@PDA-Glu-NPs.
[0017] This embodiment prepares a nanoparticle for treating artemisinin-resistant malaria, including a targeted nanocarrier and an artemisinin drug and a malarial parasite PI3K inhibitor encapsulated in the nanocarrier.
[0018] Nanocarriers include targeting glucosamine, polyethylene glycol-polycaprolactone, and polydopamine.
[0019] The encapsulated drugs are dihydroartemisinin and idelalisib.
[0020] Preparation of DHA+idelalisib co-loaded dual-responsive nanoparticles: The thin film dispersion method was used to prepare the co-loaded nanoparticles DI-NPs. The specific operation was as follows: DHA 4 mg, idelalisib 15 mg, and mPEG2000-PLGA1140 10 mg were accurately weighed and placed in an eggplant-shaped bottle. Methanol was added to dissolve it, and the methanol was completely evaporated at 60 ° C. A uniform film was observed to form at the bottom of the eggplant-shaped bottle. After standing overnight, 3 ml of deionized water at 60 ° C was measured, hydrated for 5 min, ultrasonicated for 5 min, and centrifuged at 13000 r / min for 5 min to obtain the supernatant to obtain the DI-NPs suspension. Tris-HCl buffer was added in an equal volume to the suspension, and DA was added to make the final concentration of DA 0.8 mg / ml. The suspension was ultrafiltered and centrifuged the next day to remove free DA and oligodopamine to obtain DI@PDA-NPs, which were then freeze-dried to form DI@PDA-NPs freeze-dried powder. 0.8 g of glucosamine was dissolved in 20 ml of PBS, and then 0.04 g of DI@PDA-NPs was added. The mixture was reacted at room temperature at 150 r / min for 24 h, and then centrifuged using an ultrafiltration centrifuge tube. The nanoparticles were washed out with water to obtain DI@PDA-Glu-NPs. DI-NPs, DI@PDA-NPs, and DI@PDA-Glu-NPs were all rounded and spherical under an electron microscope. The particle size of the three nanoparticles was about 50 nm, and the PDI was less than 0.3. Figure 1 shown.
[0021] In vitro release of DHA+idelalisib co-loaded dual-responsive nanoparticles: The dialysis method was used to determine the in vitro release of nanoparticles at different pH and GSH concentrations. The release media prepared were 20% ethanol-PBS at pH = 5.5, 20% ethanol-PBS at pH = 6.5, 20% ethanol-PBS at pH = 7.4, and a mixed solution of 10 mM GSH and 20% ethanol-PBS. 1 ml of DI-NPs, DI@PDA-NPs, and DI@PDA-Glu-NPs with a concentration of 1.0 mg / ml were placed in dialysis bags, tied, placed in the above four different release media, and placed in a constant temperature air bath oscillating box at 37°C and maintained at 100 r / min. 100 μl of the sample was taken out at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h, respectively, and fresh release media was added, and injected into the high-performance liquid chromatography to determine the content of DHA and idelalisib, respectively. The release of DHA and idelalisib in DI-NPs, DI@PDA-NPs, and DI@PDA-Glu-NPs had similar effects. The release of DHA and idelalisib from DI-NPs under different pH and GSH conditions was not much different, indicating that DI-NPs were insensitive to pH and GSH; after PDA modification, DHA and idelalisib showed different release effects at different pH (7.4, 6.5, 5.5) and different concentrations of GSH (0 mM, 10 mM). When pH was 5.5, the cumulative release of DHA and idelalisib in DI@PDA-NPs and DI@PDA-Glu-NPs was the largest, and the final cumulative release of DHA in DI@PDA-Glu-NPs was 70.7%; the final cumulative release of idelalisib in DI@PDA-Glu-NPs was 85.02%; when pH was 6.5, the release was second; when GSH was 10 mM, the release was better than that under the conditions of pH 7.4 and GSH 0 mM. The preparation can be responsively released in an acidic environment or a high GSH concentration environment to achieve a better release effect, such as Figure 2 shown.
[0022] Targeting (co-localization) of DHA+idelalisib co-loaded dual-responsive nanoparticles: Hoechst can penetrate the cell membrane and produce blue fluorescence after binding to double-stranded DNA, which can be used to mark the nucleus of malarial parasites. C6 nanoparticles show yellow-green fluorescence. In this experiment, three nanoparticles C6-NPs, C6@PDA-NPs and C6@PDA-Glu-NPs were prepared and incubated with infected red blood cells to observe the entry of nanoparticles into infected red blood cells. The specific operation is as follows: 5 μl of clean infected red blood cell sediment was aspirated and added to an EP tube containing 100 μl 1640 culture medium, 1 μl of fluorescent nanoparticles (C6-NPs, C6@PDA-NPs and C6@PDA-Glu-NPs) was added and incubated at 37°C for 45 minutes, followed by 1 μl of Hoechst dye solution, mixed and incubated at 37°C for 20 minutes. After incubation, centrifuge at 5000 r / min for 3 min, discard the supernatant, add 200 μl PBS, vortex for 30 s, centrifuge at 5000 r / min for 3 min, repeat 3-4 times to remove the fluorescence and Hoechst adhering to the surface of erythrocytes. Prepare blood smears and dry them, and observe the colocalization using the ImageXpress Pico automatic cell imaging analysis system. In order to verify whether the nanoparticles enter the infected erythrocytes through NPPs, GLUT1 and HT, furosemide (NPPs inhibitor), phloretin (GLUT1 inhibitor) or MMV (HT inhibitor) was incubated with infected erythrocytes for 1 h before the addition of nanoparticles, and then subsequent treatment was performed to verify whether the nanoparticles can enter the erythrocytes infected with Plasmodium through NPPs and glycosyl transport channels. When the prepared C6@PDA-Glu-NPs were incubated with red blood cells infected with K173 resistant strains, Hoechst was added. Only the Hoechst channel after incubation had blue fluorescence, and it appeared in the infected red blood cells, indicating that Hoechst can be used to mark the cell nucleus. The results of incubation of fluorescent nanoparticles with infected red blood cells can be seen from the figure that C6@PDA-Glu-NPs have a better targeting effect in the resistant strains, indicating that the glucose modification of nanoparticles is beneficial for the nanoparticles to target drug-resistant malarial parasites. When furosemide was added during incubation with red blood cells infected with K173 resistant strains, the NPPs channel was inhibited, the intake of nanoparticles was reduced, and the targeting ability was weakened. When phloretin was added during incubation, the GLUT1 channel was inhibited, the intake of glucose-modified nanoparticles was reduced, and the targeting ability was weakened. When MMV was added during incubation, the HT channel was inhibited, the intake of glucose-modified nanoparticles into malarial parasites was reduced, and the targeting ability was weakened. When furosemide, phloretin, and MMV were all added during incubation, C6@PDA-Glu-NPs could not overlap with infected erythrocytes, and the targeting ability was smaller compared with the sensitive strain.It was confirmed that C6@PDA-Glu-NPs entered erythrocytes infected with drug-resistant strains through these three channels, and this targeting effect was more prominent in drug-resistant strains, such as. Figure 3 shown.
[0023] In vivo studies: 90 mice were randomly divided into 18 groups, 5 mice in each group. They included a normal blank group (0.2 ml saline), an artemisinin-resistant malaria mouse model group (0.2 ml saline), three groups of DI-NPs, DI@PDA-NPs, and DI@PDA-GLU-NPs at DHA doses of 1.1, 2.2, 8.8, and 17.6 μmol / kg; and five groups of DHA solution group, DHA+idealisib solution group, DI-NPs, DI@PDA-NPs, and DI@PDA-GLU-NPs at DHA doses of 4.4 μmol / kg. The drug was administered using the Pearson four-day inhibition method, and each mouse was inoculated with 10 7 Infected red blood cells were administered by tail vein 2 hours later. The drugs were administered for four consecutive days at the same time, and the inhibition rate on the fifth day after the mice stopped taking the drugs was calculated. Compared with the treatment with DHA solution alone, the growth inhibition rate of malarial parasites in drug-resistant malarial mice increased significantly after adding idealisib. After the two drugs were co-encapsulated into nanoparticles, the inhibition rate of the preparation group was higher than that of the solution group; especially on the basis of the initial preparation, the growth inhibition effect of malarial parasites was further enhanced after modification with dopamine and glucosamine, such as Figure 4 shown.
[0024] The present invention designs a nanoparticle capable of encapsulating two drugs with different physical properties, and uses a nano-delivery system to change the dosage form and administration method of the drug, reduce the particle size of the drug, increase the solubility of the drug, avoid the first-pass effect, and encapsulate the antimalarial drug and Pf PI3K inhibitors are delivered to infected red blood cells. In vivo, the polydopamine coating allows the nanoparticles to remain stable in vivo and delay the release of the drug, improving the short-term effect of traditional antimalarial drugs until they enter the infected red blood cells under high GSH and slightly acidic conditions, where they are rapidly cleaved and released. At the same time, the glucose derivatives on the surface of the carrier enable the nanoparticles to better target infected red blood cells, improve the bioavailability of the drug, and develop a safe and efficient method for treating malaria.
[0025] The two drugs provided by the present invention can act together on red blood cells infected with malarial parasites: dihydroartemisinin destroys the structure of malarial parasites by interfering with the surface membrane, mitochondria, etc. of malarial parasites, thereby killing malarial parasites, while Pf The PI3K inhibitor idealisib can effectively inhibit PfPI3K-mediated phosphatidylinositol phosphorylation reduces the expression level of PI3P and increases the sensitivity of malarial parasites to dihydroartemisinin. In in vitro experiments, the two drugs have a synergistic effect, and the combination of the two drugs has the effect of killing malarial parasites against both artemisinin-sensitive and -resistant strains.
Claims
1. A nanoparticle preparation targeting malarial parasites, characterized by: These include two drugs with antimalarial efficacy and the ability to reverse drug resistance, targeted glucose, polyethylene glycol-polycaprolactone copolymer, and the high molecular weight polymer polydopamine.
2. The nano preparation targeting malarial parasites according to claim 1, characterized in that: The targeted glucose includes glucosamine, a pH-GSH dual-responsive polyethylene glycol-polycaprolactone polymer, and a high-molecular polymer polydopamine, which can achieve specific targeted infection of red blood cell glucose transporters.
3. A method for preparing a nanoparticle preparation targeting malarial parasites, characterized in that: The following steps are included: Step 1: dissolving dihydroartemisinin, idealisib, and polyethylene glycol-polycaprolactone copolymer in methanol at room temperature; Step 2: The methanol solution obtained in step 1 is subjected to rotary evaporation to remove the solution to obtain a uniformly dispersed film; Step 3: adding ultrapure water to the film obtained in step 2 for hydration at 60°C; Step 4: centrifuging the nanosuspension obtained in step 3 to remove uncoated drugs to obtain a nanoparticle suspension; Step 5: Add the same volume of pH 8.5 Tris-HCl solution to the suspension obtained in step 4 to create an alkaline environment, add dopamine, and incubate at 37°C in the dark for 12 hours; Step 6: The suspension obtained in step 5 is subjected to ultrafiltration centrifugation to remove the Tris-HCl solution and free dopamine to obtain a DI@PDA-NPs suspension; Step 7: Mix DI@PDA-NPs and glucosamine and incubate at 37 °C for 24 h; Step 8: The suspension obtained in step 7 is subjected to ultrafiltration and centrifugation to obtain a nanosuspension.
4. The method for preparing a nano preparation targeting malarial parasites according to claim 3, characterized in that: The step seven is: the suspension obtained in step six is stirred to form DI@PDA-NPs freeze-dried powder, glucosamine is dissolved in phosphate buffer, and then the DI@PDA-NPs freeze-dried powder is added, and the reaction is carried out at a speed of 150 r / min and 37° C. for 24 hours.