CN112168963A - Nano photothermal medicine and its preparing method - Google Patents
Nano photothermal medicine and its preparing method Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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Abstract
The invention discloses a nano photothermal therapeutic medicine and a preparation method thereof. The nano photothermal therapeutic medicine is composed of a nano medicine particle inner core and an outer cancer cell membrane; wherein the nano-drug system is composed of a heat shock protein inhibitor and polydopamine. The medicine can play roles of cancer cell membrane immune escape, tumor part self-targeting, poly-dopamine photo-thermal treatment and heat shock protein inhibitor sensitization low-temperature photo-thermal treatment, and can perform striking treatment aiming at the heat resistance of tumor cells; the heat shock protein inhibitor can be quickly released, and the hepatotoxicity of the heat shock protein inhibitor can be reduced; the compound has excellent photothermal imaging and fluorescence imaging properties, and can be used for cancer targeted imaging and phototherapy; high accumulation is formed at the self-targeting tumor part, and the low-dose HI can achieve the ideal effect, thereby avoiding the possible toxicity of the excessive use to healthy organs.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a nano photothermal treatment medicine and a preparation method thereof.
Background
Statistics of data indicate that cancer has become a leading cause of death worldwide over heart disease. The current cancer treatment methods mainly comprise surgery, radiotherapy and chemotherapy, but the three treatments can kill normal cells, destroy the immune system and increase the risk of secondary attack of the cancer while treating the cancer. Photothermal therapy mainly depends on various artificially synthesized materials (such as gold nanorods, carbon nanomaterials, copper sulfide, palladium nanosheets, near-infrared fluorescent dyes, high molecular polymers and the like) to absorb near-infrared light and convert the near-infrared light into heat so as to kill tumor cells. Photothermal therapy has gradually become an important technology for cancer treatment due to its advantages of high selectivity, low invasiveness, easy operation, rapid recovery, few complications, and the like.
However, the inorganic nano photothermal material has poor biocompatibility, and has in vivo metabolic problems and severe long-term toxicity, which makes it difficult to obtain approval from international medical approval authorities (such as FDA) and thus difficult to apply clinically. The polydopamine material has good biocompatibility, is degradable in vivo, has good photothermal conversion performance, and is a good choice for photothermal treatment reagents. However, the polydopamine material serving as an exogenous material enters a human body and causes the recognition of an immune system and is rapidly eliminated, so that the concentration of an effective material reaching a tumor part is greatly reduced. Research has shown that tumor cells rapidly produce large amounts of Heat Shock Proteins (HSPs) to protect them from thermal damage caused by high temperature when exposed to high temperature, which is a self-protection mechanism of tumor cells.
Therefore, the inventor of the invention prepares a nano photothermal therapy medicament, which can inhibit the synthesis of heat shock protein, effectively reduce the heat resistance of tumor cells, improve the effect of tumor photothermal therapy, effectively avoid the recognition and removal of conventional nanoparticles by an immune system, improve the in vivo circulation retention time, and specifically target homologous tumor cells through a homologous adhesion mechanism, so that the medicament can be efficiently absorbed, the use amount is reduced, and the toxicity generated by excessive medicament is avoided. The medicine of the invention has wide application prospect.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a nano photothermal therapeutic drug.
The invention also aims to provide a preparation method of the nano photothermal treatment medicine.
The purpose of the invention is realized by the following technical scheme: a nanometer medicinal preparation for treating photothermal diseases comprises nanometer medicinal granule core and outer cancer cell membrane; the nano-drug particles are a nano-drug system consisting of a heat shock protein inhibitor and polydopamine nanospheres loaded with the heat shock protein inhibitor.
The cancer cell membrane is a homologous tumor cell membrane of a cancer in logarithmic growth phase in need of treatment.
The heat shock protein inhibitor is geldanamycin.
The polydopamine nanosphere is obtained by stirring and reacting dopamine hydrochloride and ammonia water in deionized water.
The concentration of the ammonia water is 25-28%.
The dosage of the dopamine hydrochloride and the ammonia water is preferably 1-10 mg: 0.5-5 μ L; more preferably 1 mg: 5 μ L.
The preferable dosage of the deionized water is 1-4 mL according to the proportion of the deionized water to the dopamine hydrochloride: 2-10 mg.
The polydopamine nanosphere and the heat shock protein inhibitor are respectively dissolved and dispersed by adopting an organic solvent and deionized water to obtain a heat shock protein inhibitor solution and a polydopamine nanosphere solution.
The organic solvent is dimethyl sulfoxide (DMSO).
The volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 10-5: 5-1, wherein the mass ratio of the solute is 10-5: 6-3; the volume ratio of the solution is preferably 9:1, the mass ratio of the solute is preferably 7: 6.
the dosage of the nano-drug particles and the cancer cell membrane is 1: 1-2; preferably, the mass ratio is 1: 1 to 1.2.
The preparation method of the nano photothermal treatment medicine comprises the following steps:
(1) synthesis of polydopamine nanospheres (PDA): mixing dopamine hydrochloride, ammonia water and a solvent, stirring for reaction, centrifuging, collecting precipitate, and washing to obtain a polydopamine nanosphere;
the concentration of the ammonia water in the step (1) is preferably 25-28%.
The dosage of the dopamine hydrochloride and the ammonia water in the step (1) is preferably 1-3 mg: 0.5-2.5 muL; more preferably 1 mg: 1 μ L.
The solvent in step (1) is preferably deionized water.
The dosage of the solvent in the step (1) is preferably 1-4 mL according to the ratio of the solvent to the dopamine hydrochloride: 2-10 mg; preferably, the ratio of the dopamine hydrochloride to the dopamine hydrochloride is 1 mL: 2 mg.
The stirring reaction in the step (1) is carried out at room temperature at 250-1000 rpm for 12-48 h; preferably, the reaction is carried out for 20 to 28 hours at the temperature of 20 to 25 ℃ and the rpm of 400 to 600.
The centrifugation in the step (1) is 8000 rpm-16000 rpm for 5 min-20 min; preferably 10000rpm to 12000rpm for 8min to 12 min.
The washing in step (1) is to wash the precipitate until the supernatant is clear and transparent.
(2) Preparation of a heat shock protein inhibitor-loaded nano-drug system (PDA/HI): mixing a heat shock protein inhibitor and the polydopamine nanospheres prepared in the step (1), stirring for reaction, and centrifuging to obtain a nano-drug system;
respectively adopting an organic solvent and deionized water to dissolve and disperse the heat shock protein inhibitor and the polydopamine nanospheres before mixing to obtain a heat shock protein inhibitor solution and a polydopamine nanosphere solution.
The organic solvent is dimethyl sulfoxide (DMSO).
The volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 10-5: 5-1, wherein the mass ratio of the solute is 10-5: 6-3; the volume ratio is preferably 9:1, the mass ratio of the solute is preferably 7: 6.
the heat shock protein inhibitor in the step (2) is geldanamycin.
Stirring reaction in the step (2) is carried out for 12-48 h at 100-1000 rpm under the condition of 20-25 ℃; preferably, the stirring is carried out for 20 to 28 hours at the temperature of 25 ℃ and at the rpm of 300 to 500.
The centrifugation in the step (2) is 8000 rpm-16000 rpm for 5 min-20 min; centrifugation is preferably carried out at 12000rpm for 10 min.
(3) Preparation of polydopamine drug (PDA/HI @ CCM) wrapped by cancer cell membrane and loaded with heat shock protein inhibitor: and (3) blending and extruding the nano-drug system obtained in the step (2) and the cancer cell membrane fragments to obtain the nano-photothermal therapeutic drug.
The dosage of the nano-drug system and the cancer cell membrane fragments in the step (3) is 1: 1-2; preferably, the mass ratio is 1: 1 to 1.2.
The extrusion in the step (3) comprises the following steps: mixing the nanometer medicine system and the cancer cell membrane fragments uniformly in proportion, and then filtering by adopting a polycarbonate microporous filter membrane to obtain the nanometer photothermal treatment medicine.
The uniform mixing is realized by adopting vortex mixing.
The polycarbonate microporous filter membrane filtration is sequentially polycarbonate microporous filter membranes with the specifications of 1 micron, 0.85 micron and 0.45 micron, and the filtration is carried out for 8-15 times respectively.
The cancer cell membrane fragments in step (3) are fragments obtained by treating cell membranes of homologous tumor cells of the cancer to be treated, which grow in the logarithmic phase.
The preparation method of the cancer cell membrane fragment in the step (3) comprises the following steps: extracting to obtain cell membrane fragments, dissolving in pure water or PBS, and performing ultrasonic treatment to obtain uniform nanoscale cell membrane fragments; the whole process is carried out under ice bath condition.
And (3) and (2) are carried out under the condition of keeping out of the sun.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention prepares polydopamine with isoelectric point similar to cancer cell membrane, loads heat shock protein inhibitor and wraps the cancer cell membrane, when the obtained nano photothermal therapy medicine PDA/HI @ CCM is in the acidic environment in the tumor cell, the surface charge of PDA and the cancer cell membrane wrapped on the surface tend to be zero, and the electrostatic interaction between the PDA and the cancer cell membrane is weakened, thereby rapidly releasing the heat shock protein inhibitor.
2. The PDA/HI @ CCM prepared by the invention has excellent photothermal imaging and fluorescence imaging characteristics, and can be used for cancer targeted imaging and phototherapy.
3. The PDA/HI @ CCM prepared by the invention can automatically target tumor parts and form high accumulation. Therefore, a low dose of HI may achieve the desired therapeutic effect, avoiding possible toxicity to healthy organs due to overuse. In addition, the PDA/HI @ CCM can well reduce the hepatotoxicity of the heat shock protein inhibitor.
4. The preparation method is simple and convenient to operate, the product is easy to obtain, the method is stable and reliable, the prepared medicine has the advantages of good biocompatibility, specific tumor targeting, small toxic and side effects on organisms and the like, the immune escape of cancer cell membranes and the self-targeting of tumor parts, the photothermal treatment of polydopamine and the sensitization low-temperature photothermal treatment of heat shock protein inhibitors can be exerted in the aspect of tumor treatment, and the hit treatment is carried out aiming at the heat resistance of tumor cells.
Drawings
Fig. 1 is a particle size diagram of polydopamine nanospheres (PDA), cancer cell membrane fragments (CCM), and a polydopamine drug loaded with a heat shock protein inhibitor (PDA/HI @ CCM) encapsulated by cancer cell membranes.
FIG. 2 is a transmission electron microscope image of polydopamine nanospheres (PDA) and a polydopamine drug loaded with a heat shock protein inhibitor (PDA/HI @ CCM) wrapped by a cancer cell membrane; wherein, A is PDA and B is PDA/HI @ CCM.
FIG. 3 is a graph of the potential change of cancer cell membranes (CCM, 4T1), polydopamine nanospheres (PDA), gold Nanostars (NSs) and Lignin (LNPs) under different pH conditions; wherein, A is cancer cell membrane, B is gold nano star, C is polydopamine nanosphere, and D is lignin.
FIG. 4 is a graph showing the results of targeting analysis of polydopamine drug (PDA/HI @ CCM) loaded with heat shock protein inhibitor encapsulated by cancer cell membrane.
FIG. 5 is a graph showing the results of in vitro drug release analysis of a polydopamine drug (PDA/HI @ CCM) loaded with a heat shock protein inhibitor encapsulated by a cancer cell membrane.
FIG. 6 is a graph showing the results of in vitro dark cytotoxicity assay of polydopamine drug loaded with heat shock protein inhibitor (PDA/HI @ CCM) encapsulated in cancer cell membrane.
FIG. 7 is a graph showing the results of in vitro cytotoxicity assay of polydopamine drug loaded with heat shock protein inhibitor (PDA/HI @ CCM) encapsulated in cancer cell membrane.
FIG. 8 is a graph showing the change in tumor volume of mice in each experimental group throughout the treatment period.
FIG. 9 is a photograph showing the isolated tumors of mice in each experimental group on the 24 th day of treatment.
FIG. 10 is a graph of H & E staining of heart, liver, spleen, lung, and renal paraffin sections of mice in each experimental group on day 24 of treatment.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Dopamine hydrochloride was purchased from Shanghai Maxlin Biotechnology, Inc., heat shock protein inhibitor was purchased from Guangzhou Lenke Biotechnology, Inc., and cell membrane protein and cytoplasmic protein extraction kit was purchased from Biyuntian Biotechnology, Inc.
Example 1 Synthesis of Polydopamine nanospheres (PDA)
Adding 50mg of dopamine hydrochloride and 250 mu L of 25% (v/v) concentrated ammonia water into 25mL of deionized water, reacting for 20-28 h at the room temperature and the rotating speed of 500rpm, then centrifuging for 10min at 12000rpm, collecting the precipitate, repeatedly washing and centrifuging by using the deionized water, and collecting the precipitate until the supernatant is clear and transparent, wherein the precipitate product is the polydopamine nanosphere.
Example 2 preparation of Heat shock protein inhibitor loaded Polydopamine Nanoparticulate pharmaceutical System (PDA/HI)
6mg of the heat shock protein inhibitor is dissolved in 1mL of organic DMSO solution to obtain a heat shock protein inhibitor solution. 7mg of the polydopamine nanosphere obtained in example 1 was dispersed in 9mL of deionized water to obtain a polydopamine nanosphere solution. Mixing two solutions, wherein the volume ratio of the solutions is 9:1, and the mass ratio of solutes is 7: 6. stirring at 25 deg.C and 300rpm for 24h, centrifuging at 12000rpm for 10min, and collecting polydopamine nanometer medicinal system loaded with heat shock protein inhibitor.
EXAMPLE 3 extraction of Cancer Cell Membranes (CCM)
Cell membrane protein and cytoplasm protein extraction kit are adopted to extract cell membrane. In order to avoid the change of membrane protein caused by protease contained or adsorbed on the membrane, the following operation should be performed under ice bath condition.
In order to obtain cell membrane fragments, mouse breast cancer cells 4T1 are cultured in a cell culture dish with the diameter of 25cm, then the cells are collected by a cell scraper, the cells are centrifuged for 5min at 700g to obtain cell precipitates, the cell precipitates are resuspended in precooled 1 XPBS (0.01M, pH 7.4) and centrifuged for 5min at 700g, the obtained cell precipitates are resuspended in hypotonic cell lysate (containing cell membrane proteins and a membrane protein extraction reagent A and 1mM PMSF in a cell plasma protein extraction kit, the dosage of 2000-5000 ten thousand cells are lysed by 990 muL of cell membrane extraction reagent +10 muL of PMSF), and the cell precipitates are placed in an ice water mixture for 10-15 min. Then, the cells are broken up by means of cell freeze thawing, the supernatant is carefully collected after centrifugation at 700g for 10min at 4 ℃, and the precipitate obtained is the cell membrane fragment after centrifugation at 14,000g for 30 min. Freeze-drying, weighing, and storing in-80 deg.C ultra-low temperature refrigerator. The lyophilized membrane material was dissolved and dispersed in ultrapure water or 1 × PBS before use.
Example 4 preparation of Heat shock protein inhibitor-loaded NanoMedicine System (PDA/HI @ CCM) wrapped with cancer cell Membrane
The cell membrane fragments extracted in example 3 are dissolved in pure water, and are subjected to ice-bath ultrasonic treatment by an ultrasonic cell disruptor to obtain uniformly dispersed nano-scale cell membrane fragments (the ultrasonic power is 97.5W, and the working time is 30 min). Then, the polydopamine nanospheres prepared in example 2 and the cancer cell membrane fragments are mixed according to the mass ratio of 1: 1.2, and then sequentially filtering the mixture for 10 times through 1 micron, 0.85 micron and 0.45 micron polycarbonate microporous filter membranes respectively to obtain the polydopamine drug (PDA/HI @ CCM) wrapped by the cancer cell membrane and loaded with the heat shock protein inhibitor. The particle size of the nanoparticles is measured by a Malvern nanometer particle sizer, and the result is shown in figure 1. The morphology is characterized by Transmission Electron Microscopy (TEM) as shown in FIG. 2. The figure shows that the medicine has a regular spherical structure, and the particle size is about 150-200 nm.
Example 5 change of surface potential of cancer cell membrane, polydopamine nanospheres, gold nanostars and lignin under different pH conditions.
1mL of four materials (polydopamine nanospheres synthesized in example 1, Cancer cell membranes synthesized in example 3 from disrupted cell membranes, Lignin having photothermal conversion activity purchased from Sigma, Germany) were prepared according to literature procedures (Zhu et al.2018. using the method of measuring the photothermal interaction Area of Nanomedicine Enhances types, Homotypic Cancer efficiency [ J. ACS NANO.2020,14(3):2557-2560.) were ultrasonically dispersed in 1.5mL of deionized water, the pH of the solution was adjusted to 2, 3, 4, 5, 6, 7, respectively, using 0.1M hydrochloric acid (HCl), the change in surface Potential was measured using a Marwin nano-particle size analyzer, and the pH at which the surface Potential was 0 was obtained, three sets of repeated experiments were performed, the isoelectric points of polydopamine were all 4 to 355, while the isoelectric points of lignin and gold nano-star do not meet the interval. Therefore, polydopamine is selected as the core of the nano-drug, the nano-drug can be disassembled and released in the slightly acidic environment of tumor tissues, and lignin or gold nanostar as the core of the nano-drug cannot be disassembled and released in the acidic environment to release the drug.
Example 6 Targeted analysis of Polydopamine drug (PDA/HI @ CCM) loaded with heat shock protein inhibitor and wrapped by cancer cell membrane
Experiments were performed with mouse breast cancer cell 4T1, and mouse melanoma B16F-10 cells and smooth muscle cell L929 as controls. The operation steps are as follows: 4T1 cells, B16F-10 cells and L929 cells were seeded into confocal dishes at 1X 10 cells/dish, respectively5For each cell, 1.5mL of complete medium was added to each dish. After 24h of Cell culture after attachment, fresh medium containing PDA/HI @ CCM (obtained in example 4) labeled with the fluorescent dye Cy5 was added and the culture was continued for 4h, after which the old medium containing material was aspirated off and excess material was washed away with fresh 1 XPBS, followed by Cell nucleus staining with Hochest 33342 dye and Cell MaskTMGreen Plasma Membrane Stain dye of living cells, dyeing for 10min at 37 ℃, then washing off redundant dye and flooding with fresh 1 XPBS, adding paraformaldehyde for fixing for 40min, washing off fixative with fresh 1 XPBS after fixing, and finally adding 1mL of 1 XPBS solution to prevent cell adhesion. The confocal culture dish is placed under laser confocal observation and photographs are taken. The tumor targeting results are shown in FIG. 4, where blue represents the fluorescence of Cell nucleus Hochest 33342, red represents the fluorescence of PDA/HI @ CCM, and green represents the Cell Mask of live Cell membraneTMFluorescence of Green Plasma Membrane Stain. As the PDA/HI @ CCM is coated by the shell of the cell membrane of the cancer cell 4T1, the targeting property of the drug to the same tumor cell can be effectively improved, and as can be seen from figure 4, the PDA/HI @ CCM is specifically combined with the 4T1 cell (the fluorescence intensity is stronger), while the PDA/HI @ CCM is poor in combination with the B16F-10 cell and the L929 cell (the fluorescence intensity is weak).
Example 7 in vitro drug Release assay for Heat shock protein inhibitor-loaded Polydopamine drugs (PDA/HI @ CCM) encapsulated in cancer cell membranes
The in vitro drug release behavior of the nanosystem was evaluated by dialysis with phosphate buffer (1 × PBS) at pH 7.4 and acetate buffer (1 × ABS) at pH 5.2 as release media. The PDA/HI @ CCM obtained in example 4 was filled in a dialysis bag (molecular weight cut-off: 1000kDa), which was then added to a solution containing phosphate buffer and acetate buffer, respectively, and placed in a 37 ℃ thermostat water bath for dialysis, and the release behavior of PDA/HI @ CCM was investigated with 160rpm shaking. The buffer was aspirated at time points 0,1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96h, respectively, to 5mL, followed by the addition of 5mL of fresh buffer, maintaining the buffer volume constant, and the withdrawn buffer was measured with a uv spectrophotometer and the cumulative release rate was calculated. As can be seen from FIG. 5, the cumulative release of PDA/HI @ CCM is significantly higher in the acetate buffer environment at pH 5.0 than in the 1 XPBS environment at pH 7.4. Therefore, in the acidic environment of the tumor cells, the PDA/HI @ CCM can quickly and effectively release the heat shock protein inhibitor, prevent the tumor cells from expressing the heat shock protein when the tumor cells are heated by illumination, and improve the killing effect of photothermal therapy on the tumor cells.
Example 8 cytotoxicity assay of Polydopamine (PDA/HI @ CCM) loaded with Heat shock protein inhibitor encapsulated by cancer cell Membrane
(1) The PDA/HI @ CCM obtained in example 4 was evaluated for in vitro dark toxicity against 4T1 cells and L929 cells using the CCK8 method for measuring cell activity. The specific operation steps are as follows: first, 4T1 cells and L929 cells were cultured at 1X 10 cells, respectively4The density of each well was inoculated in a 96-well plate, which was then placed in a carbon dioxide incubator for overnight anchorage (4T1 cells in RPMI1640 medium and L929 cells in DMEM high-sugar medium). Subsequently, the original medium was aspirated and replaced with fresh complete medium containing different concentrations of PDA/HI @ CCM. The entire experimental process was without laser irradiation. The concentration of the selected PDA/HI @ CCM is based on the concentration of HI, the range of HI concentration is 5-80 mu g/mL (gradient setting: 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 60 mu g/mL and 80 mu g/mL), and each concentration has 3 parallels; pure cell setIs a positive control group. The cells were incubated with the material in a cell incubator for 24h, after incubation, the medium was removed, the cells were washed twice with 1 × PBS and 100 μ L of fresh medium (containing 10% by volume of CCK-8) was added to each well. Placing the mixture in an incubator for incubation for a period of time, finally detecting and recording the absorbance at the wavelength of 450nm by using a microplate reader, and calculating the cell survival rate by the following formula:
(％)＝(OD450sample-OD450background)/(OD450control-OD450background)×100％；
wherein OD450sample is the absorbance value of cells with PDA/HI @ CCM added, OD450 control is the absorbance value of cells without material added, and OD450 background is the absorbance value of blank background without material and cells added.
Data are based on three independent replicates and are expressed as mean ± Standard Deviation (SD).
(2) The method for detecting cell activity by using the CCK-8 method is used for evaluating the in vitro phototoxicity of PDA/HI @ CCM on 4T1 cells and L929 cells. The specific operation steps are as follows: first, 4T1 cells and L929 cells were treated with 1 × 10 cells, respectively4The density of individual/well was seeded in 96-well plates and then placed in carbon dioxide incubators for overnight culture attachment. Subsequently, the original medium was aspirated and replaced with fresh complete medium containing different concentrations of PDA/HI @ CCM. After the cells were incubated with PDA/HI @ CCM for 4h, the cells were placed at 808nm and 1.0W/cm2Irradiating with near infrared light for 5 min. After the irradiation, the cells were returned to the cell incubator and cultured for 24 hours. After the incubation was complete, the medium containing the material was aspirated, the cells were washed twice with 1 × PBS and 100 μ L of fresh medium (containing 10% CCK-8) was added to each well. Placing in an incubator, incubating for 20-40min, and detecting and recording the absorbance at 450nm by using a microplate reader. The concentration of PDA/HI @ CCM was chosen based on the concentration of HI, which ranged from 5 to 80. mu.g/mL (gradient setup: 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL). Each concentration setting was 3 replicates; the pure cell group was set as a positive control group. Cell viability was calculated by the following formula:
(％)＝(OD450sample-OD450background)/(OD450control-OD450background)×100％；
wherein OD450sample is the absorbance value of cells with PDA/HI @ CCM added, OD450 control is the absorbance value of cells without material added, and OD450 background is the absorbance value of blank background without material and cells added.
Data are based on three independent replicates and are expressed as mean ± Standard Deviation (SD).
The results of dark toxicity analysis are shown in FIG. 6, where PDA/HI @ CCM had low in vitro dark toxicity to both 4T1 cells and L929 cells.
Phototoxicity results as shown in fig. 7, PDA/HI @ CCM produced dose-dependent cytotoxicity on both 4T1 and L929 cells under laser irradiation, showing more pronounced cytotoxicity in 4T1 cells compared to L929 cells, indicating that PDA/HI @ CCM has excellent cell selectivity towards (homologously targeted) homotypic cancer cell 4T1 cells.
Comparing fig. 6 and 7, the heat shock protein inhibitor was significantly released and cytotoxicity was significantly increased under the stimulation of near infrared light.
Example 9 in vivo anti-tumor Activity study of Polydopamine Nano Material loaded with Heat shock protein inhibitor (PDA/HI @ CCM) wrapped by cancer cell Membrane
The establishment steps of the mouse 4T1 tumor model are as follows: the breast cancer cells (4T1) cultured in advance were digested with trypsin, centrifuged, resuspended in an appropriate amount of 1 XPBS and resuspended at 5X 105Each 100. mu.L cell density was inoculated to the right hind limb of BALB/C mice (purchased from Beijing Wittingerihua laboratory animal technology Co., Ltd.) and the tumor size was observed daily until the tumor volume of the mice reached 100mm3On the left and right, the next experiment was performed.
4T1 tumor model mice were randomly divided into 5 groups of 5 mice each. Each experimental group was: 1 XPBS, PDA/HI @ CCM + NIR, PDA/HI @ CCM, PDA @ CCM + NIR, PDA + NIR; NIR stands for 1.0W/cm at 808nm2Irradiating with near infrared light for 5 min. The injection amount of the drug injected into the tail vein of the experimental group is based on the injection amount of the heat shock protein inhibitor HI of 10mg/kg of mice. The administration was started on the first day, and was administered and irradiated once every other day for a total of 3 treatments. At the same time, tumors were recorded every two daysVolume. Tumor volume was defined as: w ═ V2L/2, where W and L refer to the longest and shortest diameters of the tumor, respectively.
Observation was continued for 19 days after the end of treatment, and then all mice were euthanized, tumors collected, weighed and photographed. And major organs such as heart, liver, spleen, lung and kidney were collected, immersed in a tissue fixative, subjected to conventional paraffin embedding, tissue sectioned, and then the sections were analyzed by hematoxylin-eosin (H & E) staining and photographed using an optical microscope. The change in tumor volume of each group of mice throughout the treatment period is shown in figure 8. Tumors from each group of mice ex vivo after the entire treatment cycle was completed are shown in fig. 9. As can be seen from fig. 8, both the PDA + NIR group and the PDA @ CCM + NIR group showed a certain degree of tumor suppression capability, compared to the PBS group, mainly due to the EPR effect and the PTT effect. As expected, the PDA/HI @ CCM + NIR treatment group has the best treatment effect, even completely eliminates part of tumors of mice, and proves that the PDA/HI @ CCM can achieve good tumor inhibition effect under laser irradiation. As shown in fig. 9, the isolated tumors of the mice of each experimental group after 24 days met the expected results, confirming that the PDA/HI @ CCM + NIR group has excellent anti-tumor efficacy. The results of H & E staining of major organs (heart, liver, spleen, lung, kidney) of each group of mice are shown in fig. 10, and no pathological changes of major organs were observed in the PDA/HI @ CCM + NIR group compared to the control group. It was further confirmed that the systemic toxicity of PDA/HI @ CCM was negligible and that the hepatotoxicity of HI was greatly improved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A nanometer photothermal therapeutic medicine is characterized by comprising a nanometer medicine particle inner core and an outer cancer cell membrane;
the nano-drug particles are a nano-drug system consisting of a heat shock protein inhibitor and polydopamine nanospheres loaded with the heat shock protein inhibitor;
the cancer cell membrane is a homologous tumor cell membrane of a cancer in logarithmic growth phase in need of treatment.
2. The nano photothermal therapy drug according to claim 1,
the heat shock protein inhibitor is geldanamycin;
the polydopamine nanosphere is obtained by stirring and reacting dopamine hydrochloride and ammonia water in deionized water;
the polydopamine nanosphere and the heat shock protein inhibitor are respectively dissolved and dispersed by adopting an organic solvent and deionized water to obtain a heat shock protein inhibitor solution and a polydopamine nanosphere solution.
3. The nano photothermal therapy drug according to claim 2,
the dosage of the nano-drug particles and the cancer cell membrane is 1: 1-2;
the concentration of the ammonia water is 25-28%;
the dosage of the dopamine hydrochloride and the ammonia water is 1-10 mg: 0.5-5 μ L;
the dosage of the deionized water is 1-4 mL according to the proportion of the deionized water to the dopamine hydrochloride: 2-10 mg;
the organic solvent is dimethyl sulfoxide;
the volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 10-5: 5-1, wherein the mass ratio of the solute is 10-5: 6-3.
4. The nano photothermal therapy drug according to claim 3,
the dosage of the nano-drug particles and the cancer cell membrane is 1: 1.2;
the dosage of the dopamine hydrochloride and the ammonia water is 1 mg: 5 mu L of the solution;
the volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 9:1, the mass ratio of solute is 7: 6.
5. the method for preparing a nano photothermal therapeutic agent according to any one of claims 1 to 4, characterized by comprising the steps of:
(1) synthesis of polydopamine nanospheres: mixing dopamine hydrochloride, ammonia water and a solvent, stirring for reaction, centrifuging, collecting precipitate, and washing to obtain a polydopamine nanosphere;
(2) preparing a nano-drug system loaded with a heat shock protein inhibitor: mixing a heat shock protein inhibitor and the polydopamine nanospheres prepared in the step (1), stirring for reaction, and centrifuging to obtain a nano-drug system;
(3) preparing a polydopamine drug wrapped by a cancer cell membrane and loaded with a heat shock protein inhibitor: and (3) blending and extruding the nano-drug system obtained in the step (2) and the cancer cell membrane fragments to obtain the nano-photothermal therapeutic drug.
6. The method for preparing a nano photothermal therapeutic agent according to claim 5,
the dosage of the dopamine hydrochloride and the ammonia water in the step (1) is 1-3 mg: 0.5-2.5 muL;
the dosage of the solvent in the step (1) is 1-4 mL according to the ratio of the solvent to the dopamine hydrochloride: 2-10 mg;
respectively dissolving and dispersing the heat shock protein inhibitor and the polydopamine nanospheres by using an organic solvent and deionized water before mixing to obtain a heat shock protein inhibitor solution and a polydopamine nanosphere solution;
the volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 10-5: 5-1, wherein the mass ratio of the solute is 10-5: 6-3;
the heat shock protein inhibitor in the step (2) is geldanamycin;
the dosage of the nano-drug system and the cancer cell membrane fragments in the step (3) is 1: 1-2;
the cancer cell membrane fragments in step (3) are fragments obtained by treating cell membranes of homologous tumor cells of the cancer to be treated, which grow in the logarithmic phase.
7. The method for preparing a nano photothermal therapeutic agent according to claim 6,
the dosage of the dopamine hydrochloride and the ammonia water in the step (1) is 1 mg: 1 mu L of the solution;
the volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 9:1, the mass ratio of solute is 7: 6;
the dosage of the nano-drug system and the cancer cell membrane fragments in the step (3) is 1: 1 to 1.2.
8. The method for preparing a nano photothermal therapeutic agent according to claim 6,
the concentration of the ammonia water in the step (1) is 25-28%;
the solvent in the step (1) is deionized water;
the stirring reaction in the step (1) is carried out at room temperature at 250-1000 rpm for 12-48 h;
the centrifugation in the step (1) is 8000 rpm-16000 rpm for 5 min-20 min;
the washing in the step (1) is to wash the precipitate until the supernatant is clear and transparent;
the organic solvent is dimethyl sulfoxide;
stirring reaction in the step (2) is carried out for 12-48 h at 100-1000 rpm under the condition of 20-25 ℃;
the centrifugation in the step (2) is 8000 rpm-16000 rpm for 5 min-20 min;
and (3) and (2) are carried out under the condition of keeping out of the sun.
9. The method for preparing a nano photothermal therapeutic agent according to claim 8,
the stirring reaction in the step (1) is a reaction at a temperature of between 20 and 25 ℃ and at a speed of between 400 and 600rpm for 20 to 28 hours;
the centrifugation in the step (1) is 10000-12000 rpm for 8-12 min;
the stirring reaction in the step (2) is stirring at 300-500 rpm for 20-28 h at 25 ℃;
the centrifugation in the step (2) is carried out at 12000rpm for 10 min.
10. The method for preparing a nano photothermal therapeutic agent according to claim 6,
the extrusion in the step (3) comprises the following steps: mixing the nano-drug system and the cancer cell membrane fragments uniformly according to a proportion, and then filtering by adopting a polycarbonate microporous filter membrane to obtain the nano photothermal therapy drug;
the uniform mixing is vortex mixing;
the polycarbonate microporous filter membrane filtration is sequentially polycarbonate microporous filter membranes with the specifications of 1 micron, 0.85 micron and 0.45 micron, and the filtration is carried out for 8-15 times respectively;
the preparation method of the cancer cell membrane fragment in the step (3) comprises the following steps: extracting to obtain cell membrane fragments, dissolving in pure water or PBS, and performing ultrasonic treatment to obtain uniform nanoscale cell membrane fragments; the whole process is carried out under ice bath condition.
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