A novel D-lactic acid-loaded biomimetic targeted nanoplatform for the treatment of in-situ liver cancer and its preparation method

Abstract

The present invention discloses a novel D-lactic acid-loaded biomimetic targeted nanopreparation for the treatment of in-situ liver cancer and its preparation method, which relates to the technical field of drug preparation. In view of the pharmacological effects and physicochemical properties of D-lactic acid, the present invention designs polylactic acid-glycolic acid nanoparticles loaded with D-lactic acid, and prepares polylactic acid nanoparticles with uniform controllability, small particle size and good dispersibility that can encapsulate and load D-lactic acid by ultrasonic emulsification combined with solvent evaporation method; further, the tumor cell membrane is coated on the surface of the polylactic acid nanoparticles loaded with D-lactic acid to form a biomimetic nanopreparation; the biomimetic nanopreparation is vertically suspended with a targeting polypeptide to form a polylactic acid biomimetic targeted nanopreparation. The novel D-lactic acid-loaded biomimetic targeted nanopreparation for the treatment of in-situ liver cancer of the present invention has the functions of regulating the phenotype of M2-type tumor-associated macrophages and reversing the tumor microenvironment, and has good therapeutic effects on antigenic in-situ liver cancer and spontaneous liver cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and particularly to a novel D-lactic acid-loaded bionic targeted nano preparation for treating primary liver cancer and a preparation method thereof. Background Art

[0002] In recent years, studies have shown that the presence of intestinal flora and their metabolites interfere with the formation of the liver immune tolerance microenvironment and are also closely related to the occurrence, development, metastasis and drug resistance of tumors. According to literature reports, the types and numbers of intestinal flora can predict the anti-tumor efficacy of immune checkpoint inhibitors, and specific flora can regulate the efficacy of ICIs. Utilizing auxiliary ICI flora helps to overcome the drug resistance of ICI treatment. In addition, the metabolites produced by intestinal flora can enter the blood circulation and regulate the physiological functions of the host.

[0003] For example, a study reported in the journal Science in 2018 found that Clostridium can control the intestinal bile metabolism of mice, and the generated secondary bile acids can enhance the expression of the liver chemokine C-X-C motif ligand 16 (CXCL16) protein, and CXCL16 can recruit natural killer T cells to inhibit the occurrence and development of liver cancer.

[0004] A study reported in the journal Science in 2020 found that Pseudomonas in the small intestine can release metabolites inosine phosphate and hypoxanthine, which can penetrate the intestinal barrier and be distributed in the circulatory system, and activate the T cell adenosine receptor-related signaling pathway in the presence of other co-stimulatory adjuvants, further activating the anti-tumor T cell response and inhibiting the growth of colorectal cancer, bladder cancer and melanoma. It can be seen that regulating the intestinal flora and their metabolites to improve the tumor microenvironment is crucial for enhancing the efficacy of anti-tumor immunotherapy.

[0005] A study reported in the journal Cell Host & Microbe in 2020 found that the intestinal flora metabolite D-lactic acid is an important immunomodulator. It enters the liver through the hepatic portal vein, changes the morphology of Kupffer cells, increasing their surface area and volume; and the Kupffer cells with changed morphology enhance the ability to capture and kill pathogens, improving the clearance efficacy of pathogens in the liver.

[0006] However, the level of D-lactic acid produced by the gut microbiota is extremely low, and its entry into the blood circulation via the hepatic portal vein leads to its non-specific wide distribution. The D-lactic acid accumulated in the liver is not sufficient to activate the anti-tumor immune response. Therefore, targeted delivery of exogenous D-lactic acid into the liver to increase its accumulation at the tumor site is a feasible solution to exert its induction of TAM transformation and improvement of the immunosuppressive tumor microenvironment. In recent years, nanoparticles have shown advantages such as improving drug solubility, reducing toxic side effects, increasing targeted delivery, and sustained release. Therefore, the use of nanocarriers can achieve the specific delivery of D-lactic acid to TAM in liver tumors.

[0007] Poly (lactic-co-glycolic acid) is a biodegradable polymer organic compound with good biocompatibility, low immunogenicity, and good film-forming and encapsulation properties. It has been widely used in the fields of drug delivery and tumor immunotherapy. In recent years, the biomimetic modification of nanoparticles by biological membranes has also added advantages to the application of nanoparticles. The modification of biological membranes can make nanoparticles disguise as endogenous substances, reduce the uptake by the reticuloendothelial system, prolong their blood circulation time, and increase their tissue-targeted delivery performance. For example, nanoparticles coated with tumor cell membranes have the natural property of tumor cell homing, which can increase the accumulation of nanoparticles at the tumor lesion. At the same time, the tumor cell membrane surface carries tumor-associated antigens, which can mediate the tumor vaccine effect. Therefore, the use of biomimetic nanoparticles to promote long-circulation and targeted delivery of exogenous D-lactic acid is expected to become an effective strategy for realizing the immunotherapy of liver cancer with gut microbiota metabolites. Summary of the Invention

[0008] The purpose of the present invention is to provide a novel biomimetic targeted nanoplatform loaded with D-lactic acid for the treatment of orthotopic liver cancer and its preparation method to solve the problems in the background technology.

[0009] To achieve the above purpose, the present invention provides the following technical solutions:

[0010] A novel biomimetic targeted nanoplatform loaded with D-lactic acid for the treatment of orthotopic liver cancer, comprising the following raw materials: polylactic acid materials, organic solvents, D-lactic acid, phosphate buffer solution, and surfactants.

[0011] On the basis of the above technical solutions, the present invention also provides the following optional technical solutions:

[0012] In an optional solution: the concentration of the polylactic acid materials is 10%-20% and the molecular weight is 20,000-100,000.

[0013] In an optional solution: the polylactic acid materials include polylactic acid, poly (lactic-co-glycolic acid), and poly (lactic-co-vinyl alcohol).

[0014] In an alternative embodiment: the organic solvent is any one or a mixture of several of dichloromethane, ethyl acetate, and acetone.

[0015] In an alternative embodiment: the surfactant is any one of polyvinyl alcohol, sodium cholate, and emulsifier T-20; the concentration of the surfactant is 0.1% - 2%.

[0016] A method for preparing a novel D-lactic acid-loaded biomimetic targeted nanoformulation for treating in-situ liver cancer as described above, comprising the following steps: Step 1: Dissolve a polylactic acid-based material in an organic solvent as the oil phase, dissolve D-lactic acid in phosphate buffer as the inner aqueous phase, and dissolve the surfactant in ultrapure water as the outer aqueous phase; Step 2: Pour the inner aqueous phase into the oil phase, and ultrasonically prepare a water-in-oil primary emulsion under ice bath conditions. Pour the primary emulsion into a part of the outer aqueous phase containing a certain amount of surfactant, and ultrasonically prepare a water-in-oil-in-water pre-complex emulsion under ice bath conditions. Then pour the pre-complex emulsion into another part of the outer aqueous phase containing a certain amount of surfactant to obtain a complex emulsion; Step 3: Further evaporate the solvent of the complex emulsion to solidify it to obtain polylactic acid nanoparticles loaded with D-lactic acid; Step 4: Extract the liver cancer tumor cell membrane, and repeatedly extrude it through a liposome extruder to coat the cell membrane on the surface of the polylactic acid nanoparticles loaded with D-lactic acid to form a polylactic acid biomimetic nanoformulation loaded with D-lactic acid coated with the liver cancer tumor cell membrane; Step 5: Vertically suspend the solution containing the targeting polypeptide with the biomimetic nanoformulation coated with the cell membrane, and insert the targeting polypeptide into the lipid bilayer of the cell membrane through the post-insertion method to form a targeted polypeptide-modified polylactic acid biomimetic targeted nanoformulation loaded with D-lactic acid.

[0017] In an alternative embodiment: the volume ratio of the inner aqueous phase to the oil phase in Step 2 is 1:5 - 1:20; the ultrasonic time of the primary emulsion is 20 - 80 s, the ultrasonic intensity is 50%, and the ultrasonic mode is pulsed.

[0018] In an alternative embodiment: the ultrasonic time of the complex emulsion is 60 - 180 s, the ultrasonic intensity of the complex emulsion is 50%, and the ultrasonic mode is pulsed.

[0019] In an alternative embodiment: the solidification method of solvent evaporation and solidification in Step 3 is the solvent evaporation method or the solvent diffusion method; in Step 4, the cell membrane is the liver cancer cell membrane, and the pore size of the polycarbonate membrane is 100 nm - 1 μm.

[0020] In an alternative embodiment: the targeting polypeptide in Step 5 is a tumor-associated macrophage targeting ligand, and the vertical suspension time is 1 - 4 h.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] By adjusting the ratio of the oil phase to the water phase and preparing the primary emulsion by ultrasonic treatment, the primary emulsion can be fused during the formation of the nano - preparation to form an inner cavity for encapsulating D - lactic acid. Through cell - membrane biomimetic modification, the homing effect of cancer cells is utilized to endow the nano - preparation with the ability to target tumor sites. Further, M2 - type TAM - targeting polypeptides are used to enable precise targeting of tumor - associated macrophages.

[0023] Compared with non - targeted nano - preparations, the ability of the D - lactic acid - loaded biomimetic - targeted nano - preparation to target M2 - type macrophages is significantly improved.

[0024] And it can effectively reverse the phenotype of M2 - type macrophages, reduce M2 - type macrophage markers such as Arg - 1, CD206, and IL - 10, and increase M1 - type macrophage markers such as TNF - α, IL - 1β, and IL - 12.

[0025] In the in - vivo anti - tumor experiment, this nano - preparation can exert powerful pharmacodynamic effects in orthotopic liver cancer and spontaneous liver cancer, reverse the phenotype of M2 - type tumor - associated macrophages, reshape the tumor microenvironment, inhibit the occurrence and development of orthotopic liver cancer and spontaneous liver cancer, and prolong the survival period of tumor - bearing mice, demonstrating the good anti - liver - cancer immunotherapeutic efficacy of the D - lactic acid - loaded biomimetic - targeted nano - preparation.

[0026] In view of the pharmacological effects and physicochemical properties of D - lactic acid, poly (lactic - co - glycolic acid) nanoparticles loaded with D - lactic acid were designed in this invention. The poly (lactic - co - glycolic acid) nanoparticles that can encapsulate and load D - lactic acid with uniform controllability, small particle size, and good dispersibility were prepared by ultrasonic emulsification combined with solvent evaporation method. The surface of the nanoparticles was coated with liver cancer tumor cell membranes and modified with M2 - type tumor - associated macrophage (TAM) - targeting polypeptides to form a biomimetic - targeted nano - preparation, improve the targeted delivery of D - lactic acid, enhance the immunogenicity of the nano - preparation, activate the anti - liver - cancer immune response of the D - lactic acid - loaded nano - preparation, and effectively inhibit the occurrence and development of orthotopic liver cancer and spontaneous liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the morphology and particle - size distribution diagram of the D - lactic acid - loaded biomimetic - targeted nano - preparation prepared in this invention. Among them, A is the scanning electron microscope image and particle - size distribution diagram of the PLGA nano - preparation loaded with D - lactic acid, and B is the transmission electron microscope image and particle - size distribution diagram of the D - lactic acid - loaded biomimetic - targeted nano - preparation.

[0028] Figure 2 It is the display diagram of the uptake of the targeted - modified nano - preparation and non - targeted - modified nano - preparation by M2 - type macrophages after the D - lactic acid - loaded biomimetic - targeted nano - preparation prepared in this invention is co - incubated with M2 - type macrophages.

[0029] Figure 3The figure shows the mRNA expression levels of M2 macrophage markers such as Arg-1, CD206, and IL-10, and M1 macrophage markers such as TNF-α, IL-1β, and IL-12 after co-incubation of the D-lactic acid-loaded biomimetic targeted nanoformulation prepared by the present invention with M2 macrophages.

[0030] Figure 4 It shows the expression of M2 tumor-associated macrophage markers (A) and M1 tumor-associated macrophage markers in the immune fluorescence quantitative in-situ liver cancer tumor microenvironment after intravenous injection of the D-lactic acid-loaded biomimetic targeted nanoformulation.

[0031] Figure 5 It is a statistical chart of the results of inhibiting the development of in-situ liver cancer after intravenous injection of the D-lactic acid-loaded biomimetic targeted nanoformulation; among them, A is the bioluminescence intensity of liver cancer tumor cells in the liver, B is the number of necrotic cells in the tumor tissue, and C is the survival rate of mice.

[0032] Figure 6 Statistical chart of the results of inhibiting the development of spontaneous liver cancer after intravenous injection of the D-lactic acid-loaded biomimetic targeted nanoformulation; among them, A is the weight of liver cancer tumors and B is the survival rate of mice. Detailed implementation mode

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The various embodiments listed in the present invention are only used to illustrate the present invention and are not used to limit the scope of the present invention. Any obvious modification or change made to the present invention does not depart from the spirit and scope of the present invention.

[0034] The following technical solutions are adopted:

[0035] In the first aspect, the present invention provides a polylactic acid nanoformulation loaded with D-lactic acid. The polylactic acid nanoformulation loaded with D-lactic acid has the characteristics of small and uniform particle size and good dispersibility; the surface of the nanoformulation is coated with liver cancer tumor cell membranes and modified with a targeting polypeptide targeting M2 TAMs, having the ability to target M2 TAMs in the tumor microenvironment and the effect of activating the immune response against liver cancer.

[0036] The particle size of the polylactic acid-based biomimetic targeted nanoformulation is 70-130 nm, and for example, it can be 70 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm or 130 nm.

[0037] When the particle size of the polylactic acid-based nanoformulation is smaller, it is more easily taken up by cells. For example, when the particle size is 80 - 130 nm, the amount of nanoformulation taken up by cells is larger. Moreover, the smaller the nanoparticles, the more they can enter tumor tissues through the EPR effect, and the greater the possibility for the nanoformulation to exert its immune anti-hepatocellular carcinoma effect.

[0038] The polydispersity index range of the D-lactic acid-loaded polylactic acid-based biomimetic targeted nanoformulation is 0.1 - 0.3, and it can be, for example, 0.1, 0.15, 0.2, 0.25, or 0.3.

[0039] The biomimetic modification of the D-lactic acid-loaded polylactic acid-based biomimetic targeted nanoformulation is the hepatocellular carcinoma tumor cell membrane, and it can be, for example, Hepa1-6, H22, Huh7, or HepG2 cells.

[0040] The targeted modification ligands of the D-lactic acid-loaded polylactic acid-based biomimetic targeted nanoformulation targeting M2-type TAMs include, but are not limited to, galactose, mannose, M2pep polypeptide, or scavenger receptor.

[0041] In a second aspect, a preparation method of a D-lactic acid-loaded polylactic acid-based biomimetic targeted nanoformulation is also provided. The method includes the following steps:

[0042] (1) Dissolve the polylactic acid-based material in an organic solvent as the oil phase, dissolve D-lactic acid in phosphate buffer solution (PBS) as the inner aqueous phase, and dissolve the surfactant in ultrapure water as the outer aqueous phase;

[0043] (2) Pour the inner aqueous phase into the oil phase, and ultrasonically prepare a water-in-oil primary emulsion under ice bath conditions. Pour the inner aqueous phase into the oil phase, and ultrasonically prepare a water-in-oil primary emulsion under ice bath conditions. Pour the primary emulsion into a part of the outer aqueous phase containing a certain amount of surfactant, and ultrasonically prepare a water-in-oil-in-water pre-complex emulsion under ice bath conditions. Then pour the pre-complex emulsion into the other part of the outer aqueous phase containing a certain amount of surfactant to obtain a complex emulsion;

[0044] (3) Further evaporate the solvent of the complex emulsion to solidify it to obtain polylactic acid nanoparticles loaded with D-lactic acid.

[0045] (4) Extract the hepatocellular carcinoma tumor cell membrane, and use a liposome extruder to repeatedly extrude through polycarbonate membranes with different pore sizes to coat the cell membrane on the surface of the polylactic acid nanoparticles loaded with D-lactic acid, forming a polylactic acid biomimetic nanoformulation loaded with D-lactic acid coated with the hepatocellular carcinoma tumor cell membrane;

[0046] (5) Vertically suspend the solution containing the targeting polypeptide with the biomimetic nanoformulation coated with the cell membrane, and insert the targeting polypeptide into the lipid bilayer of the cell membrane through the post-insertion method to form a D-lactic acid-loaded polylactic acid biomimetic targeted nanoformulation modified with the targeting polypeptide.

[0047] In step (1), the polylactic acid material includes, but is not limited to, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid-co-vinyl alcohol) (PELA), etc., and is preferably poly(lactic-co-glycolic acid) (PLGA); the concentration of the polylactic acid material is 10%-20%; the molecular weight of the polylactic acid-based material is 20,000-100,000.

[0048] In step (1), the organic solvent is any one or a combination thereof of dichloromethane, ethyl acetate, and acetone, and is preferably dichloromethane.

[0049] The surfactant is any one of polyvinyl alcohol (PVA), sodium cholate, or emulsifier T-20; the concentration of the surfactant is 0.1%-2%, and can be, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2%, and is preferably 0.5%-2%.

[0050] In step (2), the volume ratio of the inner aqueous phase to the oil phase is 1:5-1:20, and can be, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, and is preferably 1:10.

[0051] In step (2), the ultrasonic time of the primary emulsion is 20-80 s, and can be, for example, 20 s, 40 s, 60 s, 80 s, and is preferably 60 s.

[0052] The ultrasonic intensity of the primary emulsion is 50%, and the ultrasonic mode is pulsed.

[0053] In step (2), the ultrasonic time of the double emulsion is 60-180 s, and is preferably 120 s.

[0054] The ultrasonic intensity of the double emulsion is 50%, and the ultrasonic mode is pulsed.

[0055] In step (3), the curing method is solvent evaporation or solvent diffusion.

[0056] In step (4), the cell membrane is a liver cancer cell membrane, and can be, for example, Hepa1-6, H22, Huh7, or HepG2 cells.

[0057] In step (4), the pore size of the polycarbonate membrane is 100 nm-1 μm, and is preferably 100 nm-400 nm; the number of extrusion times is 9-15 times.

[0058] In step (5), the targeting polypeptide is a tumor-associated macrophage targeting ligand, for example, it can be galactose, mannose, M2pep polypeptide, or scavenger receptor.

[0059] In step (5), the vertical suspension time is 1 - 4 h.

[0060] Example 1

[0061] Prepare a certain amount of PBS aqueous solution of D-lactic acid as the inner aqueous phase (W1), 0.5% sodium cholate aqueous phase as the outer aqueous phase (W3), prepare a certain amount of PBS aqueous solution of baicalin as the inner aqueous phase (W1), weigh a certain amount of poly(lactic-co-glycolic acid) (PLGA) and dissolve it in 1 mL of dichloromethane as the oil phase (O). Add 100 μL of the dissolved inner aqueous phase containing D-lactic acid to 1 mL of the oil phase, and prepare a water-in-oil primary emulsion (W1 / O) by ultrasonic emulsification in an ice bath. Then pour it into 6 mL of the sodium cholate aqueous phase (W2), and prepare a pre-complex emulsion (W1 / O / W2) by ultrasonic emulsification for 60 s in an ice bath. Pour the pre-complex emulsion into 9 mL of the 0.5% sodium cholate aqueous phase (W3), and ultrasonicate for 120 s in an ice bath with an ultrasonic intensity of 50% to prepare a complex emulsion (W2 / O / W3) with small and uniform particle size. Subsequently, place it in a fume hood and stir to solidify overnight. The solidified nano-preparation is centrifuged and washed with ultrapure water 3 - 5 times to obtain the poly(lactic-co-glycolic acid) (PLGA) nano-preparation loaded with D-lactic acid;

[0062] As Figure 1 shown in A, the PLGA nanoparticles loaded with D-lactic acid have good dispersibility, PDI 0.131, particle size 105 nm, encapsulation efficiency of D-lactic acid glycoside of 22%, and loading rate of D-lactic acid of 6.7 μg / mg.

[0063] Example 2

[0064] Use the PLGA nano-preparation loaded with D-lactic acid in Example 1 as the core and the hepatoma cell membrane as the shell to coat on its surface. Accurately weigh 5 mg of the PLGA nano-preparation loaded with D-lactic acid, take 1 mg of the hepatoma cell membrane solution, mix the two at a ratio of 1:1, and then extrude them through polycarbonate membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm successively to coat the cell membrane on the surface of the nano-preparation. Then mix the solution containing 10 μL of the targeting polypeptide with 1 mL of the biomimetic nano-preparation coated with the cell membrane and vertically suspend it at 4°C for 4 h to insert the targeting polypeptide into the lipid bilayer of the cell membrane, forming a biomimetic targeting nano-preparation of poly(lactic acid) loaded with D-lactic acid modified with the targeting polypeptide, as Figure 1 shown in B.

[0065] Example 3

[0066] Confocal laser scanning microscopy (CLSM) was used to qualitatively characterize the cellular uptake of D-lactic acid-loaded poly(lactic acid) biomimetic targeted nanoparticles. In the study of cellular uptake, macrophages were co-incubated with 5 mg / mL nanoparticles for a certain period of time, and then the cellular uptake of the biomimetic nanoparticles was observed by CLSM, with non-targeted nanoparticles as the control. First, macrophages were seeded in confocal dishes at a density of 5×10 4 cells / well, and 1 mL of culture medium was added. After 12 hours, when the cell growth state was stable, rhodamine-labeled nanoparticles were added, and co-incubation was continued for 12 h. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS, DAPI dye buffer was added, and the cell nuclei were stained for 20 min. Finally, the cells were washed twice with PBS to remove unbound dye, and observed and imaged by CLSM.

[0067] The results were as Figure 2 shown. Compared with the non-targeted nanoparticles of the control group, the targeted modified nanoparticles were taken up more by M2 macrophages.

[0068] Example 4

[0069] Study on the induction of the transformation of M2 macrophages into M1 macrophages by D-lactic acid-loaded poly(lactic acid) biomimetic targeted nanoparticles. Culture medium containing nanoparticles (DL@NP@M) was added to the collected M2 macrophages, with the PBS group and free D-lactic acid (Free DL) as controls. After stimulation for 24 hours, the cells were collected and the sample mRNA was extracted. RT-qPCR technology was used to detect the mRNA expression levels of highly expressed markers related to M2 and M1 macrophages (CD206, IL-10, Arg-1, TNF-α, IL-1β, and IL-12).

[0070] The results were as Figure 3 shown. The D-lactic acid-loaded poly(lactic acid) biomimetic targeted nanoparticles (DL@NP@M) significantly down-regulated the mRNA expression levels of the highly expressed markers CD206, IL-10, and Arg-1 in M2 macrophages, while up-regulating the mRNA expression levels of the highly expressed markers TNF-α, IL-1β, and IL-12 in M1 macrophages, indicating that the D-lactic acid-loaded poly(lactic acid) biomimetic targeted nanoparticles could effectively induce the transformation of M2 macrophages into M1 macrophages.

[0071] Example 5

[0072] Study on the phenotypic transformation of M2 tumor-associated macrophages in the immune fluorescence quantitative in-situ liver cancer tumor microenvironment after intravenous injection of D-lactic acid-loaded biomimetic targeted nanoparticles. First, establish in-situ liver cancer tumor-bearing mice, and intravenously inject D-lactic acid-loaded biomimetic targeted nanoparticles (DL@NP@M) according to a certain dosing procedure. At 7-10 days after the last injection, take the tumor tissues of the mice, and detect the expression of surface markers of tumor-associated macrophages in the local tumor by immunofluorescence method; M2 tumor-associated macrophages are labeled with F4 / 80, CD206 and Arg-1 fluorescent antibodies, and M1 tumor-associated macrophages are labeled with F4 / 80, CD86 and iNOS fluorescent antibodies, and a confocal microscope is used to observe the expression of related markers. The results show that the D-lactic acid-loaded biomimetic targeted nanoparticles (DL@NP@M) significantly down-regulated the expression levels of the highly expressed markers CD206 and Arg-1 of M2 macrophages, and at the same time up-regulated the expression levels of the highly expressed markers CD86 and iNOS of M1 macrophages, indicating that the poly(lactic acid) biomimetic targeted nanoparticles loaded with D-lactic acid can also effectively induce the transformation of M2 tumor-associated macrophages in the tumor microenvironment to M1 type in vivo, as Figure 4 shown.

[0073] Example 6

[0074] Study on the inhibition of the development of in-situ liver cancer by D-lactic acid-loaded biomimetic targeted nanoparticles in vivo.

[0075] C57BL / 6 mice (6-8 weeks) were used for in vivo experiments, and the cell line Hepa1-6-LUC was used to construct syngeneic transplanted in-situ tumor-bearing mice; the development of the tumor was monitored by a live imaging instrument. When the luciferase intensity value of the tumor reached -5×10 9 p / sec / cm 2 / sr, the nanoparticles were intravenously injected according to a certain dosing procedure, and the luciferase intensity of the tumor was recorded every two days. Taking the luciferase intensity of the tumor -5×10 11 p / sec / cm 2 / sr as the end point, draw the "time-fluorescence intensity" and "time-survival rate" curves, and the necrosis rate of liver cancer cells was detected by Tunel immunofluorescence method. The results show that the D-lactic acid-loaded biomimetic targeted nanoparticles (DL@NP@M) effectively delayed the occurrence and development of in-situ liver cancer, induced more apoptosis of liver cancer cells, and significantly prolonged the survival period of in-situ liver cancer mice, as Figure 5 shown.

[0076] Example 7

[0077] Study on the Inhibition of the Development of Spontaneous Hepatocellular Carcinoma by D-Lactic Acid-Loaded Biomimetic Targeted Nanopreparations in Vivo. First, C57BL / 6 neonatal mice were selected. Two weeks after birth, diethylnitrosamine (DEN) was intraperitoneally injected at a dose of 10 mg / kg. One week later, the mice were randomly divided into cages, with 16 mice in each group, and continued to be given drinking water containing DEN. The mice were fed with sterile distilled water containing 30 μg / mL DEN every day. The mice in the normal group were fed with sterile distilled water. After continuous modeling for 5 months, the mice were randomly selected and euthanized, and their livers were taken to observe the formation of liver cancer. After the successful establishment of the liver cancer model, the nanopreparations (the D-lactic acid content was 100 mmol) were intravenously injected according to a certain dosing procedure. One week after dosing, 6 mice were euthanized in each group, and liver tissues were taken to observe the antitumor effect of the nanopreparations. The results showed that the nanopreparations reduced the weight of cancer tissues in the livers of mice, significantly inhibited the formation and development of liver cancer tissues, and significantly prolonged the survival period of mice; as Figure 6 shown.

[0078] As described above, it is only the specific implementation manner of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present disclosure can easily think of changes or substitutions, which should all be covered by the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A D-lactic acid-loaded biomimetic targeted nanoformulation for the treatment of in-situ liver cancer, characterized in that, It includes the following raw materials: a polylactic acid-based material with a concentration of 10%-20% and a molecular weight of 20,000-100,000, an organic solvent, D-lactic acid, a phosphate buffer solution, and a surfactant; Among them, the polylactic acid-based material is a poly(lactic-co-glycolic acid) copolymer; The preparation method of a D-lactic acid-loaded biomimetic targeted nanoformulation for the treatment of in-situ liver cancer includes the following steps: Step 1: Dissolve the polylactic acid-based material in an organic solvent as the oil phase, dissolve D-lactic acid in the phosphate buffer solution as the inner aqueous phase, and dissolve the surfactant in ultrapure water as the outer aqueous phase; Step 2: Pour the inner aqueous phase into the oil phase, and ultrasonically prepare a water-in-oil type primary emulsion under ice bath conditions. Pour the primary emulsion into a part of the outer aqueous phase containing a certain amount of surfactant, and ultrasonically prepare a water-in-oil-in-water type pre-complex emulsion under ice bath conditions. Then pour the pre-complex emulsion into another part of the outer aqueous phase containing a certain amount of surfactant to obtain a complex emulsion; Step 3: Further volatilize and solidify the complex emulsion to obtain polylactic acid nanoparticles loaded with D-lactic acid; Step 4: Extract the liver cancer tumor cell membrane, and through a liposome extruder, repeatedly extrude to coat the cell membrane on the surface of the polylactic acid nanoparticles loaded with D-lactic acid to form a polylactic acid biomimetic nanoformulation loaded with D-lactic acid coated with the liver cancer tumor cell membrane; Step 5: Vertically suspend the solution containing the targeting polypeptide with the biomimetic nanoformulation coated with the cell membrane, and through the post-insertion method, insert the targeting polypeptide into the lipid bilayer of the cell membrane to form a targeting polypeptide-modified polylactic acid biomimetic targeted nanoformulation loaded with D-lactic acid.

2. The D-lactic acid-loaded biomimetic targeting nanoformulation for treating in-situ liver cancer according to claim 1, wherein The organic solvent is any one or a mixture of several of dichloromethane, ethyl acetate, and acetone.

3. The D-lactic acid-loaded biomimetic targeting nanoformulation for treating in-situ liver cancer according to claim 1, wherein, The surfactant is any one of polyvinyl alcohol, sodium cholate, and emulsifier T-20; the concentration of the surfactant is 0.1%-2%.

4. A preparation method of a D-lactic acid-loaded biomimetic targeting nanoplatform for treating in situ liver cancer according to any one of claims 1-3, characterized in that, It includes the following steps: Step 1: Dissolve the polylactic acid-based material in an organic solvent as the oil phase, dissolve D-lactic acid in the phosphate buffer solution as the inner aqueous phase, and dissolve the surfactant in ultrapure water as the outer aqueous phase; Step 2: Pour the inner aqueous phase into the oil phase, and ultrasonically prepare a water-in-oil type primary emulsion under ice bath conditions. Pour the primary emulsion into a part of the outer aqueous phase containing a certain amount of surfactant, and ultrasonically prepare a water-in-oil-in-water type pre-complex emulsion under ice bath conditions. Then pour the pre-complex emulsion into another part of the outer aqueous phase containing a certain amount of surfactant to obtain a complex emulsion; Step 3: Further volatilize and solidify the complex emulsion to obtain polylactic acid nanoparticles loaded with D-lactic acid; Step 4: Extract the liver cancer tumor cell membrane, and through a liposome extruder, repeatedly extrude to coat the cell membrane on the surface of the polylactic acid nanoparticles loaded with D-lactic acid to form a polylactic acid biomimetic nanoformulation loaded with D-lactic acid coated with the liver cancer tumor cell membrane; Step 5: Vertically suspend the solution containing the targeting polypeptide with the biomimetic nanoformulation coated with the cell membrane, and through the post-insertion method, insert the targeting polypeptide into the lipid bilayer of the cell membrane to form a targeting polypeptide-modified polylactic acid biomimetic targeted nanoformulation loaded with D-lactic acid.

5. The preparation method of the D-lactic acid-loaded biomimetic targeting nanoformulation for treating in-situ liver cancer according to claim 4, characterized in that, In the second step, the volume ratio of the inner aqueous phase to the oil phase is 1:5 - 1:20; the ultrasonic time of the primary emulsion is 20 - 80 s, the ultrasonic intensity is 50%, and the ultrasonic mode is pulsed.

6. The preparation method of the D-lactic acid-loaded biomimetic targeting nanoformulation for treating in-situ liver cancer according to claim 4, wherein, The ultrasonic time of the double emulsion is 60 - 180 s, the ultrasonic intensity of the double emulsion is 50%, and the ultrasonic mode is pulsed.

7. The preparation method of the D-lactic acid-loaded biomimetic targeting nanoformulation for treating in-situ liver cancer according to claim 4, characterized in that, In the third step, the solidification method for solvent evaporation and solidification is the solvent evaporation method or the solvent diffusion method; in the fourth step, the cell membrane is a liver cancer cell membrane, and the pore size of the polycarbonate membrane is 100 nm - 1 µm.

8. The preparation method of the D-lactic acid-loaded biomimetic targeted nanoformulation for treating in-situ liver cancer according to claim 4, wherein, In the fifth step, the targeting polypeptide is a tumor-associated macrophage targeting ligand, and the vertical suspension time is 1 - 4 h.

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