CN109771376B - Tumor cell source microparticle drug-carrying preparation and preparation method thereof - Google Patents
Tumor cell source microparticle drug-carrying preparation and preparation method thereof Download PDFInfo
- Publication number
- CN109771376B CN109771376B CN201910119648.3A CN201910119648A CN109771376B CN 109771376 B CN109771376 B CN 109771376B CN 201910119648 A CN201910119648 A CN 201910119648A CN 109771376 B CN109771376 B CN 109771376B
- Authority
- CN
- China
- Prior art keywords
- drug
- preparation
- microparticles
- loaded
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The embodiment of the invention relates to the technical field of biological medicines, and provides a tumor cell-derived microparticle drug-loaded preparation and a preparation method thereof, wherein the preparation method comprises the steps of delivering photo-thermal nanoparticles and chemotherapeutic drugs into tumor cells through micropores by utilizing an electroporation technology; inducing tumor cell apoptosis by ultraviolet light or chemotherapy drugs, and releasing drug-loaded microparticles wrapping photo-thermal nanoparticles and chemotherapy drugs to obtain tumor cell-derived microparticle drug-loaded preparation. According to the tumor cell-derived microparticle drug-loaded preparation and the preparation method thereof provided by the embodiment of the invention, the limited uptake of cells to photothermal nanoparticles is overcome by adopting an electroporation technology, so that the photothermal nanoparticles can easily enter tumor cells, the integration with the microparticles is realized, the inherent in-vivo behavior of the microparticles is not influenced by the integration mode, the synergistic treatment effect can be fully exerted, meanwhile, the whole integration construction mode is simple, the efficiency is high, the cost is low, and the preparation method can be widely popularized and applied in tumor treatment.
Description
Technical Field
The embodiment of the invention relates to the technical field of biological medicines, in particular to a tumor cell derived microparticle drug-loaded preparation and a preparation method thereof.
Background
In recent years, the threat of cancer to human health has been increasingly appreciated by researchers. With the development of scientific technology, the treatment means for cancer in human beings is also increasingly diversified. However, the therapeutic effect of cancer is still unsatisfactory. Thus, the continual drive to develop new effective cancer treatment modalities is a historical mission for a large number of related researchers.
A cell-derived microparticle is a phospholipid bilayer vesicle secreted by a cell upon exogenous or endogenous stimulation. It is believed that the size of the microparticles is between 100-1000nm, and they are mainly composed of proteins, phospholipids, DNA, RNA, etc. The property of microparticles to participate in intercellular bioactive molecule transmission makes them an effective drug delivery vehicle in cancer treatment possible. Compared with the traditional artificially constructed drug-carrying system, the cell-derived microparticle drug-carrying system has the advantages of extremely low immunogenicity, excellent in-vivo circulation stability, capability of overcoming physiological barriers, easiness in being taken by tumor cells and the like. It has achieved good therapeutic effects in cancer chemotherapy, immunotherapy and gene therapy.
The nano drug-carrying particles have wide application in cancer treatment due to unique nano structures and physicochemical properties, and particularly, compared with a single treatment mode, comprehensive treatment and diagnosis and treatment integrated research based on inorganic nano particles can effectively solve the treatment difficulty of multiple causes of cancer. For example, photothermal therapy can not only utilize the sensitivity of cells to hyperthermia to kill cells, but also sensitize chemotherapy and inhibit their multidrug resistance.
Therefore, the integration of cell-derived microparticles with functional inorganic nanoparticles provides a new and effective strategy for cancer treatment. Up to now, the latest research finds that the multifunctional microparticle drug-carrying preparation can be prepared by integrating cell-derived microparticles and functional inorganic nanoparticles together by modifying donor cell membranes through gene regulation or membrane phospholipid metabolism and connecting the nanoparticles to the surfaces of the microparticles through receptor-ligand binding or chemical bonding. However, the integration method inevitably changes the surface structure of the microparticles, thereby affecting the inherent in vivo behavior of the microparticles and reducing the inherent drug-loading efficacy of the microparticles, and the integration construction method is complex, high in cost and low in efficiency, thereby further limiting the application of the microparticles in treating tumors.
Disclosure of Invention
The embodiment of the invention provides a tumor cell-derived microparticle drug-loaded preparation and a preparation method thereof, which are used for solving the problem that the inherent in-vivo behavior of microparticles is influenced by the fact that the surface structure of the microparticles is changed by a combination mode of ligand combination or chemical bonding between cell-derived microparticles and functional nanoparticles in the prior art.
The embodiment of the invention provides a preparation method of a tumor cell derived microparticle drug-loaded preparation, which comprises the following steps: using electroporation technology to deliver photothermal nanoparticles and chemotherapeutic drugs into the tumor cells through the micropores; the tumor cells are apoptotic through the treatment of ultraviolet light or chemotherapeutic drugs, and drug-loaded microparticles wrapping the photo-thermal nanoparticles and the chemotherapeutic drugs are released, so that the microparticle drug-loaded preparation is prepared.
Chemotherapy has poor targeting property in tumor treatment, large toxic and side effects and low treatment efficiency; the photothermal therapy method directly irradiates a tumor site with near infrared light and converts light energy into heat energy through a photothermal agent, thereby effectively killing tumor cells without causing systemic toxicity. The nano particles are more beneficial to the enrichment at the tumor part, penetrate through tumor blood vessels and penetrate into the deep part of the tumor, and are more effectively absorbed and killed by common tumor cells. Therefore, the photothermal-chemotherapy combination therapy is a relatively advanced tumor clinical treatment technology with high efficiency and low toxic and side effects at present.
The cell-derived microparticles are an effective drug carrier, and have the advantages of extremely low immunogenicity, excellent in-vivo circulation stability, capability of overcoming physiological barriers, easiness in being taken up by tumor cells and the like in tumor treatment. In the photothermal-chemotherapy combination treatment technology, the cell-derived microparticles are used as carriers of photothermal nanoparticles and chemotherapeutic drugs, so that the combination treatment effect can be remarkably improved. However, it is a problem how to integrate the cell-derived microparticles, the photothermal nanoparticles and the chemotherapeutic drugs and to exert their synergistic therapeutic effects.
According to the invention, the tumor cells are subjected to electroporation by using electric pulses, instantaneous micropores can be formed on the phospholipid bilayer of the tumor cell membrane under the action of the electric pulses, so that the permeability and the membrane conductance of the cell membrane are increased instantaneously, photo-thermal nanoparticles and chemotherapeutic drugs enter the tumor cells through the micropores, and then the tumor cells are induced to die by ultraviolet light or chemotherapeutic drugs and the like to release the microparticles, so that the photo-thermal nanoparticles and the chemotherapeutic drugs are wrapped in the microparticles, the integration of cell-derived microparticles, the photo-thermal nanoparticles and the chemotherapeutic drugs is realized, and a microparticle drug-carrying preparation is formed.
The invention adopts the electroporation technology to overcome the limit of limited uptake of cells to photo-thermal nanoparticles, so that the photo-thermal nanoparticles and chemotherapeutic drugs can easily enter tumor cells, thereby realizing integration with the microparticles, secondly, the integration mode of firstly delivering the drugs and then releasing the microparticles does not change the structure of the microparticles, does not influence the inherent in vivo behavior of the microparticles, can fully exert the synergistic treatment effect of the microparticles, the photo-thermal nanoparticles and the chemotherapeutic drugs, simultaneously, the tumor cells contain the photo-thermal nanoparticles, can induce apoptosis by ultraviolet light or infrared light irradiation, has simple integral construction mode, high efficiency and low cost, and can be widely popularized and applied in tumor treatment.
Preferably, the conditions under which the electrical pulses are subjected to electroporation are: the voltage is 100-500V, the capacitance is 100-300 muF, and the shock frequency is 1-6 more preferable. The hydrophilic molecules, DNA, proteins, macromolecular particles and the like can not pass through cell membranes under normal conditions, the uptake of photothermal nanoparticles by tumor cells is limited, the tumor cells can generate instant micropores through an electroporation technology so that the photothermal nanoparticles can enter the cells to be integrated with the microparticles, and under the optimized condition, the pore diameter of the micropores formed on the tumor cell membranes is between 20 and 200nm, so that a large amount of photothermal nanoparticles and chemotherapeutic drugs can be used for intracellular application, the contents of the photothermal nanoparticles and the chemotherapeutic drugs in the tumor cells are greatly improved, and the load capacity of the microparticles is improved, compared with the traditional preparation method, the load capacity of the photothermal nanoparticles and the chemotherapeutic drugs in the microparticles is improved by 2 to 4 times. Meanwhile, the irrecoverable damage to the tumor cells can be avoided under the optimized condition.
Preferably, the tumorThe concentration of the suspension of tumor cells is 1-4X 106The concentration of the photothermal nanoparticles is 0.2-0.8mg/mL, and the concentration of the chemotherapeutic drug is 0.05-0.2 mg/mL. The higher the concentration of the tumor cells is, the more the release amount of the microparticles is, the higher the concentrations of the photothermal nanoparticles and the chemotherapeutic drugs are, the higher the content of the photothermal nanoparticles and the chemotherapeutic drugs entering the tumor cells is, the higher the loading capacity of the microparticles is, however, the loading capacity has a certain limit, so the concentration is not too high, the preferred concentration of the invention is the optimum concentration range, and the loading capacity of the microparticles can reach the maximum saturation.
Preferably, before the tumor cells are electroporated, the method further comprises: and placing the suspension of the tumor cells in a cell culture medium, adding the photo-thermal nanoparticles and the chemotherapeutic drugs, and then incubating at 0-8 ℃ for 10-30 min. The optimal temperature and time can ensure better solidification of the tumor cells, and meanwhile, the photo-thermal nanoparticles and the chemotherapeutic drugs can be uniformly dispersed around the tumor cells, so that the photo-thermal nanoparticles and the chemotherapeutic drugs can rapidly enter the tumor cells in the electroporation process, and the integration efficiency is improved.
Preferably, the cell suspension prepared after delivery of the photothermal nanoparticles and chemotherapeutic drug into the tumor cells is incubated at 37 ℃ for 0.5-3 h. The preferred temperature and time ensure better tumor cell solidification and proper mechanical force.
Preferably, after the tumor cells are subjected to apoptosis to release the drug-loaded microparticles, the drug-loaded microparticles are centrifugally collected at a low temperature of 4-6 ℃ and a centrifugal force of 200-. The microparticles with the grain size of 200-600nm secreted by the tumor cells can be more effectively enriched under the preferred centrifugation condition.
Preferably, the photothermal nanoparticles comprise one or more of bismuth selenide nanodots, bismuth sulfide nanodots, gold nanoparticles, or platinum nanodots.
Good photothermal agents need to meet the requirements of safety, non-toxicity, stability and high photothermal conversion efficiency, and in particular can carry out thermal conversion on near-infrared light with strong tissue penetrability. The preferred photo-thermal nano-particles are inorganic photo-thermal reagents made of near-infrared nano materials, and high photo-thermal conversion efficiency is generated under the irradiation of low-energy near-infrared light, so that the photo-thermal treatment effect is achieved. Since the photothermal therapy induces cells to produce heat shock proteins, increases the heat tolerance thereof, and reduces the effect of thermotherapy, a better therapeutic effect can be obtained by combining it with chemotherapeutic drugs.
Preferably, the chemotherapeutic agent comprises one or more of a chemotherapeutic agent for treating liver cancer, breast cancer, lung cancer, lymphoma, skin cancer, ovarian cancer, acute leukemia, cervical cancer, bladder cancer, or chorioepithelial cancer.
Preferably, the tumor cells are derived from tumor cells in liver cancer, breast cancer, lung cancer, lymphoma, skin cancer, ovarian cancer, acute leukemia, cervical cancer, bladder cancer, or chorioepithelial cancer.
The maximum amount of chemotherapeutic agent depends on the maximum saturation of the microparticles with the chemotherapeutic agent used, so that different sizes of agent are available in the highest range. The microparticles used to encapsulate the chemotherapeutic agent may be of a different or the same origin as the tumor cells to be treated, preferably of the same origin as the tumor cells to be treated.
The embodiment of the invention also provides a tumor cell source microparticle drug-loaded preparation prepared by the method. The preparation form of the microparticle drug-loaded preparation prepared by the invention is an injection preparation.
The preparation method adopts an electroporation technology, overcomes the limitation of limited uptake of photothermal nanoparticles and chemotherapeutic drugs by cells, enables the photothermal nanoparticles and the chemotherapeutic drugs to easily enter tumor cells, realizes integration with the microparticles, delivers the nanoparticles and the drugs firstly and then releases the microparticles in an integration mode, does not change the structure of the microparticles, does not influence the inherent in vivo behavior of the microparticles, can fully play the synergistic treatment effect of the microparticles, the photothermal nanoparticles and the chemotherapeutic drugs, and can induce apoptosis by ultraviolet light or infrared light irradiation in the tumor cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is an electron microscope observation of drug-loaded microparticles prepared according to an embodiment of the present invention;
FIG. 2 is a fluorescence emission spectrum of a pure microparticle, doxorubicin, and a drug-loaded microparticle of an embodiment of the invention;
FIG. 3a is a plot of particle size distribution for both neat microparticles and drug-loaded microparticles of embodiments of the invention;
FIG. 3b is a graph of zeta potential for a neat microparticle and a drug-loaded microparticle of an embodiment of the invention;
FIG. 4 is a graph of bismuth selenide content in H22 mouse hepatoma cells after electroporation and without electroporation treatment;
FIG. 5 is a graph showing the content of bismuth selenide in drug-loaded microparticles induced by electroporation and without electroporation;
FIG. 6 is a graph showing the amount of protein induced in microparticles after electroporation and without electroporation;
FIG. 7 is a graph of in vitro photothermal performance of drug-loaded microparticles according to embodiments of the invention;
figure 8 is a graph of the effect of different drug-loaded microparticles on the growth of H22 subcutaneous tumor mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The term "microparticle" used in the present invention is a cell vesicle produced by apoptotic tumor cells, and is not coated with a chemotherapeutic drug, and "drug-loaded microparticle" is formed by coating a chemotherapeutic drug and a photothermal nanoparticle with a cell vesicle. The chemotherapy drugs wrapped in the microparticles can be clinically applied chemotherapy drugs or effective components of the clinically applied chemotherapy drugs, and the drug dosage related in the invention is understood as the effective component amount of the drug.
The micro-particles are obtained by inducing apoptosis release of tumor cells, and chemotherapeutic drugs and photo-thermal nano-particles can be wrapped in the micro-particles by a method known by a person skilled in the art to obtain drug-loaded micro-particles. Apoptosis can be considered based on criteria well known to those skilled in the art, such as the observation of tumor cell shrinkage, refractive darkening. For collection of drug-loaded microparticles, high-speed centrifuges can be used for separation under cryogenic conditions.
Various tumor cells, drugs and experimental animals were used in the following examples:
h22 mouse liver cancer cell, purchased from China center for type Collection CCTCC;
BALB/C mice, purchased from the center of experimental animals in Hubei province, and weighing 18-20 g;
doxorubicin, purchased from Sigma.
Example 1: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps:
(1) h22 mouse liver cancer cells were cultured in RPMI 1640 cell culture medium.
(2) H22 mouse liver cancer cell 2.5X 106Suspending the suspension in RPMI 1640 serum-free cell culture medium at a concentration of 0.4mg/mL, respectively adding bismuth selenide nanodots and chemotherapy drug adriamycin at a concentration of 0.1mg/mL, and incubating at 4 ℃ for 10 min. 400 μ L of the above suspension was added to a 0.4cm electric rotor of a set prescribed by an electroporator,electroporation was performed at 300V and a capacitance of 150. mu.F. After electroporation was complete, the cell suspension was incubated at 37 ℃ for 2 h.
(3) Placing the cell suspension obtained in the step (2) at the strength of 300J m-2For 12 hours under ultraviolet light.
(4) After 12h of UV irradiation, the residual cells were removed by centrifugation at 200g at 4 ℃. The supernatant was further subjected to stepwise centrifugation at 600g for 10min and 14000g for 1min at 6 ℃ to remove cell debris. Centrifuging the centrifuged supernatant for 1H by 20000g to obtain drug-loaded microparticles generated by H22 mouse hepatoma cells. Stored at-80 ℃ for further use.
Example 2: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: electroporation was performed at 500V and 100. mu.F capacitance.
Example 3: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: electroporation was performed at 100V and 300 μ F capacitance.
Example 4: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: the concentration of the suspension of tumor cells was 4X 106The concentration of the photothermal nanoparticles is 0.8mg/mL, and the concentration of the chemotherapeutic drug is 0.1 mg/mL.
Example 5: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: the concentration of the suspension of tumor cells was 4X 106The concentration of the photothermal nanoparticles is 0.6mg/mL, and the concentration of the chemotherapeutic drug is 0.2 mg/mL.
Example 6: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: the concentration of the suspension of tumor cells was 1X 106The concentration of the photothermal nanoparticles is 0.2mg/mL, and the chemotherapy is performedThe concentration of the drug was 0.05 mg/mL.
Example 7: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: adding bismuth selenide nanodots and chemotherapeutic drug adriamycin into a cell culture medium, and then incubating for 10min at 0 ℃.
Example 8: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: adding bismuth selenide nanodots and chemotherapeutic drug adriamycin into a cell culture medium, and incubating at 8 ℃ for 30 min.
Example 9: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: after electroporation was complete, the cell suspension was incubated at 37 ℃ for 0.5 h.
Example 10: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: after electroporation was complete, the cell suspension was incubated at 357 ℃ for 3 h.
Examples 11 to 14: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: the photo-thermal nano-particles are respectively a mixture of bismuth sulfide nano-dots, gold nano-particles, platinum nano-dots, bismuth selenide nano-dots and bismuth sulfide nano-dots.
Examples 15 to 22: drug-loaded microparticles
The preparation method of the drug-loaded microparticles comprises the following steps: the difference from example 1 is that: the chemotherapy drugs are respectively chemotherapy drugs for treating breast cancer, lung cancer, lymphoma, skin cancer, ovarian cancer, acute leukemia, cervical cancer, bladder cancer or chorioepithelioma; accordingly, the tumor cells are derived from breast cancer, lung cancer, lymphoma, skin cancer, ovarian cancer, acute leukemia, cervical cancer, bladder cancer or chorioepithelial cancer, respectively.
Hereinafter, an experimental study is performed on the drug-loaded microparticles prepared according to the present invention, using example 1 as an example.
1. Transmission electron microscope observation of drug-loaded microparticles
Fixing the extracted drug-loaded microparticles with 2.5% (v/v) glutaraldehyde, carrying out negative staining with 1% uranyl acetate, and observing with a transmission electron microscope. Fig. 1 is an electron microscope observation image of the drug-loaded microparticle prepared in the embodiment of the present invention, and as can be seen from fig. 1, a large number of black dots are distributed inside the microparticle, which indicates that the bismuth selenide nanodots are successfully loaded into the microparticle.
2. Fluorescence spectrum analysis of multifunctional drug-loaded microparticles
H22 mouse liver cancer cell 2.5X 106The suspension was suspended in RPMI 1640 serum-free cell culture medium at a concentration of one/mL, and directly irradiated with ultraviolet light for 12 hours, and then centrifuged to collect pure microparticles (control group) as in example 1. Fluorescence emission spectra of the pure microparticles, doxorubicin and the drug-loaded microparticles of example 1 were respectively observed by a fluorescence spectrophotometer at an excitation wavelength of 488nm, and fig. 2 is a fluorescence emission spectrum of the pure microparticles, doxorubicin and the drug-loaded microparticles of the examples of the present invention. As shown in fig. 2, the pure microparticles did not have the characteristic emission peak of doxorubicin, whereas the drug-loaded microparticles showed the characteristic emission peak of doxorubicin. The results show that doxorubicin was successfully loaded into the multifunctional drug-loaded microparticles.
3. Dynamic light scattering monitoring particle size distribution and zeta potential of multifunctional drug-loaded microparticles
Dispersing pure microparticles and the drug-loaded microparticles of example 1 in phosphate buffer, and measuring the particle size distribution and zeta potential in a dynamic light scattering instrument, respectively, wherein fig. 3a is a particle size distribution diagram of the pure microparticles and the drug-loaded microparticles of the example of the present invention; fig. 3b is a graph of zeta potential for a pure microparticle and a drug-loaded microparticle of an embodiment of the invention. As shown in fig. 3a, the particle size distribution of the two microparticles is not significantly different, and the average particle size is 360.5 nm and 356.7 nm; as shown in FIG. 3b, the zeta potential was also not significantly different at-12.60 and-12.02 mV, respectively. The results show that no significant change in the surface properties of the microparticles produced by electroporation of the cells was observed.
4. Effect of electroporation on bismuth selenide content, drug-loaded microparticle loading and protein content taken by cells
H22 mouse liver cancer cells, bismuth selenide nanodots and adriamycin are directly incubated at 37 ℃ for 2H, then irradiated by ultraviolet light for 12H, and centrifuged as in example 1, and drug-loaded microparticles obtained by incubation and induction are collected (i.e., without electroporation treatment).
Respectively detecting the content of bismuth selenide in the same number of H22 mouse liver cancer cells after incubation or electroporation treatment and drug-loaded microparticles induced by the cells by an atomic fluorescence spectrophotometer, wherein the graph in FIG. 4 is a diagram of the content of bismuth selenide in H22 mouse liver cancer cells after electroporation treatment and without electroporation treatment, the graph in FIG. 5 is a diagram of the content of bismuth selenide in drug-loaded microparticles induced after electroporation treatment and without electroporation treatment, and the graph in FIG. 6 is a diagram of the protein content of microparticles induced after electroporation treatment and without electroporation treatment.
As shown in fig. 4, the content of bismuth selenide in the H22 mouse hepatoma cells treated by electroporation was 2.7 times that of the direct incubation. As shown in fig. 5, the amount of bismuth selenide in the drug-loaded microparticles induced by electroporation treatment was 3.7 times that of the direct incubation. The amount of microparticles is usually quantified by their membrane proteins, and by quantifying the amount of protein that produces microparticles for the same number of cells, as shown in fig. 6, the amount of microparticle protein obtained after electroporation is significantly higher than that obtained after incubation. Therefore, electroporation not only improves the bismuth selenide loading within a single microparticle, but also improves the yield of drug-loaded microparticles.
According to the invention, the influence research of the electroporation on the bismuth selenide content, the drug-loaded microparticle loading capacity and the protein content of the cell in the embodiments 1-21 is respectively carried out by the above methods, and the final experimental result can obtain that the loading capacity of the photo-thermal nanoparticles in the drug-loaded microparticles obtained by the preparation method of the invention is improved by 2-4 times compared with the traditional direct incubation mode.
5. In vitro photothermal performance evaluation of drug-loaded microparticles
In order to evaluate the photothermal performance of the drug-loaded microparticles, the drug-loaded microparticles were dispersed in a phosphate buffer solution at a concentration of 100 μ g/mL. Respectively measuring 1mL of ultrapure water and multifunctional drug-loaded microparticlesDispersing with 808nm near infrared laser at 1.5W/cm2The intensity is continuously irradiated for 10min, and the temperature change is recorded, and fig. 7 is an in vitro photo-thermal performance graph of the drug-loaded microparticles of the embodiment of the invention. As shown in fig. 7, the temperature of the drug-loaded microparticle dispersion of the present invention increased by as much as 43.5 ℃ after 10min of laser irradiation, whereas the temperature of pure water increased by only 3.1 ℃. The result shows that the drug-loaded microparticles prepared by the invention have excellent photo-thermal conversion performance.
6. Effect of drug-loaded microparticles on growth of H22 subcutaneous tumor mice
(1) Establishment of mouse liver cancer H22 subcutaneous tumor model: 100 mu L of liver cancer H22 cell suspension (the cell number is 2X 10) is inoculated to the lower right lower limb of the back of BALB/C mice under the skin6) And establishing a mouse liver cancer H22 subcutaneous tumor model.
(2) When mouse liver cancer H22 subcutaneous tumor grows to 80-100mm in volume3At this time, mice bearing H22 subcutaneous tumors of liver cancer were randomly divided into 4 groups of 8 mice each. Injecting the prepared drug-loaded microparticle tail vein of H22 mouse hepatoma cell into tumor-loaded BALB/C mouse, and injecting 808 laser (1.5W/cm) 12H later2) Irradiating for 10min to serve as an experimental group; injecting the prepared drug-loaded microparticle tail vein of the H22 mouse hepatoma cells into a tumor-loaded BALB/C mouse without laser irradiation to serve as a third control group; injecting the prepared bismuth selenide loaded micro-particle tail vein of H22 mouse hepatoma cell into tumor-bearing BALB/C mouse, and injecting 808 laser (1.5W/cm) 12H later2) Irradiating for 10min to obtain control group II; saline tail vein injection was performed to tumor bearing BALB/C mice as a first control group.
(3) The tumor length (L) and tumor width (W) of the tumor were measured daily by a vernier caliper, and the tumor volume V ═ L × W was calculated for the mice in the experimental group and the control group2/2。
Fig. 8 is a graph showing the influence of different drug-loaded microparticles on the growth of H22 subcutaneous tumor mice, and as shown in fig. 8, the drug-loaded microparticles derived from H22 mouse hepatoma cells in the experimental group can significantly inhibit the growth of H22 hepatoma in mice. Compared with the simple photothermal treatment of the third control group and the simple chemotherapy of the second control group, the experimental group shows good combined effect of photothermal treatment and chemotherapy.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (2)
1. A preparation method of a tumor cell derived microparticle drug-loaded preparation is characterized by comprising the following steps:
placing the suspension of tumor cells in cell culture medium, adding photo-thermal nanoparticles and chemotherapeutic drugs, and incubating at 0-8 deg.C for 10-30 min;
delivering the photothermal nanoparticles and chemotherapeutic drugs into the tumor cells through microwells using electroporation;
incubating the cell suspension prepared after delivery of the photothermal nanoparticles and chemotherapeutic drug into the tumor cells at 37 ℃ for 0.5-3 h;
carrying out ultraviolet treatment to ensure that the tumor cells are apoptotic, releasing drug-loaded microparticles wrapping the photothermal nanoparticles and the chemotherapeutic drugs, and centrifugally collecting the drug-loaded microparticles at the low temperature of 4-6 ℃ and the centrifugal force of 200-;
the photo-thermal nano particles are bismuth selenide nano dots;
the chemotherapeutic drug is adriamycin;
the tumor cells are derived from liver cancer cells;
the concentration of the suspension of the tumor cells is 1-4X 106The concentration of the photothermal nanoparticles is 0.2-0.8mg/mL, and the concentration of the chemotherapeutic drug is 0.05-0.2 mg/mL;
the conditions of the electroporation are as follows: the voltage is 100-500V, the capacitance is 100-300 muF, and the number of electric shocks is 1-6.
2. A tumor cell-derived microparticle drug-loaded preparation prepared by the preparation method of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910119648.3A CN109771376B (en) | 2019-02-18 | 2019-02-18 | Tumor cell source microparticle drug-carrying preparation and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910119648.3A CN109771376B (en) | 2019-02-18 | 2019-02-18 | Tumor cell source microparticle drug-carrying preparation and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109771376A CN109771376A (en) | 2019-05-21 |
| CN109771376B true CN109771376B (en) | 2021-01-01 |
Family
ID=66504449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910119648.3A Active CN109771376B (en) | 2019-02-18 | 2019-02-18 | Tumor cell source microparticle drug-carrying preparation and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109771376B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228221A (en) * | 2020-02-25 | 2020-06-05 | 湖北盛齐安生物科技股份有限公司 | Method for improving productivity of drug-loaded vesicle |
| CN111265491B (en) * | 2020-03-25 | 2022-05-20 | 江苏省中医药研究院 | Cell microparticle drug delivery system for entrapping allicin and preparation method and application thereof |
| CN111407742B (en) * | 2020-03-30 | 2021-10-22 | 西南交通大学 | Anti-tumor nano-particles and preparation method and application thereof |
| CN116919918A (en) * | 2022-04-11 | 2023-10-24 | 华中科技大学 | Succinic acid-loaded tumor cell-derived microparticles, and preparation method and application thereof |
| CN115068439A (en) * | 2022-05-26 | 2022-09-20 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of tumor microparticles encapsulating metabolic inhibitor |
-
2019
- 2019-02-18 CN CN201910119648.3A patent/CN109771376B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| Electroporation-delivered Bi2S3 QDs and doxorubicin into cell-derived microparticles for photothermal and chemotherapy;Dongdong Wang等;《Nanomedicine: Nanotechnology, Biology, and Medicine》;20181231;第14卷;参见第1876页右栏倒数第1段至第1877页左栏第1段和图1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109771376A (en) | 2019-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109771376B (en) | Tumor cell source microparticle drug-carrying preparation and preparation method thereof | |
| Mathiyazhakan et al. | A concise review of gold nanoparticles-based photo-responsive liposomes for controlled drug delivery | |
| Li et al. | Cell‐based delivery systems: emerging carriers for immunotherapy | |
| Du et al. | Extracellular vesicles: a rising star for therapeutics and drug delivery | |
| Yeo et al. | Protein corona in drug delivery for multimodal cancer therapy in vivo | |
| Su et al. | A neutrophil membrane-functionalized black phosphorus riding inflammatory signal for positive feedback and multimode cancer therapy | |
| Dhas et al. | Advancements in cell membrane camouflaged nanoparticles: a bioinspired platform for cancer therapy | |
| CN110448541B (en) | Double-functionalized nanoparticle, soluble microneedle, preparation method and application thereof | |
| CN110623939B (en) | Preparation method of cantharidin-loaded tumor cell membrane encapsulated tellurium elementary substance nanoparticles | |
| Zeng et al. | Cell membrane-coated nanomaterials for cancer therapy | |
| Fang et al. | Stimulus-responsive nanocarriers for targeted drug delivery | |
| Zhao et al. | Cell membrane-based biomimetic nanosystems for advanced drug delivery in cancer therapy: A comprehensive review | |
| Guo et al. | Construction of nanocarriers based on endogenous cell membrane and their application in nanomedicine | |
| Chen et al. | Nanomaterials: small particles show huge possibilities for cancer immunotherapy | |
| CN113041356A (en) | Hemopsin targeting drug-loading system, preparation method and application thereof | |
| Wang et al. | Tailoring viruslike mesoporous FeSe2 hedgehogs for controlled drug delivery and synergistic tumor suppression | |
| WO2021083370A1 (en) | Preparation and use of nanomaterial specifically activating immune system | |
| Li et al. | Functional core/shell drug nanoparticles for highly effective synergistic cancer therapy | |
| Zhang et al. | Nano-engineered immune cells as “guided missiles” for cancer therapy | |
| Deshmukh et al. | Extracellular nanovesicles: from intercellular messengers to efficient drug delivery systems | |
| Peng et al. | Research progress of the engagement of inorganic nanomaterials in cancer immunotherapy | |
| CN112791061B (en) | Preparation method of multi-stage bionic nano-drug carrier with targeting long circulation | |
| Liu et al. | Lipid-hybrid cell-derived biomimetic functional materials: A state-of-the-art multifunctional weapon against tumors | |
| Hu et al. | The application of nanoparticles in immunotherapy for hepatocellular carcinoma | |
| CN110711249B (en) | Preparation method of lysosome membrane-coated nanoparticles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |