Photosensitizer-entrapped tumor cell membrane bionic metal organic framework nanoparticles as well as preparation and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses photosensitizer-entrapped tumor cell membrane bionic metal organic framework nanoparticles as well as preparation and application thereof. The photosensitizer-entrapped tumor cell membrane biomimetic metal organic framework nanoparticles are prepared by adopting a tumor cell membrane biomimetic camouflage technology and coating the surface of the metal organic framework nanoparticles with the photosensitizer through repeated membrane extrusion or ultrasonic treatment. The preparation process is simple, the cost is low, and a complex synthesis process is not needed. The bionic nanoparticle disclosed by the invention has the functions of homologous targeting and immune escape, and is beneficial to prolonging the in-vivo circulation time and realizing higher tumor tissue targeting. In addition, the bionic nanoparticles can kill tumor cells through a photodynamic effect, and have the advantages of good therapy and low toxic and side effects.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to tumor cell membrane bionic metal organic framework nanoparticles encapsulating photosensitizers, and preparation and application thereof. Background Art

[0002] Malignant tumors are a global problem that seriously endangers human health, with high morbidity and mortality rates. Surgical resection and adjuvant chemotherapy are the most commonly used methods for treating tumors, which can effectively improve the survival of patients. However, the inherent disadvantages of chemotherapy drugs, such as poor water solubility, low bioavailability, and large toxic side effects, have greatly limited their clinical application. In recent years, photodynamic therapy has been proven to be a very promising treatment method. After the photosensitizer is injected into the patient's body, a fixed wavelength of light is used to irradiate the tumor site to stimulate the production of substances such as reactive oxygen species (ROS), thereby destroying tumor tissue. However, the tissue selectivity of photosensitizers is poor, and they often produce off-target effects and cause toxic side effects, which is also the main reason for the limitation of clinical application.

[0003] In order to solve the above-mentioned problems of photosensitizers, the design of intelligent tumor-targeted drug delivery carriers has attracted widespread attention from researchers in recent years. Encapsulating photosensitizers inside nano drug delivery carriers can not only improve the pharmacokinetic characteristics of photosensitizers and increase their half-life in vivo, but also specifically target and deliver them to tumor tissues, reduce the accumulation in normal tissues, and achieve the effect of increasing efficacy and reducing toxicity. Metal organic framework materials (MOFs) are considered to be one of the preferred nanomaterials for constructing drug delivery carriers. Zeolitic imidazolate frameworks 8 (ZIF8) is a relatively promising MOF material, which consists of a tetrahedral structure formed by the coordination bonds between the N atoms in dimethylimidazole and the metal Zn ions. The structure of the ZIF8 nano drug delivery carrier is porous and has a large specific surface area, so it is expected to load more drugs. In addition, the ZIF-8 nano drug delivery carrier is pH-sensitive, and its structure is highly stable under neutral physiological conditions, but can decompose under acidic conditions. Since the endosomes / lysosomes of tumor cells have an acidic environment, ZIF-8 nano drug delivery carriers can achieve controllable and precise release of encapsulated drugs inside tumor cells. Although nano drug delivery carriers can target tumor tissues using the EPR effect of tumor tissues, due to the heterogeneity of tumors and the physiological and pathological barriers of the body, the targeting of nano drug delivery carriers in tumor tissues still needs to be improved.

[0004] Natural tumor cell membrane biomimetic camouflage technology is an emerging biomimetic strategy that enables nano drug delivery carriers to inherit the homologous binding and immune escape capabilities similar to source cells. Therefore, nano drug carriers with tumor cell membrane biomimetic camouflage can not only reduce phagocytosis by the mononuclear macrophage system and prolong the in vivo circulation time, but also achieve higher tumor targeting accumulation and specific cell uptake through the dual effects of EPR effect and homologous targeting. Summary of the invention

[0005] The technical problem solved by the present invention is to overcome the problems of poor tumor targeting and toxic side effects caused by off-target in existing photosensitizers, and provide a tumor cell membrane bionic metal organic framework nanoparticles encapsulating photosensitizers, preparation and application thereof. The present invention adopts tumor cell membrane bionic camouflage technology, and coats it on the surface of metal organic framework nanoparticles by repeated membrane extrusion or ultrasound to prepare a tumor cell membrane bionic metal organic framework nanoparticles encapsulating photosensitizers. The nanoparticles can enhance the targeted accumulation of tumor tissues by using the EPR effect and homologous targeting, and then effectively inhibit the growth and proliferation of tumors through the photodynamic effect of the photosensitizer.

[0006] Specifically, the present invention is achieved through the following technical solutions:

[0007] In the first aspect of the present invention, a tumor cell membrane biomimetic metal organic framework nanoparticles encapsulating photosensitizers is provided for the first time. The nanoparticles are formed by repeatedly extruding tumor cell membranes through membranes or ultrasonically coating the surfaces of metal organic framework nanoparticles. The encapsulated photosensitizers include 5-aminolevulinic acid, chlorophyll a, temoporfin, pheophorbide a, dihydrochlorin e6, pyropheophorbide-a, pyropheophorbide a hexyl ether, preferably dihydrochlorin e6 (Chlorin e6, Ce6). The metal organic framework material is ZIF8. The tumor cell membrane is selected from one or more of breast cancer cell membranes, lung cancer cell membranes, bladder cancer cell membranes, melanoma cell membranes, cervical cancer cell membranes, lung cancer cell membranes, colorectal cancer cell membranes, and gastric cancer cell membranes.

[0008] The second aspect of the present invention provides for the first time a method for preparing tumor cell membrane biomimetic metal organic framework nanoparticles encapsulating photosensitizers, wherein the method comprises the steps of: dissolving photosensitizers and zinc nitrate hexahydrate in a dimethyl sulfoxide solution, stirring and reacting for a period of time, adding an aqueous solution containing dimethyl imidazole, reacting for a period of time, and obtaining metal organic framework nanoparticle solids encapsulating photosensitizers by centrifugal separation, washing, and drying. The solids and tumor cell membranes are mixed in a certain proportion, subjected to membrane extrusion or ultrasonic treatment, centrifuged, and washed to obtain tumor cell membrane biomimetic metal organic framework nanoparticles encapsulating photosensitizers.

[0009] The method for preparing the biomimetic metal organic framework nanoparticles, wherein the mass ratio of the photosensitizer to zinc nitrate hexahydrate is 1:5-1:50, preferably 1:10; the molar ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:5 to 1:72, preferably 1:36; the reaction time is 5-120 min, preferably 40 min; the mass ratio of the metal organic framework material nanoparticles to the tumor cell membrane is 1:1-10:1, preferably 1:1.

[0010] The preparation method of the bionic metal organic framework nanoparticles, wherein the preparation of the tumor cell membrane comprises the steps of: lysing cancer cells, ultrasonically destroying them, taking the supernatant after gradient centrifugation to obtain tumor cell membrane fragments, and obtaining the tumor cell membrane after membrane extrusion filtration.

[0011] The preparation method of the biomimetic metal organic framework nanoparticles, when the photosensitizer is dihydrochlorin e6 (Ce6), the preferred preparation method is:

[0012] Dissolve 2 mg of dihydrochlorin e6 (Ce6) and 20 mg of zinc nitrate hexahydrate in 2 ml of dimethyl sulfoxide and stir for 15 minutes. Add 2 ml of dimethylimidazole aqueous solution containing 720 mg at room temperature and react for 40 minutes. Centrifuge at 10000 rpm / min for 20 minutes. Wash three times with DMSO to obtain Ce6@ZIF8; disperse 1 mg of Ce6@ZIF8 and 1 mg of tumor cell membrane in aqueous solution, squeeze through 200 nm membrane 12 times, centrifuge at 10000 rpm / min for 20 minutes, and wash with PBS to obtain tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer.

[0013] The present invention also provides an application of tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer in anti-tumor. The method and steps are as follows:

[0014] The tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer were diluted with serum-containing culture medium to a solution of predetermined concentration. Six replicate wells were set for each concentration, and a control group and a zero adjustment group were set. An appropriate amount of breast cancer 4T1 cells in the logarithmic growth phase were inoculated in a 96-well plate. After culturing overnight, tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizers at different concentrations were added. After continuing to culture for 4 hours, light was irradiated and continued to culture for 24 hours. 200 μl of 10% CCK-8 reagent was added to each well. After incubation for 1 hour, the absorption value was measured at 450nm. Cell viability was calculated using the following formula:

[0015] Cell viability (%) = [(OD experimental - OD zero adjustment) / (OD control - OD zero adjustment)] × 100

[0016] The beneficial effects of the present invention are as follows:

[0017] 1 The preparation process of the bionic nanoparticles carrying photosensitizers described in the present invention is simple, low-cost, does not require a complicated synthesis process, has a simple cell membrane encapsulation process, and has high stability and repeatability.

[0018] 2 After being coated with tumor cell membranes, the bionic nanoparticles described in the present invention have the functions of homologous targeting and immune escape, which helps to prolong the circulation time in the body and achieve higher tumor tissue targeting.

[0019] 4.3 The biomimetic nanoparticles loaded with photosensitizers described in the present invention can kill tumor cells through photodynamic effect, and have the advantages of good therapeutic effect and low toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Particle size diagram of Ce6@ZIF8

[0021] Figure 2 :Electrochemical potential diagram of Ce6@ZIF8

[0022] Figure 3 :Particle size diagram of Ce6@CM-ZIF8

[0023] Figure 4 :Electrochemical potential diagram of Ce6@CM-ZIF8

[0024] Figure 5 :Cytotoxicity of blank biomimetic nanoparticles

[0025] Figure 6 :Cell proliferation inhibition

[0026] Figure 7 :Cellular uptake DETAILED DESCRIPTION

[0027] The following examples are intended to illustrate the present invention rather than to further limit the present invention. The present invention is further described in detail below with reference to the examples, but the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art can carry out equivalent replacement, combination, improvement or modification to the present invention based on the description of the present invention, but these will all be included in the scope of the present invention.

[0028] Example 1: Preparation of tumor cell membrane biomimetic metal organic framework nanoparticles loaded with dihydrochlorin e6 (Ce6)

[0029] In a 5 ml centrifuge tube, 2 mg of dihydrochlorin e6 (Ce6) and 20 mg of zinc nitrate hexahydrate were dissolved in 2 ml of dimethyl sulfoxide and stirred for 15 min. At room temperature, 2 ml of an aqueous solution containing 720 mg of dimethylimidazole was added thereto and reacted for 40 min. Centrifuged at 10,000 rpm / min for 20 min. Washed three times with DMSO to obtain Ce6@ZIF8; 1 mg of Ce6@ZIF8 and 1 mg of tumor cell membrane were dispersed in an aqueous solution, squeezed through a 200 nm membrane 12 times, centrifuged at 10,000 rpm / min for 20 min, and washed with PBS to obtain tumor cell membrane biomimetic metal organic framework nanoparticles (Ce6@CM-ZIF8) loaded with photosensitizer.

[0030] The physical properties of the bionic nanoparticles are as follows: Figure 1 and 2 As shown, the particle size of Ce6@ZIF8 measured by dynamic light scattering is 107.3nm, PDI is 0.100, and Zeta potential is 24.0V; Figure 3 and 4 , the particle size of Ce6@CM-ZIF8 is 146.2 nm, the PDI is 0.077, and the Zeta potential is -16.2 V. The drug loading of the nanoparticles was determined to be 7.32% by HPLC.

[0031] Example 2: Preparation of tumor cell membrane biomimetic metal organic framework nanoparticles loaded with dihydrochlorin e6 (Ce6)

[0032] In a 5 ml centrifuge tube, 1 mg of dihydrochlorin e6 (Ce6) and 40 mg of zinc nitrate hexahydrate were dissolved in 2 ml of dimethyl sulfoxide and stirred for 15 min. At room temperature, 2 ml of an aqueous solution containing 800 mg of dimethylimidazole was added thereto and reacted for 40 min. Centrifuged at 10,000 rpm / min for 20 min. Washed three times with DMSO to obtain Ce6@ZIF8; 1 mg of Ce6@ZIF8 and 1 mg of tumor cell membrane were dispersed in an aqueous solution, sonicated under ice water for 5 min, centrifuged at 10,000 rpm / min for 20 min, and washed with PBS to obtain tumor cell membrane bionic metal organic framework nanoparticles (Ce6@CM-ZIF8) loaded with photosensitizer.

[0033] Example 3: Material cytotoxicity study

[0034] The blank nanoparticle (CM-ZIF8) solution was diluted with culture medium to a solution of a predetermined concentration. Six replicate wells were set for each concentration, and a control group and a zero adjustment group were set. An appropriate amount of breast cancer 4T1 cells in the logarithmic growth phase were inoculated in a 96-well plate. After overnight culture, tumor cell membrane biomimetic metal organic framework nanoparticles containing photosensitizers at different concentrations were added and cultured for 24 hours. 200 μl of 10% CCK-8 reagent was added to each well. Incubate for 1 hour, measure the absorbance at 450 nm to calculate the cell viability, and the formula is as follows:

[0035] Cell viability (%) = [(OD 实验 -OD 调零 ) / (OD 对照 -OD 调零 )]×100

[0036] Depend on Figure 5 The results show that within the concentration range of the blank nanoparticles, the viability of 4T1 cells is above 90%, indicating that the material itself has low cytotoxicity. This indicates that the nanoparticles provided by this patent are non-toxic and safe drug delivery materials.

[0037] Example 4: Cell proliferation inhibition study

[0038] Free dihydrochlorin e6 (Ce6) and Ce6@CM-ZIF8 were diluted to the preset concentrations with culture medium. Six replicate wells were set for each concentration, and a control group and a zero adjustment group were set. An appropriate amount of breast cancer 4T1 cells in the logarithmic growth phase were inoculated in a 96-well plate. After culturing overnight, tumor cell membrane biomimetic metal organic framework nanoparticles containing photosensitizers at different concentrations were added. After continuing to culture for 4 hours, they were illuminated and continued to be cultured for 24 hours. 200 μl of 10% CCK-8 reagent was added to each well. After incubation for 1 hour, the absorbance was measured at 450 nm. Cell viability was calculated using the following formula:

[0039] Cell viability (%) = [(OD 实验 -OD 调零 ) / (OD 对照 -OD 调零 )]×100

[0040] Figure 6 The results showed that within the drug concentration range of 0.01-10 μg / mL, the inhibitory effect of Ce6@CM-ZIF8 on cell proliferation was significantly dose-dependent and significantly stronger than that of Ce6@ZIF8 and free dihydrochlorin e6 (Ce6).

[0041] Example 5: Flow cytometry observation of cellular uptake of biomimetic nanoparticles

[0042] 4T1 cells were cultured at 2 × 105 Cells / well were seeded in a 24-well plate with a cell slide pre-placed on the bottom and incubated at 37°C and 5% CO 2 After culturing overnight in an incubator, free dihydrochlorin e6 (Ce6), Ce6@ZIF8 and Ce6@CM-ZIF8 were added for incubation, and finally the cells in the culture plate were digested and collected, and the cellular uptake of biomimetic nanoparticles was observed by flow cytometry.

[0043] The results are as follows Figure 7 As shown, Ce6@CM-ZIF8 exhibited a higher uptake efficiency than other groups due to its homologous targeting effect after being coated with the tumor cell membrane.

Claims

1. A tumor cell membrane biomimetic metal organic framework nanoparticle loaded with photosensitizer, characterized in that: Tumor cell membrane bionic camouflage technology is used to coat the metal organic framework nanoparticles on the surface of the nanoparticles through repeated membrane extrusion or ultrasound to prepare tumor cell membrane bionic metal organic framework nanoparticles carrying photosensitizers.

2. The tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer according to claim 1, characterized in that: The metal organic framework material is ZIF8.

3. The tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer according to claim 1, characterized in that: The tumor cell membrane is selected from one or more of breast cancer cell membrane, lung cancer cell membrane, bladder cancer cell membrane, melanoma cell membrane, cervical cancer cell membrane, lung cancer cell membrane, colorectal cancer cell membrane and gastric cancer cell membrane.

4. The tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer according to claim 1, characterized in that: The photosensitizer is selected from one or more of 5-aminolevulinic acid, chlorophyll a, temoporfin, pheophorbide a, chlorin e6, pyropheophorbide-a, pyropheophorbide a hexyl ether, preferably chlorin e6 (Chlorin e6, Ce6).

5. The method for preparing the tumor cell membrane biomimetic metal organic framework nanoparticles carrying photosensitizer according to claim 1, characterized in that: The preparation method is as follows: dissolving a photosensitizer and zinc nitrate hexahydrate in a dimethyl sulfoxide solution, stirring and reacting for a period of time, adding an aqueous solution containing dimethyl imidazole, reacting for a period of time, and obtaining a metal organic framework nanoparticle solid containing a photosensitizer by centrifugal separation, washing, and drying. The solid and the tumor cell membrane are mixed in a certain proportion, subjected to membrane extrusion or ultrasonic treatment, centrifuged after completion, and washed to obtain the tumor cell membrane biomimetic metal organic framework nanoparticles containing a photosensitizer.

6. The preparation method according to claim 5, characterized in that: The photosensitizer and zinc nitrate hexahydrate are dissolved in a dimethyl sulfoxide solution, the mass ratio of the photosensitizer to zinc nitrate hexahydrate is 1:5-1:50, preferably 1:10; the molar ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:5 to 1:72, preferably 1:36; an aqueous solution containing dimethylimidazole is added, and the reaction is carried out for a period of time, the time being 5-120 minutes, preferably 40 minutes; the mass ratio of metal organic framework material nanoparticles to tumor cell membranes is 1:1-10:1, preferably 1:

1.

7. The preparation method according to claim 5, characterized in that: The preparation of the tumor cell membrane comprises the steps of: lysing cancer cells, ultrasonically destroying them, taking the supernatant after gradient centrifugation to obtain tumor cell membrane fragments, and obtaining the tumor cell membrane after membrane extrusion filtration.

8. The preparation method according to claim 5, characterized in that: When the encapsulated photosensitizer is dihydrochlorin e6 (Ce6), the preparation method thereof includes: dissolving 2 mg dihydrochlorin e6 (Ce6) and 40 mg zinc nitrate hexahydrate in 2 ml dimethyl sulfoxide, stirring for 15 minutes. At room temperature, adding 2 ml of an aqueous solution containing 800 mg of dimethylimidazole, reacting for 40 minutes. Centrifuging at a speed of 10000 rpm / min for 20 minutes. Washing three times with DMSO to obtain Ce6@ZIF8; dispersing 1 mg Ce6@ZIF8 and 1 mg tumor cell membrane in an aqueous solution, extruding through a 200 nm membrane 12 times, centrifuging at a speed of 10000 rpm / min for 20 minutes, and washing with PBS to obtain tumor cell membrane bionic metal organic framework nanoparticles encapsulating photosensitizer.

9. Use of the tumor cell membrane biomimetic metal organic framework nanoparticles loaded with photosensitizer according to claim 1 in the preparation of drugs for treating malignant tumors.

10. The use according to claim 9, characterized in that The tumor includes one or more of breast cancer, lung cancer, bladder cancer, melanoma, cervical cancer, lung cancer, colorectal cancer and gastric cancer.

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