Bionic nano-liposome for tumor photodynamic therapy, preparation method of bionic nano-liposome and application of bionic nano-liposome in preparation of medicine for treating tumors

Abstract

The invention relates to the technical field of medicines, in particular to a bionic nano-liposome for tumor photodynamic therapy, a preparation method of the bionic nano-liposome and application of the bionic nano-liposome in preparation of medicines for treating tumors. The bionic nano liposome comprises a macrophage membrane and Ce6 which is combined with the macrophage membrane. The bionic nano-liposome provided by the invention has good stability and biocompatibility, can reduce the side effect of a drug on human, can target GBM cells through a macrophage membrane aiming at an inflammatory microenvironment of brain glioma, and can generate active oxygen under a laser condition, so that the bionic nano-liposome is used for photodynamic therapy of tumors.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical technology, and particularly to a biomimetic nanoliposome for tumor photodynamic therapy, a preparation method thereof, and an application thereof in the preparation of a drug for treating tumors. Background Art

[0002] Glioblastoma multiforme (GBM) is one of the most common primary intracranial tumors, originating from the malignant transformation of glial cells in the brain and spinal cord, and is associated with poor prognosis, high recurrence rate and increased mortality. GBM rapidly invades healthy brain tissue by rapid proliferation, promoting angiogenesis and suppressing the immune system, which is a key factor leading to its high mortality. Despite the progress of diagnostic techniques and treatment modalities, including surgical resection combined with chemotherapy and radiotherapy, the prognosis of patients remains very poor, with a 5-year survival rate of only 5%.

[0003] Photodynamic (PDT) therapy uses a laser with a specific wavelength to excite photosensitizers in tissues, resulting in the generation of reactive oxygen species (ROS), thereby inducing oxidative damage and apoptosis of tumor cells. ROS can also block the vascular system of tumors, thus disrupting tumor metabolism and energy supply to tumor cells.

[0004] In addition, with the rapid development of nano-bionic technology, a cell membrane (CM)-coated photosensitizer delivery system has become a promising alternative. Compared with traditional photosensitizer carriers such as liposomes or polymer-based nanoparticles, the bionic CM photosensitizer carrier has advantages such as better targeting, biocompatibility and lower immunogenicity.

[0005] However, most photosensitizers are small molecule substances, which often do not have sufficient tissue targeting, short blood circulation time and poor biocompatibility. In particular, the blood-brain barrier (BBB) severely hinders the delivery of photosensitizers to gliomas, limiting the efficacy of PDT. Among them, tumor cell membranes exhibit strong targeting effects, but they may cause immune stimulation. Therefore, developing a carrier with higher targeting, biocompatibility and lower immunogenicity is crucial for tumor PDT. In addition to delivery challenges, the uncertainty of GMB localization and potential light-dose-dependent cytotoxicity pose another challenge to the application of GMB in PDT. Therefore, real-time monitoring of the location and treatment efficacy of tumors is crucial for optimizing light targeting and adjusting treatment regimens. Summary of the Invention

[0006] In order to solve at least one of the above technical problems, the present application proposes a biomimetic nanoliposome for tumor photodynamic therapy, a preparation method thereof, and an application thereof in the preparation of a drug for treating tumors.

[0007] In a first aspect, a biomimetic nanoliposome for tumor photodynamic therapy is provided. The biomimetic nanoliposome comprises a macrophage cell membrane and chlorine e6 (Ce6) bound to the macrophage cell membrane.

[0008] In some possible embodiments, the Ce6 binds to the macrophage cell membrane in a manner that encapsulates the macrophage cell membrane.

[0009] In a second aspect, a method for preparing the biomimetic nanoliposome as described in the first aspect is provided, comprising:

[0010] Providing a lipid membrane containing Ce6;

[0011] Drying the lipid membrane under vacuum to obtain a dehydrated phospholipid mixture;

[0012] Hydrating the phospholipid mixture in an aqueous PBS solution;

[0013] Uniformly mixing the hydrated phospholipid mixture with the macrophage cell membrane in a PBS solution to obtain a solution mixture;

[0014] Filtering the solution mixture using a filter membrane to obtain the biomimetic nanoliposome as the filtrate.

[0015] In some possible embodiments, the providing a lipid membrane containing Ce6 includes:

[0016] Dissolving 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (CHOL), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotin(polyethylene glycol)2000] (DSPE-PEG 2000) and Ce6 in chloroform to obtain a chloroform solution;

[0017] Evaporating the chloroform solution to remove the organic solvent and generate a lipid membrane containing Ce6.

[0018] In some possible embodiments, the providing a lipid membrane containing Ce6 includes:

[0019] Dissolving 3 parts by mass of DPPC, 1 part by mass of CHOL, 1 part by mass of DSPE-PEG 2000 and 2 parts by mass of Ce6 in 10 parts by volume of chloroform, wherein the ratio of the parts by mass to the parts by volume is mg / ml, to obtain a chloroform solution;

[0020] Using a rotary evaporator to evaporate the chloroform solution to remove the organic solvent and generate a lipid membrane containing Ce6.

[0021] In some possible embodiments, the hydration of the phospholipid mixture in an aqueous PBS solution includes:

[0022] Hydrating the phospholipid mixture in 4 parts by volume of an aqueous PBS solution.

[0023] In some possible embodiments, the uniform mixing of the hydrated phospholipid mixture with macrophage cell membranes in a solution includes:

[0024] Adding the hydrated phospholipid mixture and RAW264.7 macrophage cell membranes to the solution at a mass ratio of 30:1 and uniformly mixing.

[0025] In some possible embodiments, the filtration of the solution mixture using a filtration membrane includes:

[0026] Filtering the solution mixture back and forth through a 200 nm polycarbonate membrane multiple times to obtain uniform biomimetic nano-liposomes.

[0027] In some possible embodiments, after obtaining the biomimetic nano-liposomes as the filtrate, it further includes:

[0028] Dialyzing and purifying the biomimetic nano-liposomes through an ultrafiltration membrane to obtain purified biomimetic nano-liposomes.

[0029] In a third aspect, there is provided an application of the biomimetic nano-liposomes as described in claim 1 in the preparation of a drug for treating tumors.

[0030] The biomimetic nano-liposomes provided according to the present application are loaded with Ce6 (Chlorin e6) and contain macrophage cell membranes, have good stability and biocompatibility, can reduce the side effects of the drug itself on humans, and can target GBM cells through macrophage cell membranes in response to the inflammatory microenvironment of glioblastoma. Moreover, it can generate reactive oxygen species under laser conditions, thereby being used for photodynamic therapy of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the technical solutions of the embodiments of the present application, the accompanying drawings of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only relate to some embodiments of the present application and do not limit the present application.

[0032] Figure 1 It is a flowchart of the preparation method of the biomimetic nano-liposomes provided by the embodiments of the present application.

[0033] Figure 2 It is a TEM image of the biomimetic nano-liposomes prepared in this embodiment. The horizontal bright line in the lower right corner of the figure represents a scale of 100 nm.

[0034] Figure 3 The particle size distribution of the SonoVue contrast agent prepared in this example.

[0035] Figure 4 The comparative graph of the spectral absorption curves of the biomimetic nanoliposomes (MLCNPs), Ce6, and LCNPs prepared in this example.

[0036] Figure 5 The relationship between the ROS production amount and the wavelength of the irradiated laser when the biomimetic nanoliposomes prepared in this example are irradiated with a laser for different durations. Detailed implementation manners

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of this application. Obviously, the described embodiments are some but not all of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of this application without creative efforts fall within the scope of protection of this application. It can be understood that, without conflict, some technical means described in the various embodiments herein may be replaced or combined with each other.

[0038] In the description of this application, references to "one embodiment" or "some embodiments" etc. mean that a specific feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of this application. Thus, the phrases "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments", etc. that appear in different places in this specification are not necessarily all referring to the same embodiment, but mean "one or more but not all of the embodiments", unless otherwise specifically emphasized in other ways.

[0039] Figure 1 A specific embodiment of the preparation method of the biomimetic nanoliposomes (MLCNPs) is shown. In this embodiment, the preparation method includes the following steps 1) to 7):

[0040] 1) Dissolve 3 mg of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 mg of cholesterol (CHOL), 1 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotin(polyethylene glycol)2000] (DSPE-PEG 2000), and 2 mg of Ce6 in 10 ml of chloroform to obtain a chloroform solution.

[0041] 2) Evaporate the chloroform solution obtained in step 1) using a rotary evaporator to remove the organic solvent therein and generate a lipid film containing Ce6.

[0042] 3) Dry the lipid membrane obtained in step 2) under vacuum for 2 hours to obtain a dehydrated phospholipid mixture.

[0043] It is understood that the phospholipid mixture contains Ce6.

[0044] 4) Hydrate the dehydrated phospholipid mixture in 4 mL of aqueous PBS solution.

[0045] 5) Add the hydrated phospholipid mixture and RAW264.7 macrophage cell membrane to a PBS solution at a mass ratio of 30:1, and mix evenly to obtain a solution mixture.

[0046] 6) Extrude and filter the solution mixture back and forth 10 times using a 200 nm polycarbonate membrane (an example of a filtration membrane) to obtain uniform biomimetic nanoliposomes with a particle size of less than 200 nm.

[0047] 7) Purify the biomimetic nanoliposomes obtained in step 6) by overnight dialysis through an ultrafiltration membrane (MWCO 1000) to remove unencapsulated components, thereby obtaining purified biomimetic nanoliposomes.

[0048] Figure 2 This is a TEM image of the biomimetic nanoliposomes prepared in this example. It can be seen from this figure that the particle size of the obtained biomimetic nanoliposomes is mainly around 100 nm. And, Figure 3 More specifically shows the particle size distribution of the biomimetic nanoliposomes prepared in this example.

[0049] Figure 4 This is a comparative graph of the spectral absorption curves of the biomimetic nanoliposomes (MLCNPs) prepared in this example, Ce6, and Ce6 nanoparticles encapsulated in liposomes (LCNPs). Among them, LCNPs correspond to the phospholipid mixture obtained in the aforementioned step 3). It can be seen from this figure that both MLCNPs and LCNPs have successfully loaded Ce6.

[0050] Under 690 nm laser irradiation, the MLCNPs exhibit significant photothermal and photodynamic (PDT) effects and can induce a large number of cell apoptosis. The inventors studied the targeting ability and PDT effect of MLCNPs in an orthotopic glioma mouse model. During in vivo PDT, we used high-resolution photoacoustic (PA) imaging to monitor the changes in the structure and function of the tumor vascular system. The combination of MLCNPs and high-resolution PA imaging provides a new strategy for the diagnosis, treatment, and monitoring of GBM tumors.

[0051] Figure 5The figure shows the relationship between the amount of ROS generated and the wavelength of the irradiated laser under laser irradiation of the biomimetic nanoliposomes prepared in this example for different durations. It can be seen from this figure that as the irradiation time prolongs, the amount of ROS generated increases accordingly.

[0052] As can be known from the previous introduction, the biomimetic nanoliposomes (MLCNPs) prepared in the above examples contain macrophage membranes and Ce6 bound to the macrophage membranes. In application, the macrophage membrane, as a carrier, can deliver the photosensitizer Ce6 to the inflammatory microenvironment of GBM. Once localized at the tumor site, the biomimetic nanoliposomes will selectively target glioma cells, and the accumulation of the MLCNPs can be monitored through the fluorescence (FL) and photoacoustic imaging (PAI) emitted by Ce6. To observe the changes in blood vessels and the efficacy of PDT, in vivo PA imaging technology can be used to monitor the effect of MLCNP-induced PDT on tumor blood vessels in real time at the microscopic level. Moreover, in these MLCNPs, Ce6 is encapsulated and bound to the macrophage membrane, so its photosensitive properties can be well demonstrated. In addition, the inventors found that in a mouse model with orthotopic glioma, the use of these MLCNPs for PDT treatment showed significant anti-cancer effects.

Claims

1. A biomimetic nanoliposome for photodynamic therapy of tumors, characterized in that: The biomimetic nanoliposome comprises a macrophage membrane and Ce6 combined with the macrophage membrane.

2. The biomimetic nanoliposome according to claim 1, characterized in that: The Ce6 is combined with the macrophage membrane in a manner of wrapping the macrophage membrane.

3. A method for preparing the biomimetic nanoliposome as claimed in claim 1, characterized in that: include: providing a lipid film containing Ce6; drying the lipid film under vacuum to obtain a dehydrated phospholipid mixture; hydrating the phospholipid mixture in a PBS aqueous solution; The hydrated phospholipid mixture and the macrophage membrane are uniformly mixed in a PBS solution to obtain a solution mixture; The solution mixture is filtered using a filter membrane to obtain biomimetic nanoliposomes as a filtrate.

4. The method according to claim 3, characterized in that The lipid membrane containing Ce6 is provided, comprising: 2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (CHOL), 1,2-distearoyl sn-glycero-3-phosphoacetamide-N-[biotin(polyethylene glycol) 2000] (DSPE-PEG 2000) and Ce6 were dissolved in chloroform to obtain a chloroform solution; The chloroform solution is evaporated to remove the organic solvent and generate a lipid film containing Ce6.

5. The method according to claim 4, characterized in that The lipid membrane containing Ce6 is provided, comprising: Dissolve 3 parts by mass of DPPC, 1 part by mass of CHOL, 1 part by mass of DSPE-PEG 2000 and 2 parts by mass of Ce6 in 10 parts by volume of chloroform, wherein the ratio of the parts by mass to the parts by volume is mg / ml, to obtain a chloroform solution; The chloroform solution was evaporated by a rotary evaporator to remove the organic solvent and generate a lipid film containing Ce6.

6. The method according to claim 3, characterized in that The step of hydrating the phospholipid mixture in a PBS aqueous solution comprises: The phospholipid mixture was hydrated in 4 parts by volume of PBS aqueous solution.

7. The method according to claim 3, characterized in that The step of uniformly mixing the hydrated phospholipid mixture with the macrophage membrane in a solution comprises: The hydrated phospholipid mixture and RAW264.7 macrophage membrane were added to the solution at a mass ratio of 30:1 and mixed evenly.

8. The method according to claim 3, characterized in that The method of filtering the solution mixture by using a filter membrane comprises: The solution mixture was squeezed and filtered back and forth for multiple times using a 200 nm polycarbonate membrane to obtain uniform biomimetic nanoliposomes.

9. The method according to claim 3, characterized in that: After obtaining the biomimetic nanoliposome as the filtrate, the method further comprises: The bionic nanoliposomes are purified by dialysis through an ultrafiltration membrane to obtain purified bionic nanoliposomes.

10. Use of the bionic nanoliposome as claimed in claim 1 in preparing a drug for treating tumors.

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