CRGD / PDA co-modified dihydroartemisinin prodrug liposome as well as preparation method and application thereof

Abstract

The invention discloses a cRGD / PDA co-modified dihydroartemisinin prodrug liposome as well as a preparation method and application of the cRGD / PDA co-modified dihydroartemisinin prodrug liposome. The liposome comprises a polydopamine (PDA) shell, a cRGD target head and a dihydroartemisinin prodrug containing a disulfide bond. The preparation method comprises the following steps: synthesizing a dihydroartemisinin prodrug with reduction sensitivity through cyclization reaction of dithiodipropionic acid; dissolving the prodrug and auxiliary materials, and injecting into a water phase to form liposome; the lipidosome is wrapped through PDA self-deposition; and modifying with the cRGD peptide to obtain the dihydroartemisinin prodrug liposome. The disulfide bond dihydroartemisinin prodrug carried in the liposome has GSH and pH sensitivity at the same time, the lipid solubility of the drug is effectively enhanced, the defect of low drug loading capacity of a nano preparation is overcome, oral administration can be met, and dihydroartemisinin is triggered and released in plasmodium. The cRGD modified lipidosome with stable gastrointestinal tracts can specifically target intestinal M cell mediated lymphatic circulation, promote the lipidosome to be transferred into blood through lymph, reduce liver-intestine first-pass metabolism and improve the oral curative effect.

Description

Technical Field

[0001] The present invention discloses cRGD / PDA co-modified dihydroartemisinin prodrug liposomes, a preparation method and application thereof; specifically, the present invention discloses a pH- and glutathione-responsive dihydroartemisinin prodrug and a synthesis method; a preparation method of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes; and application of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes in the oral administration of malaria. Background Art

[0002] Malaria is a major global public health challenge. Dihydroartemisinin, as the first-line treatment for malignant malaria and multidrug-resistant malaria, produces reactive oxygen free radicals through the cleavage of intramolecular peroxide bridges, causing oxidative damage to malarial parasites, exerting antimalarial efficacy and significantly reducing the mortality rate of severe malaria. However, dihydroartemisinin has problems such as a large first-pass effect, a short half-life, and a lack of targeting, which together affect its oral bioavailability and antimalarial efficacy. In addition, drug-resistant strains escape drug pressure by regulating the dormancy period of malarial parasites and repairing oxidative damage, allowing them to spread rapidly, which has seriously threatened the long-term application of dihydroartemisinin.

[0003] Malaria parasites possess a specific acidic lysosomal microenvironment (pH 5.2-5.8), and their glutathione content is 10 times that of normal red blood cells. This concentration is further elevated in drug-resistant strains. pH- and glutathione-responsive drug release, coupled with simultaneous glutathione consumption, can achieve targeted drug release and enhance the drug's oxidative stress response.

[0004] Oral delivery requires overcoming the first-pass effect simultaneously. The M cell-targeted delivery strategy based on gut-associated lymphoid tissue shows unique advantages. M cells lack microsomal enzymes and a dense mucus layer. Nanocarriers can enter the lymphatic circulation directly through efficient endocytosis, avoiding the first-pass effect. Integrin receptor αvβ3 is highly expressed on the surface of M cells. The use of arginine-glycine-asparagine peptide (RGD) can effectively recognize and bind to M cell-specific integrins to achieve M cell-targeted delivery. However, due to the presence of aspartic acid residues in the RGD structure, RGD is very sensitive to chemical degradation in the gastrointestinal tract. Cyclic RGD peptide (cRGD) is resistant to enzymatic degradation because the exposed N-terminus and C-terminus are removed. Summary of the Invention

[0005] To address the significant first-pass effect, short half-life, and lack of targeting of conventional dihydroartemisinin, as well as the oxidative damage defenses against drug-resistant strains, this paper proposes two synergistic strategies: molecular structure optimization and the development of an oral delivery system. A pH- and glutathione-responsive dihydroartemisinin prodrug was designed and delivered via cRGD / PDA co-modified liposomes. This approach avoids the first-pass effect, prolongs circulation time, and targets drug release in red blood cells infected with Plasmodium parasites, increasing oxidative stress on the parasites and enhancing antimalarial efficacy.

[0006] The present invention adopts the following technical solution: cRGD / PDA co-modified dihydroartemisinin prodrug liposomes, including a polydopamine shell, a cRGD target head and a dihydroartemisinin prodrug.

[0007] Furthermore, soybean lecithin and cholesterol are included, the mass ratio of dihydroartemisinin prodrug to soybean lecithin is 1:15, and the mass ratio of soybean lecithin to cholesterol is 10:1.

[0008] Furthermore, a stabilizer is included, and the mass percentage of the stabilizer is 5%.

[0009] Furthermore, the stabilizer is vitamin E polyethylene glycol succinate.

[0010] Furthermore, the dihydroartemisinin prodrug contains a reduction-responsive disulfide bond.

[0011] The preparation method of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes comprises the following steps: Step 1: Weigh dithiodipropionic acid, add acetic anhydride to react and form a ring; weigh hexadecanol and 4-dimethylaminopyridine, dissolve them in dichloromethane, and add them to the above reaction flask for reaction; separate and purify the product to obtain an intermediate product; weigh the intermediate product, dihydroartemisinin, 4-dimethylaminopyridine and carbodiimide hydrochloride, dissolve them in dichloromethane for reaction, and separate and purify the product to obtain a dihydroartemisinin prodrug; Step 2: Take the prescribed amount of dihydroartemisinin prodrug, soybean lecithin, cholesterol and vitamin E polyethylene glycol succinate, accurately weigh and set aside; Step 3: Accurately measure the final volume of anhydrous ethanol to dissolve the drug weighed in step 2; Step 4: Slowly inject the mixed solution obtained in step 3 into the aqueous solution below the liquid surface and stir evenly to prepare dihydroartemisinin prodrug liposomes; the dihydroartemisinin prodrug liposome solution prepared above is mixed with a Tris-HCl buffer solution, and dopamine hydrochloride is added, stirred and mixed, incubated, and ultrafiltration and centrifugation are performed to obtain polydopamine-coated dihydroartemisinin prodrug liposomes; the dihydroartemisinin prodrug liposomes prepared above are mixed with a Tris-HCl buffer solution, and cRGD peptide is added, stirred and mixed, incubated in the dark, and ultrafiltration and centrifugation are performed to obtain cRGD / PDA-modified dihydroartemisinin prodrug liposomes.

[0012] Application of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes for oral administration in the treatment of malaria.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The disulfide-bonded dihydroartemisinin prodrug is both GSH- and pH-sensitive, and effectively enhances the drug's lipid solubility, allowing it to be effectively loaded into liposomes. This overcomes the drawback of low drug loading in nanoformulations, allows for oral administration, and triggers the release of dihydroartemisinin in Plasmodium.

[0014] 2. Utilizing gastrointestinal stable cRGD-modified liposomes, we can specifically target the lymphatic circulation mediated by intestinal M cells, promote the transport of liposomes into the blood via lymph, reduce first-pass metabolism in the liver and intestine, and improve oral efficacy.

[0015] 3. The PDA coating ensures drug stability in the gastrointestinal tract and triggers drug release within the parasite, prolonging its circulation time and achieving parasite-targeted action. Furthermore, the disulfide bond of the prodrug and PDA enhance oxidative stress within the parasite by consuming GSH, further enhancing antimalarial efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of different mass ratios of dihydroartemisinin prodrug to soybean lecithin, soybean lecithin to cholesterol, and stabilizer content on the particle size, PDI, and zeta potential of prodrug liposomes; Figure 2 Transmission electron microscopy images of dihydroartemisinin prodrug liposomes, PDA-modified dihydroartemisinin prodrug liposomes, and the liposomes of the present invention; Figure 3 The pH and GSH sensitivity results of the dihydroartemisinin prodrug of the present invention are as follows; Figure 4 The in vitro release results of PDA-modified dihydroartemisinin prodrug liposomes; Figure 5 This is the in vitro release result of the liposome of the present invention; Figure 6The in vitro hemolysis results of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention; Figure 7 The effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on Caco-2 cell activity; Figure 8 The blood drug concentration-time curves of dihydroartemisinin suspension, PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention are shown; Figure 9 The pharmacokinetic parameters of dihydroartemisinin suspension, PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention are shown in FIG. Figure 10 The results of fluorescence imaging of the gastrointestinal tract in mice using PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention; Figure 11 The results of in vivo liver fluorescence imaging of mice using PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention; Figure 12 The results of fluorescence quantification of the liver of mice in vivo for PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention are shown; Figure 13 The fluorescence imaging results of the PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention in mice are shown in FIG. Figure 14 The fluorescence quantitative results of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention in mice containing Peyer's colon segments (PP) and non-Peyer's colon segments (NPP); Figure 15 The effects of the non-intervention group, dihydroartemisinin solution, prodrug solution, unloaded PDA-modified liposomes, PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on reactive oxygen species in infected erythrocytes were compared. Figure 16 The effects of the non-intervention group, dihydroartemisinin solution, prodrug solution, unloaded PDA-modified liposomes, PDA-modified dihydroartemisinin prodrug liposomes, and the liposomes of the present invention on reduced glutathione in Plasmodium; Figure 17 The in vivo antimalarial results of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention; Figure 18 The effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on ALT function in the liver of malaria mice; Figure 19 The effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on AST in the liver function of malaria mice; Figure 20 The effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on the liver and spleen coefficients of malaria mice; Figure 21 The effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on the blood routine of malaria mice; Figure 22 The present invention shows the effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on pathological sections of major organs of malaria mice.

[0017] Figure 23 The present invention discloses the effects of PDA-modified dihydroartemisinin prodrug liposomes and the liposomes of the present invention on the survival rate of malaria mice. DETAILED DESCRIPTION

[0018] For the convenience of description, some drugs in this embodiment and the drawings of the specification are abbreviated in common English, as follows: Dihydroartemisinin: DHA Disulfide-linked dihydroartemisinin prodrug: DSSC Dihydroartemisinin prodrug liposomes: NLs PDA-modified dihydroartemisinin prodrug liposomes: PNLs cRGD / PDA co-modified dihydroartemisinin prodrug liposomes (liposomes of the present invention): cRPNLs Vitamin E polyethylene glycol succinate: TPGS Soybean lecithin: SP Cholesterol: CH Dihydroartemisinin solution: DHA-sol Dihydroartemisinin suspension: DHA-sus Unloaded PDA-modified liposomes: BPNLs Glutathione: GSH Step 1: Synthesis of dihydroartemisinin prodrug Weigh 1.50 mmol of dithiodipropionic acid, accurately weighed, and place in a 100 mL eggplant-shaped flask. Add 3.00 mL of acetic anhydride and react at 30°C for 3 h. Remove excess acetic anhydride with toluene. Weigh 1.00 mmol of hexadecanol and 0.15 mmol of 4-dimethylaminopyridine, accurately weighed, dissolved in dichloromethane, and added to the above eggplant-shaped flask. React at 35°C for 24 h. The product is purified by thin-layer chromatography to obtain a white intermediate. Weigh 1.00 mmol of the white intermediate, accurately weighed, and place in a 100 mL eggplant-shaped flask. Dissolve the reaction solution in dichloromethane to obtain a reaction solution. Weigh 1.50 mmol of dihydroartemisinin, 0.15 mmol of 4-dimethylaminopyridine, and 1.00 mmol of carbodiimide hydrochloride, accurately weighed, and place in the above reaction solution. React at 35°C for 48 h. Purify by thin-layer chromatography to obtain the dihydroartemisinin prodrug.

[0019] Step 2: Screening of preparation formula The drug-to-lipid ratio, soybean phospholipid-cholesterol ratio, and stabilizer content percentage were investigated by single-factor optimization method, and the best formulation was the one with small and stable particle size. Figure 1 As shown, the drug-lipid ratio is 1:5, 1:10, 1:15, 1:20, preferably 1:15; the soybean lecithin-cholesterol ratio is 20:1, 15:1, 10:1, 5:1, preferably 10:1; the stabilizer content is 5%, 10%, 15%, 20% (w / w), preferably 5%.

[0020] Step 3: Preparation of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes First, prepare unmodified prodrug liposomes. Accurately weigh 2.00 mg of DSSC, 30.00 mg of soybean lecithin, 3.00 mg of cholesterol, and 1.75 mg of TPGS. Dissolve these substances in 400.00 μL of anhydrous ethanol and mix thoroughly. Slowly inject the mixed solution subsurface into a uniformly stirred aqueous solution (1200 rpm, 60°C). Continue stirring for 2 h after injection to obtain NLs.

[0021] The NLs solution prepared above was mixed with equal volumes of 20 mmol / L Tris-HCl (pH 8.5) solution, and dopamine hydrochloride was added at a concentration of 0.6 mg / mL, stirred and mixed. The mixture was incubated at 37°C and 100 rpm for 12 h, and then ultrafiltration and centrifugation were performed to obtain PNLs.

[0022] The PNLs solution prepared above was mixed with equal volumes of 20 mmol / L Tris-HCl (pH 8.5) solution, and cRGD was added at a concentration of 2 mg / mL and stirred thoroughly. After stirring overnight at room temperature in the dark, the mixture was ultrafiltrated and centrifuged to obtain cRPNLs.

[0023] Take 0.5 mL of cRPNLs solution, dilute it 10 times with purified water, and drop it onto the copper grid with carbon film. After it evaporates naturally, place the sample under a transmission electron microscope to observe the morphology of the nanoparticles, as shown in the following figure: Figure 2 shown.

[0024] Validation trial 1: sensitivity of dihydroartemisinin prodrug To verify the dual sensitivity of the DSSCs to GSH and pH, they were placed in 20% ethanol-PBS (pH 1.2), 20% ethanol-PBS (pH 5.5), 20% ethanol-PBS (pH 7.4), and 20% ethanol-PBS (pH 7.4) containing 10 mM GSH, and shaken continuously at 37°C and 100 rpm. At 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours, 100 μL of sample was taken, 400 μL of acetonitrile was immediately added, the mixture was vortexed, and the supernatant was centrifuged at 13,000 rpm for 5 minutes. The results are shown in Table 1. Figure 3 DSSCs degrade rapidly in an acidic environment. At pH 1.2 and pH 5.5, DHA conversion within 24 h was 100% and 86.5%, respectively, compared to 25% in a pH 7.4 buffer. At pH 7.4, the addition of 10 mM GSH significantly accelerated DSSC degradation, reaching 100% DHA conversion within 24 h. These results demonstrate that DSSCs exhibit dual sensitivity to GSH and pH.

[0025] Validation experiment 2: in vitro release of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes To verify the dual GSH and pH responsiveness of the liposomes, dialysis was used to measure DHA release from the liposomes. 1 mL of RPNLs and cRPNLs were placed in a 3500 KD dialysis bag, clamped at both ends, and placed in 20% ethanol-PBS (pH 1.2), 20% ethanol-PBS (pH 5.5), 20% ethanol-PBS (pH 7.4), 20% ethanol-PBS (pH 5.5) containing 10 mM GSH, and 20% ethanol-PBS (pH 7.4) containing 10 mM GSH. The bags were shaken continuously at 37°C and 100 rpm. At 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h, 200 μL of dialysate was collected and ultrasonically demulsified with 800 μL of acetonitrile for 5 minutes. The supernatant was then centrifuged at 13,000 rpm for 5 minutes, and the supernatant was analyzed. The same volume of release medium was then added. 200 μL of blank release medium was added to 800 μL of acetonitrile and vortexed to mix thoroughly. The mixture was centrifuged at 13,000 r / min for 5 min, and the supernatant was measured as a blank control. 1 mL of RPNLs and cRPNLs were added to 9 mL of release medium and vortexed to mix thoroughly. 200 μL of the mixture was added to 800 μL of acetonitrile and ultrasonically demulsified for 5 min. The mixture was centrifuged at 13,000 r / min for 5 min, and the supernatant was measured as a complete release control. Release results are shown in Table 1. Figure 4 、 5 At pH 1.2 and 7.4, RPNLs and cRPNLs released almost no drug. However, at pH 5.5, drug release from RPNLs and cRPNLs increased significantly, with cumulative DHA release rates reaching 74.23% ± 1.81% and 84.86% ± 1.45%, respectively. Adding GSH further increased the cumulative DHA release rates, reaching 82.58% ± 2.85% and 88.60% ± 0.99%, respectively, significantly higher than at pH 7.4 + 10 mM GSH. These release results demonstrate the dual-responsive release characteristics of the liposomes of the present invention.

[0026] Verification experiment 3: In vitro hemolysis results of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes PNLs and cRPNLs solutions with DSSC concentrations of 5, 10, 50, 100, and 200 μg / mL were prepared, respectively, and mixed with 2% red blood cell suspension at a ratio of 1:1 (v / v) as the experimental group. Distilled water and 2% red blood cell suspension were mixed at a ratio of 1:1 (v / v) as the positive control group, and normal saline and 2% red blood cell suspension were mixed at a ratio of 1:1 (v / v) as the negative control group. The mixed solutions of all groups were placed in a constant temperature water bath at 37°C for 3 hours, centrifuged at 3500 r / min for 10 minutes, and the absorbance (Optical Density, OD) was measured at 540 nm using a microplate reader. The hemolysis rate was calculated to evaluate the effect of different concentrations of liposomes on red blood cells. The results of the hemolysis test are shown in Figure 2. Figure 6 As shown in the results, the hemolysis rates of PNLs and cRPNLs at all dose concentrations did not exceed 5%, and no significant hemolysis was caused. These results indicate that PNLs and cRPNLs have good biocompatibility.

[0027] Validation experiment 4: Cellular safety of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes The CCK-8 assay was used to investigate the cytotoxicity of RPNLs and cRPNLs to Caco-2 cells. 4 Cells were seeded / well in a 96-well plate, and 100 μL of complete culture medium was added to each well. The plates were cultured at 37°C and 5% CO2 for 48 h. RPNLs and cRPNLs solutions with DSSC concentrations of 10, 25, 50, 75, and 100 μg / mL were prepared using complete culture medium. 100 mL of these solutions were added to the cells, with three replicates for each concentration. Complete culture medium was used as a blank control, and the plates were incubated at 37°C and 5% CO2 for 24 h. 10 μL of CCK-8 solution was added, and the plates were incubated in a 37°C, 5% CO2 incubator for 2 h. The absorbance of each well at 450 nm was measured using a microplate reader. The results are shown in Table 1. Figure 7 After incubation with Caco-2 cells at varying concentrations of RPNLs and cRPNLs, the viability of the Caco-2 cells remained above 80%. According to the ISO 10993-5:2009 standard, substances that maintain cell viability above 80% are considered non-toxic, indicating that RPNLs and cRPNLs have good biosafety.

[0028] Validation experiment 5: cRGD / PDA dual-modified dihydroartemisinin prodrug liposomes increase reactive oxygen species levels in infected erythrocytes A malaria mouse model was constructed. When the infection rate reached 10%, fresh red blood cells were used to prepare a 2% red blood cell suspension with RPMI1640 culture medium. In a 12-well plate, 1 mL of red blood cell suspension containing malarial parasites was added to each well, and DHA-sol, DSSC-sol, BPNLs, PNLs and cRPNLs were added respectively. After repeated pipetting to mix, the well plate was placed in an incubator for incubation. After 24 hours, the cell suspension in the well plate was transferred to an EP tube, centrifuged at 1000 r / min for 10 min, and the supernatant was removed. Subsequently, 1 mL of PBS solution was added for washing, and the washing was repeated 2 to 3 times. Finally, the obtained precipitate was treated and measured using a reactive oxygen species (ROS) detection kit, and 3 replicate wells were set up in each group to ensure the reliability of the experimental results. From Figure 15 The results show that the ROS content of the DHA-sol and DSSC-sol groups was significantly higher than that of the control group, and the DSSC-sol group was significantly higher than that of the DHA-sol group, indicating that the introduction of disulfide bonds in DSSC can enhance oxidative stress. The ROS content of the PNLs group was significantly higher than that of the DSSC-sol and BPNLs groups, indicating that the effect of PNLs on enhancing oxidative stress is better than that of DSSC or PDA alone. In addition, there was no significant difference in ROS content between the cRPNLs group and the PNLs group, indicating that the modification of cRGD does not affect the oxidative stress of PNLs.

[0029] Validation Experiment 6: cRGD / PDA co-modified dihydroartemisinin prodrug liposomes reduce the content of reduced glutathione in Plasmodium A malaria mouse model was constructed. When the infection rate reached 10%, fresh red blood cells were used to prepare a 4% red blood cell suspension with RPMI1640 medium. According to the steps in the "Verification Test 5" section, the drug was incubated with the red blood cell suspension, centrifuged, the precipitate was resuspended with PBS, and an equal volume of saponin PBS was added. The precipitate was allowed to stand on ice for 15 minutes to lyse the parasites. After that, the solution was centrifuged at 5000 r / min for 10 minutes, the supernatant was removed, and a gray-black precipitate was obtained. The total glutathione (T-GSH) and oxidized glutathione (GSSG) in the precipitate were determined using a kit, and the amount of GSH was calculated from the difference between the two. Each group was set up with 3 replicates, and the results of the determination of GSH / GSSG in malarial parasites are shown in Figure 2. Figure 16 The results showed that compared with the untreated group, DHA-sol significantly reduced the GSH / GSSG ratio in malarial parasites, and the effect of DSSC-Sol was further enhanced, indicating the necessity of prodrug synthesis. The effect of PNL in reducing the GSH / GSSG ratio was significantly enhanced compared with DSSC-sol and BPNLs alone, and there was no significant difference with cRPNLs, indicating that PDA-modified liposomes can reduce the amount of GSH in malarial parasites and enhance oxidative stress, while modification with cRGD has no effect on this effect.

[0030] Validation Experiment 7: Pharmacokinetic Study of cRGD / PDA Dual-Modified Dihydroartemisinin Prodrug Liposomes in Rats Twenty-one male Sprague-Dawley rats were randomly divided into three groups. They were fasted for 12 hours before administration and then orally administered with 10 mg / kg DHA to DHA-sus, RPNLs, and cRPNLs. At 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 hours after administration, 0.5 ml of blood was collected from a sodium heparin-soaked centrifuge tube and centrifuged at 13,000 rpm for 10 minutes. The supernatant plasma was collected and stored at -80°C. Plasma was processed as follows: 50 μL of rat plasma was added to 50 μL of acetonitrile and 50 μL of artemisinin internal standard solution (500 ng / mL). The mixture was vortexed for 5 minutes, followed by 1 mL of ether, vortexed for 5 minutes, and centrifuged at 13,000 rpm for 10 minutes. The supernatant was taken, dried with nitrogen in a 37°C water bath, reconstituted with 50 μL of acetonitrile, vortexed for 5 min, centrifuged at 13,000 r / min for 10 min, and the supernatant was taken for determination. The plasma concentration-time curve of DHA is shown in Figure 2. Figure 8 The pharmacokinetic parameters are shown in Figure 9 Compared with the DHA-sus group, the AUC of the RPNLs and cRPNLs groups were 0-t and C max Both increased significantly, MRT 0-t The in vivo exposure and mean residence time of DHA in the cRPNLs group were significantly prolonged, and the in vivo exposure and mean residence time of DHA in the cRPNLs group were significantly higher than those in the RPNLs group. In summary, cRPNLs significantly improved the pharmacokinetic properties of DHA, prolonged the circulation time of DHA in the blood, and increased the oral bioavailability of DHA.

[0031] Validation experiment 8: In vivo M cell targeting study of cRGD / PDA co-modified dihydroartemisinin prodrug liposomes DiR fluorescently labeled PNLs and cRPNLs were prepared to obtain DiR-PNLs and DiR-cRPNLs, respectively. 18 ICR mice, 20 ± 2 g, were adaptively fed for 7 days and fasted for 12 h before administration. The mice were randomly divided into 3 groups and given 2 mg / kg of Free DiR, DiR-PNLs and DiR-cRPNLs by gavage according to DiR calculation. They were killed by cervical dislocation 1 and 3 h after administration, with 3 mice in each group. The entire gastrointestinal segment and liver of each group of mice were removed, and fluorescent images were obtained using a small animal in vivo imaging device. The intestinal segment was divided into a 1 cm segment containing Peyer's colon (PP) and a segment without Peyer's colon (NPP) to obtain fluorescence quantitative data. Figure 10As shown in the figure, 1 h and 3 h after administration, the fluorescence signal in the gastrointestinal tract of the liposome group was stronger than that of the free group, indicating that the liposome can effectively protect the stability of the drug in the gastrointestinal environment. Figure 11 and Figure 12 As shown in the figure, the fluorescence intensity of the Free-DiR, DiR-PNLs, and DiR-cRPNLs groups in the liver decreased in sequence, and the difference was significant 1 hour after administration. This indicates that liposomes can effectively reduce the tissue distribution of drugs in the liver, thereby reducing the first-pass effect in the liver, and cRGD modification can further enhance this effect. Figure 13 、 14 As shown, 1 hour after administration, the PP fluorescence signal in the DiR-cRPNLs group was stronger than that in the NPP group. The PP fluorescence signal in the DiR-cRPNLs group was significantly stronger than that in the Free-DiR and DiR-PNLs groups, indicating that DiR-cRPNLs can effectively target M cells and accumulate in Peyer's lymph nodes. In summary, cRPNLs have the ability to enhance drug gastrointestinal stability, reduce first-pass effects in the liver, and target M cell-mediated lymphatic transport.

[0032] Validation Experiment 9: In Vivo Antimalarial Efficacy and Safety Evaluation of cRGD / PDA Modified Dihydroartemisinin Prodrug Liposomes 136 mice were randomly divided into 17 groups, with 8 mice in each group. The specific groups were as follows: normal group, malaria mouse model group, and intervention group with a series of concentrations of DHA-sus, PNLs, and cRPNLs. The dosages calculated as DHA were 13.7, 27.4, 54.9, 109.8, and 219.4 μmol / kg, respectively. The antimalarial efficacy was evaluated by the Pearson four-day inhibition dosing method, and the infection rate, inhibition rate, and ED rate on the first day of drug withdrawal were calculated. 50 The values ​​and infection rates on the 7th day after drug withdrawal were recorded. On the 1st day after drug withdrawal, whole blood samples were collected from mice for determination of liver function indicators and blood routine tests. At the same time, heart, liver, spleen, lung and kidney tissues were dissected and removed for pathological analysis. The 21-day survival rate of mice was recorded. The efficacy results of all preparations are shown in Figure 17 The results showed that on the first day after drug withdrawal, the infection rate of the cRPNLs group was significantly lower than that of the DHA-Sus group and the PNLs group at the same concentration, effectively inhibiting the growth of Plasmodium in malaria mice; on the seventh day after drug withdrawal, the infection rate of each group increased, but the infection rate of the cRPNLs group was still lower than that of the DHA-sus group. At the same time, with the increase of the dosage, the infection rate of malaria mice gradually decreased, which was dose-dependent. At the same dose, the inhibitory effect of the cRPNLs group was significantly better than that of the DHA-sus and PNLs groups. The ED of the cRPNLs group 50 The value was 2.65 ± 1.61 µmol / kg / day, which was significantly lower than that of DHA-sus and PNLs.

[0033] The liver function indexes of mice in each group were Figure 18 、 Figure 19 In the experiment, the liver function index of the mice in the model group was significantly higher than that of the healthy mice. As the dose increased, the liver function index of the mice in the preparation group gradually approached the index of normal mice. Figure 20 , showing a trend similar to that of the normal group. Figure 21 The red blood cell (RBC), white blood cell (WBC), hematocrit (HCT) and hemoglobin (HGB) of mice in the PNLs and cRPNLs groups were maintained within the normal reference range, indicating that the two treatment groups maintained a good hematological status. Figure 22 In the model group, mice showed accumulation of inflammatory cells in the liver tissue, reflecting the damage and metabolic abnormalities of liver cells. In the drug-treated group, the degree of liver and spleen damage was reduced, and there was almost no damage in the cRPNLs group. No obvious abnormalities were observed in the slices of other organs. Figure 23 The survival curves show that at the same dose, the average survival days of mice in the cRPNLs, PNLs, and DHA-sus groups decreased in order, with the cRPNLs group showing a stronger survival advantage in resisting malaria infection. In summary, the liposomes of the present invention have good antimalarial efficacy and formulation safety.

Claims

1. cRGD / PDA co-modified dihydroartemisinin prodrug liposomes, characterized by: It includes a polydopamine shell, a cRGD target head and a dihydroartemisinin prodrug.

2. The cRGD / PDA co-modified dihydroartemisinin prodrug liposome according to claim 1, characterized in that: It also includes soybean lecithin and cholesterol, the mass ratio of the dihydroartemisinin prodrug to soybean lecithin is 1:15, and the mass ratio of soybean lecithin to cholesterol is 10:

1.

3. The cRGD / PDA co-modified dihydroartemisinin prodrug liposome according to claim 2, characterized in that: The composition further comprises a stabilizer, the mass percentage of which is 5%.

4. The cRGD / PDA co-modified dihydroartemisinin prodrug liposome according to claim 3, characterized in that: The stabilizer is vitamin E polyethylene glycol succinate.

5. The cRGD / PDA co-modified dihydroartemisinin prodrug liposome according to any one of claims 1 to 4, characterized in that: The dihydroartemisinin prodrug comprises a reduction-responsive disulfide bond.

6. The method for preparing the cRGD / PDA co-modified dihydroartemisinin prodrug liposomes according to any one of claims 1 to 5, characterized in that: The following steps are included: Step 1: Weigh dithiodipropionic acid, add acetic anhydride to react and form a ring; weigh hexadecanol and 4-dimethylaminopyridine, dissolve them in dichloromethane, and add them to the above reaction flask for reaction; separate and purify the product to obtain an intermediate product; weigh the intermediate product, dihydroartemisinin, 4-dimethylaminopyridine and carbodiimide hydrochloride, dissolve them in dichloromethane for reaction, and separate and purify the product to obtain a dihydroartemisinin prodrug; Step 2: Take the prescribed amount of dihydroartemisinin prodrug, soybean lecithin, cholesterol and vitamin E polyethylene glycol succinate, accurately weigh and set aside; Step 3: Accurately measure the final volume of anhydrous ethanol to dissolve the drug weighed in step 2; Step 4: Slowly inject the mixed solution obtained in step 3 into the aqueous solution below the liquid surface and stir evenly to prepare dihydroartemisinin prodrug liposomes; the dihydroartemisinin prodrug liposome solution prepared above is mixed with a Tris-HCl buffer solution, and dopamine hydrochloride is added, stirred and mixed, incubated, and ultrafiltration and centrifugation are performed to obtain polydopamine-coated dihydroartemisinin prodrug liposomes; the dihydroartemisinin prodrug liposomes prepared above are mixed with a Tris-HCl buffer solution, and cRGD peptide is added, stirred and mixed, incubated in the dark, and ultrafiltration and centrifugation are performed to obtain cRGD / PDA-modified dihydroartemisinin prodrug liposomes.

7. Use of the cRGD / PDA co-modified dihydroartemisinin prodrug liposome according to any one of claims 1 to 5 for oral administration to treat malaria.

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