MaR1 nano-composite as well as preparation method and application thereof

Abstract

The invention discloses a MaR1 nano-composite and a preparation method and application thereof, and belongs to the technical field of biological medicine, the MaR1 nano-composite comprises a liposome, a targeting peptide, and MaR1 or pharmaceutically acceptable salt thereof entrapped in the liposome. According to the invention, the effective drug MaR1 and the myocardial targeting peptide are connected by adopting lipidosome, and nano precipitation is carried out by using a microfluid mixing channel, so that the nano drug has high bioavailability and stability. The MaR1 nano-liposome drug prepared by the invention has the characteristics of high bioavailability, stable property, quick response, low side effect and the like, and has a remarkable protective effect on myocardial reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a MaR1 nanocomposite and a preparation method thereof, and an application thereof in preparing a drug for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemia-reperfusion (MI / R) injury is a common complication of revascularization therapy after myocardial infarction, and its mechanism involves immune inflammatory response and oxidative stress. It may lead to cardiac dysfunction and seriously affect the quality of life and prognosis of patients, but there is still a lack of effective drugs for the prevention and treatment of myocardial ischemia-reperfusion injury in clinical practice.

[0003] The family of specialized pro-resolving mediators (SPMs) is a class of bioactive metabolites synthesized by the decomposition of polyunsaturated fatty acids (PUFA) during tissue repair. They have the functions of limiting the recruitment of inflammatory granulocytes, regulating macrophage polarization, mediating the active resolution of inflammation, and do not cause negative effects such as immunosuppression. Maresin 1 (MaR1) is a newly discovered member of the SPMs family in recent years. It is mainly catalyzed by macrophages through lipoxygenase to convert docosahexaenoic acid (DHA) into a class of stereoselective anti-inflammatory mediators through a series of lipid oxygenation reactions. MaR1 is the fastest synthesized and earliest resolving inflammatory mediator in the SPMs family that responds to inflammatory signals at the site of injury. It has received widespread attention based on its powerful anti-inflammatory effect. However, the clinical application of MaR1 is also limited by its poor stability, easy degradation, low targeting efficiency and potential toxicity. Currently, there is no relevant research on the effect of MaR1 on myocardial ischemia-reperfusion injury.

[0004] Therefore, there is an urgent need to provide effective targeted drugs for treating myocardial ischemia-reperfusion injury in order to improve the therapeutic effect of myocardial ischemia-reperfusion injury. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a MaR1 nanocomplex connected with MaR1 and myocardial targeting peptide using liposomes, so as to provide a targeted myocardial nanocomplex with good stability, non-degradability and high targeting efficiency, so as to improve the therapeutic effect of myocardial ischemia-reperfusion injury.

[0006] The technical problem that the present invention needs to solve is to provide a method for preparing the above-mentioned MaR1 nanocomposite.

[0007] The final technical problem to be solved by the present invention is to provide the application of the above MaR1 nanocomplex in the preparation of a drug for treating myocardial ischemia-reperfusion injury.

[0008] Technical solution: To solve the above technical problems, the present invention provides the following technical solutions:

[0009] A MaR1 nanocomplex, comprising a liposome, a targeting peptide, and MaR1 or a pharmaceutically acceptable salt thereof encapsulated in the liposome;

[0010] The structural formula of MaR1 is shown in formula (I):

[0011]

[0012] The liposomes are prepared from cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal;

[0013] The myocardial targeting peptide is an antibody coupled to the surface of liposomes, and its amino acid sequence is shown in SEQ ID NO.1.

[0014] Furthermore, the mass ratio of cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal is 20:5:7:5.

[0015] Furthermore, the mass ratio of the liposome to MaR1 or a pharmaceutically acceptable salt thereof is (2:1)-(2:0.1).

[0016] Furthermore, the volume ratio of the liposome to the targeting peptide is 16:5.

[0017] The preparation method of the MaR1 nanocomposite comprises the following steps:

[0018] (1) mixing cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal in a mass ratio of 20:5:7:5 to obtain a lipid mixture;

[0019] (2) dissolving the lipid mixture in anhydrous ethanol to obtain a carrier solution;

[0020] (3) taking a carrier solution and a PBS buffer, preheating them to 60° C. respectively, and mixing the preheated carrier solution and the PBS buffer to obtain a liposome mixture;

[0021] (4) The liposome extruder was then heated to 63°C and extruded 21 times using a polycarbonate membrane with a pore size of 100 nm to obtain a liposome solution with a particle size of 100 nm;

[0022] (5) Mixing the above liposome solution with the MaR1 drug solution, freezing the mixture in liquid nitrogen for 10 seconds until the liquid is completely frozen, and then heating it in a 40°C water bath for 3 minutes for 10 cycles to form drug-loaded liposomes containing MaR1;

[0023] Take 150 μL of targeting peptide solution and 480 μL of drug-loaded liposomes, incubate at 37°C for 1 hour, and then ultrafilter and centrifuge at 4500 rpm for 30 minutes. Take the upper layer of liquid in the ultrafiltration tube and rinse the inner wall of the ultrafiltration tube with PBS to 500 μL, and finally obtain the MaR1 nanocomplex targeting the myocardium.

[0024] In step (2), the concentration of the carrier solution is 20 mg / mL.

[0025] In step (3), the volume ratio of the carrier solution to the PBS buffer is 1:9; the parameters of the PBS buffer are 10 mM and pH 7.4.

[0026] In step (5), the mass ratio of the liposome solution to the MaR1 drug solution is 2:1;

[0027] The preparation method of the MaR1 drug solution is as follows: 100 μg of Maresin1 powder is dissolved in 1 ml of ethanol.

[0028] In step (5), the preparation method of the targeting peptide solution is as follows:

[0029] 1 mg myocardial targeting peptide was mixed with 200 μL PBS and the pH was adjusted to 7.4 with NaOH;

[0030] The amino acid sequence of the myocardial targeting peptide is shown in SEQ ID NO.1.

[0031] The use of the above MaR1 nanocomplex in the preparation of a drug for treating myocardial ischemia-reperfusion injury is within the protection scope of the present invention.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0033] The nanomedicine provided by the present invention can effectively deliver MaR1 to the heart, can enhance the phagocytic ability and scavenging effect of local macrophages, and promote the transformation of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type, thereby reducing the formation of reactive oxygen species in the myocardium. Further, we found that MaR1 nano can effectively improve myocardial function, inhibit myocardial cell apoptosis, reduce the degree of cardiac fibrosis, and thus play a role in improving myocardial ischemia-reperfusion injury. At the same time, MaR1 nanomedicine showed good in vivo safety and biocompatibility in vivo, confirming the rationality of nanomedicine design. Therefore, the MaR1 nanomedicine we prepared has high bioavailability, stable properties, fast onset, low side effects, and has a significant protective effect on myocardial reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of the cardiac-targeted nano-drug complex of the present invention;

[0035] Figure 2 This is a distribution diagram of the nano drug of the present invention in mice at a short-term time point after injection;

[0036] Figure 3 This is a distribution diagram of the nanomedicine of the present invention in mice at a long-term time point after injection;

[0037] Figure 4 This is a comparison diagram of the cardiac function of the experimental mice of the present invention;

[0038] Figure 5 This is a comparison chart of the inflammation level and ROS level of the experimental mice of the present invention;

[0039] Figure 6 This is a comparison diagram of apoptosis levels in cardiac tissue of experimental mice of the present invention;

[0040] Figure 7 This is a comparison diagram of the degree of cardiac fibrosis in the experimental mice of the present invention;

[0041] Figure 8 This is a comparison chart of liver and kidney function index levels of experimental mice of the present invention. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0043] Example 1: Preparation of medicine

[0044] like Figure 1As shown, cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal are mixed in a mass ratio of 20mg:5mg:7mg:5mg by microfluidics to obtain a lipid mixture; the lipid mixture is dissolved in anhydrous ethanol to obtain a carrier solution with a concentration of 20mg / ml; 1mL of the carrier solution is taken, added to a 15mL centrifuge tube and preheated to 60°C; 9mL of 60°C PBS buffer is prepared, the parameters of the PBS buffer are 10mM, pH 7.4, added to another 15mL centrifuge tube, and also preheated to 60°C; the preheated carrier solution and PBS buffer are mixed to form a final 10mL liposome mixture; after the first step of mixing, the liposome particle size is 200-300nm, and then the liposome extruder is heated to the phase transition temperature of DSPC (63°C), and a polycarbonate membrane with a pore size of 100nm is used to repeatedly extrude 21 times to obtain a liposome solution with a particle size of 100nm; the above 6μg liposome solution is mixed with 3ug The MaR1 drug solution was mixed, and the mixture was frozen in liquid nitrogen for 10 seconds. After the liquid was completely frozen, it was placed in a 40°C water bath and heated for 3 minutes, and then cycled 10 times to form drug-loaded liposomes containing MaR1. Subsequently, 1 mg of myocardial targeting peptide (sequence: CSTSMLKAC) + 200uL PBS was mixed, and the pH was adjusted to 7.4 with NaOH, and then 150uL of targeting peptide solution + 480uL of drug-loaded liposomes were taken. After incubation at 37°C for 1h, ultrafiltration centrifugation was performed at 4500rpm for 30min, and the upper layer of the ultrafiltration tube was taken. The inner wall of the ultrafiltration tube was rinsed with PBS to 500uL, and finally the myocardial-targeted MaR1 nanoliposomes were obtained.

[0045] Transmission electron microscopy and dynamic light scattering were used to measure the particle size and distribution of the nanomedicine. The results showed that the particle size of the nanomedicine was about 100nm and was evenly distributed.

[0046] Example 2: Exploration of the appropriate concentration and targeting of MaR1 nanodrugs in myocardial ischemia-reperfusion injury models

[0047] Animal model construction: 6-8 week old male C57BL / 6 mice were used to establish the myocardial ischemia-reperfusion injury (MI) model.

[0048] Animal groups: control group (Control), MI group (ischemia-reperfusion only), MaR1 nanodrug group (MI+nano-MaR1).

[0049] Drug administration: For the nanodrug group, different concentrations of MaR1 nanodrugs (2μM, 20μM, 40μM) were injected through the tail vein immediately after the myocardial ischemia-reperfusion model was established. The targeting of the drug in vivo was evaluated 12 hours, 1 day, 2 days, and 3 days after treatment.

[0050] Experimental results:

[0051] Attached Figure 2 : Imaging technology was used to observe the distribution of drugs in cardiac tissue: The results showed that MaR1 nanodrug (2μM, 20μM, 40μM) could reach the local area of ​​the heart 12 hours after injection, and had good targeting.

[0052] Example 3: Long-term residence of MaR1 nanodrug in myocardial ischemia-reperfusion injury model

[0053] Animal model construction: Myocardial MI model was established using 6-8 week old male C57BL / 6 mice.

[0054] Animal groups: control group (Control), nanomedicine group (IR+nano-MaR1).

[0055] Drug administration: Immediately after the myocardial ischemia-reperfusion model was established, 20 μM MaR1 nanodrug was injected through the tail vein. The long-term retention of the drug was evaluated 7, 14, 21, and 28 days after treatment.

[0056] Experimental results:

[0057] Attached Figure 3 : Imaging technology was used to observe the distribution of drugs in cardiac tissue: The results showed that MaR1 nanodrugs could reach local areas of the heart 28 days after injection and had good retention.

[0058] Example 4: Short-term efficacy of MaR1 nanomedicine in myocardial ischemia-reperfusion injury model

[0059] Animal model construction: Myocardial MI model was established using 6-8 week old male C57BL / 6 mice.

[0060] Animal groups: control group (Control), MI group (ischemia-reperfusion only), MI+MaR1 group (ischemia-reperfusion mice given MaR1 drug not encapsulated with nanomaterials), and MI+nano-MaR1 group (ischemia-reperfusion mice given MaR1 nano-drugs).

[0061] Drug administration: Immediately after the myocardial ischemia-reperfusion model was established, 20 μM MaR1 nanodrug was injected through the tail vein. Samples were taken 3 days after treatment to evaluate the short-term efficacy of the drug.

[0062] Experimental results:

[0063] Attached Figure 4 : Cardiac function assessment: After nano-MaR1 treatment of myocardial ischemia-reperfusion injury, the EF value was significantly improved.

[0064] Attached Figure 5:Inflammation and ROS: After nano-MaR1 treatment of myocardial ischemia-reperfusion injury, M1 macrophages decreased significantly and M2 macrophages increased relatively. At the same time, nano-MaR1 can reduce the production of ROS, indicating its potential therapeutic role in regulating inflammatory response.

[0065] Attached Figure 6 :Myocardial apoptosis: nano-MaR1 reduced myocardial cell apoptosis after treating myocardial ischemia-reperfusion injury.

[0066] The above results indicate that MaR1 nanomedicine can significantly improve cardiac function and inflammation levels after myocardial ischemia-reperfusion injury.

[0067] Example 5: Long-term therapeutic effect of MaR1 nanomedicine on myocardial ischemia-reperfusion injury

[0068] Animal model construction: Myocardial MI model was established using 6-8 week old male C57BL / 6 mice.

[0069] Animal groups: control group (Control), MI group (ischemia-reperfusion only), MI+MaR1 group (ischemia-reperfusion mice given MaR1 drug not encapsulated with nanomaterials), and MI+nano-MaR1 group (ischemia-reperfusion mice given MaR1 nano-drugs).

[0070] Drug administration: Immediately after the myocardial ischemia-reperfusion model was established, 20 μM MaR1 nanodrug was injected through the tail vein. The long-term efficacy of the drug was evaluated 14 days after treatment.

[0071] Experimental results:

[0072] Attached Figure 7 : Anti-fibrosis: After nano-MaR1 treatment of myocardial ischemia-reperfusion injury, the degree of fibrosis was significantly alleviated.

[0073] Example 6: Safety of MaR1 nanomedicine for myocardial ischemia-reperfusion injury

[0074] Animal model construction: Myocardial MI model was established using 6-8 week old male C57BL / 6 mice.

[0075] Animal groups: control group (Control), MI group (ischemia-reperfusion only), MI+MaR1 group (ischemia-reperfusion mice given MaR1 drug not encapsulated with nanomaterials), and MI+nano-MaR1 group (ischemia-reperfusion mice given MaR1 nano-drugs).

[0076] Drug administration: Immediately after the myocardial ischemia-reperfusion model was established, 20 μM MaR1 nanodrug was injected through the tail vein. The liver and kidney functions and pathological morphology of various tissues and organs were evaluated 14 days after treatment.

[0077] Experimental results:

[0078] Attached Figure 8 :MaR1 nanodrug has no effect on liver and kidney function.

[0079] The above results indicate that MaR1 nanomedicine has good biosafety.

[0080] The embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that a person skilled in the art may make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A MaR1 nanocomposite, characterized in that: The MaR1 nanocomplex comprises a liposome, a targeting peptide, and MaR1 or a pharmaceutically acceptable salt thereof encapsulated in the liposome; The structural formula of MaR1 is shown in formula (I): The liposomes are prepared from cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal; The myocardial targeting peptide is an antibody coupled to the surface of liposomes, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The MaR1 nanocomposite according to claim 1, characterized in that The mass ratio of cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal is 20:5:7:

5.

3. The MaR1 nanocomposite according to claim 1, characterized in that The mass ratio of the liposome to MaR1 or a pharmaceutically acceptable salt thereof is 2:1-2:0.

1.

4. The MaR1 nanocomposite according to claim 1, characterized in that The volume ratio of the liposome to the targeting peptide is 16:

5.

5. A method for preparing a MaR1 nanocomposite, characterized in that: The steps include: (1) mixing cholesterol, DSPC, DSPE-PEG2k and DSPE-PEG2k-mal in a mass ratio of 20:5:7:5 to obtain a lipid mixture; (2) dissolving the lipid mixture in anhydrous ethanol to obtain a carrier solution; (3) taking a carrier solution and a PBS buffer, preheating them to 60° C. respectively, and mixing the preheated carrier solution and the PBS buffer to obtain a liposome mixture; (4) The liposome extruder was then heated to 63°C and extruded 21 times using a polycarbonate membrane with a pore size of 100 nm to obtain a liposome solution with a particle size of 100 nm; (5) Mixing the above liposome solution with the MaR1 drug solution, freezing the mixture in liquid nitrogen for 10 seconds until the liquid is completely frozen, and then heating it in a 40°C water bath for 3 minutes for 10 cycles to form drug-loaded liposomes containing MaR1; Take 150 μL of targeting peptide solution and 480 μL of drug-loaded liposomes, incubate at 37°C for 1 hour, and then ultrafilter and centrifuge at 4500 rpm for 30 minutes. Take the upper layer of liquid in the ultrafiltration tube and rinse the inner wall of the ultrafiltration tube with PBS to 500 μL, and finally obtain the MaR1 nanocomplex targeting the myocardium.

6. The method for preparing the MaR1 nanocomposite according to claim 5, characterized in that: In step (2), the concentration of the carrier solution is 20 mg / mL.

7. The method for preparing the MaR1 nanocomposite according to claim 5, characterized in that: In step (3), the volume ratio of the carrier solution to the PBS buffer is 1:9; the parameters of the PBS buffer are 10 mM and pH 7.

4.

8. The method for preparing the MaR1 nanocomposite according to claim 5, characterized in that: In step (5), the mass ratio of the liposome solution to the MaR1 drug solution is 2:1; The preparation method of the MaR1 drug solution is as follows: Take 100ug Maresin1 powder and dissolve it in 1ml ethanol.

9. The method for preparing the MaR1 nanocomposite according to claim 5, characterized in that: In step (5), the preparation method of the targeting peptide solution is as follows: 1 mg myocardial targeting peptide was mixed with 200 μL PBS and the pH was adjusted to 7.4 with NaOH; The amino acid sequence of the myocardial targeting peptide is shown in SEQ ID NO.

1.

10. Use of the MaR1 nanocomplex according to any one of claims 1 to 4 in the preparation of a drug for treating myocardial ischemia-reperfusion injury.

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