CN112168963B - Nanometer photothermal therapeutic medicine and preparation method thereof - Google Patents
Nanometer photothermal therapeutic medicine and preparation method thereof Download PDFInfo
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- CN112168963B CN112168963B CN202010984054.1A CN202010984054A CN112168963B CN 112168963 B CN112168963 B CN 112168963B CN 202010984054 A CN202010984054 A CN 202010984054A CN 112168963 B CN112168963 B CN 112168963B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention discloses a nanometer photothermal therapeutic drug and a preparation method thereof. The nanometer photothermal therapeutic medicine consists of cancer cell membrane in the inner layer and the outer layer of nanometer medicine particle; wherein the nano-drug system consists of a heat shock protein inhibitor and polydopamine. The medicine can play roles in cancer cell membrane immune escape and tumor part self-targeting, photo-thermal treatment of polydopamine, sensitization of heat shock protein inhibitors and low-temperature photo-thermal treatment, and strike treatment is carried out aiming at the heat resistance of tumor cells; can rapidly release heat shock protein inhibitor, and reduce liver toxicity of heat shock protein inhibitor; has excellent photothermal imaging and fluorescent imaging characteristics, and can be used for cancer targeted imaging and phototherapy; high accumulation from targeted tumor sites and low doses of HI can achieve the desired effect, avoiding the potential toxicity of excessive use to healthy organs.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a nano photothermal therapeutic drug and a preparation method thereof.
Background
Data statistics indicate that cancer has become a leading cause of death worldwide over heart disease. The current treatment methods of cancer are mainly surgery, radiotherapy and chemotherapy, but these three treatments kill normal cells while treating cancer, destroy the immune system and increase the risk of secondary cancer. Photothermal therapy mainly relies on various synthetic 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 become an important technique for cancer treatment due to its advantages of high selectivity, low invasiveness, simple operation, rapid recovery, few complications, and the like.
However, inorganic nano-photothermal materials have poor biocompatibility and have in vivo metabolic problems and serious long-term toxicity, which makes it difficult to obtain approval by international medical authorities (such as FDA) and thus difficult to be applied clinically. The polydopamine material has good biocompatibility, is degradable in vivo, has good photo-thermal conversion performance, and is a good choice of photo-thermal therapeutic agents. However, polydopamine material, as an exogenous material, enters the human body to cause recognition of the immune system and is rapidly cleared, resulting in a substantial reduction in the concentration of effective material reaching the tumor site. Studies have shown that tumor cells, when exposed to high temperature environments, rapidly produce large amounts of heat shock proteins (Heat Shock Protein, HSP) to combat thermal damage from high temperatures, a self-protecting mechanism for tumor cells.
Therefore, the inventor prepares a nanometer photothermal therapeutic drug which can inhibit the synthesis of heat shock protein, effectively reduce the heat resistance of tumor cells, promote the photothermal therapeutic effect of the tumor, effectively avoid the recognition and the elimination of the immune system to conventional nanometer particles, improve the internal circulation residence time, and can specifically target homologous tumor cells through a homologous adhesion mechanism, thereby being efficiently absorbed, reducing the use amount and avoiding the toxicity generated by excessive drugs. The medicine of the invention has wide application prospect.
Disclosure of Invention
The invention aims at overcoming the defects and shortcomings of the prior art and providing a nanometer photo-thermal therapeutic drug.
The invention also aims at providing a preparation method of the nanometer photothermal therapeutic drug.
The aim of the invention is achieved by the following technical scheme: a nanometer photothermal therapeutic agent comprises cancer cell membrane at inner and outer layers of nanometer medicinal granule; the nano-drug particles are nano-drug systems composed of heat shock protein inhibitors and polydopamine nanospheres loaded with the heat shock protein inhibitors.
The cancer cell membrane is a homologous tumor cell membrane of the cancer in the logarithmic phase and needs to be treated.
The heat shock protein inhibitor is geldanamycin.
The polydopamine nanospheres are obtained by stirring dopamine hydrochloride and ammonia water in deionized water for reaction.
The concentration of the ammonia water is 25% -28%.
The consumption of the dopamine hydrochloride and the ammonia water is preferably 1-10 mg: 0.5-5 mu L; more preferably 1mg: 5. Mu.L.
The dosage of the deionized water is preferably 1-4 mL according to the proportion of the deionized water and the dopamine hydrochloride: 2-10 mg.
And the polydopamine nanospheres 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 to 3; the volume ratio of the solution is preferably 9:1, the mass ratio of solute is preferably 7:6.
the dosage of the nano drug particles and the cancer cell membrane is 1:1 to 2; preferably, the mass ratio is 1:1 to 1.2.
The preparation method of the nanometer photothermal therapeutic drug comprises the following steps:
(1) Synthesis of polydopamine nanospheres (PDA): mixing dopamine hydrochloride and ammonia water with a solvent, stirring for reaction, centrifuging, collecting precipitate, and washing to obtain polydopamine nanospheres;
the concentration of the ammonia water in the step (1) is preferably 25% -28%.
The usage amount of the dopamine hydrochloride and the ammonia water in the step (1) is preferably 1-3 mg: 0.5-2.5 mu L; more preferably 1mg:1 mul.
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 proportion of the solvent to the dopamine hydrochloride: 2-10 mg calculated; preferably, the ratio of the dopamine hydrochloride to the dopamine hydrochloride is 1mL:2mg calculated.
The stirring reaction in the step (1) is carried out at the room temperature of 250 rpm-1000 rpm for 12 h-48 h; preferably 400rpm to 600rpm at 20 to 25 ℃ for 20 to 28 hours.
The centrifugation in the step (1) is 8000-16000 rpm for 5-20 min; preferably 10000rpm to 12000rpm, for 8min to 12min.
The washing in the step (1) is to wash the precipitate to clear and transparent supernatant.
(2) Preparation of heat shock protein inhibitor-loaded nano-drug System (PDA/HI): mixing the heat shock protein inhibitor and the polydopamine nanospheres prepared in the step (1), stirring for reaction, and centrifuging to obtain a nano-drug system;
and (3) respectively adopting an organic solvent and deionized water to dissolve and disperse the heat shock protein inhibitor and the polydopamine nanospheres before mixing the heat shock protein inhibitor and the polydopamine nanospheres in the step (2) 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 to 3; the volume ratio is preferably 9:1, the mass ratio of 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 at a temperature of 25 ℃ at 300-500 rpm for 20-28 hours.
The centrifugation in the step (2) is 8000-16000 rpm for 5-20 min; preferably 12000rpm for 10min.
(3) Preparation of heat shock protein inhibitor-loaded polydopamine drug (PDA/HI@CCM) encapsulated by cancer cell membrane: and (3) blending and extruding the nano-drug system obtained in the step (2) with 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 to 2; preferably, the mass ratio is 1:1 to 1.2.
The extrusion in step (3) comprises the steps of: uniformly mixing the nano-drug system and cancer cell membrane fragments in proportion, and then filtering by using a polycarbonate microporous filter membrane to obtain the nano-photothermal therapeutic drug.
The uniform mixing is realized by vortex mixing.
The filtration with the polycarbonate microporous filter membrane is carried out by adopting the polycarbonate microporous filter membrane with the specification of 1 mu m,0.85 mu m and 0.45 mu m in sequence, and the filtration is carried out for 8 to 15 times respectively.
The cancer cell membrane fragments in the step (3) are fragments obtained by treating the cell membranes of homologous tumor cells of cancers which grow in log phase and need treatment.
The preparation method of the cancer cell membrane fragments 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 conditions.
Both steps (2) and (3) are carried out under the light-shielding condition.
Compared with the prior art, the invention has the following advantages and effects:
1. according to the invention, polydopamine with isoelectric point similar to that of cancer cell membrane is prepared, heat shock protein inhibitor is loaded on polydopamine and the cancer cell membrane is wrapped, when the obtained nanometer photothermal therapeutic drug PDA/HI@CCM is in an acidic environment inside tumor cells, the surface charge of PDA and the cancer cell membrane wrapped on the surface of PDA tend to be zero, and electrostatic interaction between PDA and HI@CCM is weakened, so that the heat shock protein inhibitor is released rapidly.
2. The PDA/HI@CCM prepared by the method has excellent photothermal imaging and fluorescent imaging characteristics, and can be used for cancer targeted imaging and phototherapy.
3. PDA/HI@CCM prepared by the method self-targets tumor sites and forms high accumulation. Thus, low doses of HI can achieve the desired therapeutic effect, avoiding the toxicity that excessive use may create to healthy organs. The PDA/HI@CCM can well reduce the hepatotoxicity of the heat shock protein inhibitor.
4. The preparation method provided by the invention 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, can play roles in tumor treatment of cancer cell membrane immune escape and tumor part self-targeting, photo-thermal treatment of polydopamine, heat shock protein inhibitor sensitization low-temperature photo-thermal treatment and impact treatment aiming at the heat resistance of tumor cells.
Drawings
FIG. 1 is a graph of particle size of polydopamine nanospheres (PDA), cancer cell membrane fragments (CCM), and heat shock protein inhibitor loaded polydopamine drug encapsulated by cancer cell membranes (PDA/HI@CCM).
FIG. 2 is a transmission electron microscopy image of polydopamine nanospheres (PDA) and heat shock protein inhibitor loaded polydopamine drug encapsulated by cancer cell membranes (PDA/HI@CCM); wherein A is PDA, B is PDA/HI@CCM.
FIG. 3 is a graph of potential change of cancer cell membranes (CCM, 4T 1), polydopamine nanospheres (PDA), gold Nanosomes (NSs), and Lignin (LNPs) at different pH conditions; wherein A is cancer cell membrane, B is gold nanostar, C is polydopamine nanospheres, and D is lignin.
FIG. 4 is a graph showing the results of a targeting assay for a heat shock protein inhibitor loaded polydopamine drug (PDA/HI@CCM) encapsulated by cancer cell membranes.
FIG. 5 is a graph showing the results of in vitro drug release analysis of heat shock protein inhibitor-loaded polydopamine drug (PDA/HI@CCM) encapsulated by cancer cell membranes.
FIG. 6 is a graph showing the results of in vitro cytotoxicity analysis of a heat shock protein inhibitor-loaded polydopamine drug (PDA/HI@CCM) encapsulated by cancer cell membranes.
FIG. 7 is a graph showing the results of in vitro cytotoxicity analysis of a heat shock protein inhibitor-loaded polydopamine drug (PDA/HI@CCM) encapsulated by cancer cell membranes.
Fig. 8 is a graph of tumor volume change for each experimental group of mice throughout the treatment period.
Fig. 9 is a photograph of an isolated tumor of each experimental group of mice on day 24 of treatment.
Fig. 10 is an H & E staining pattern of paraffin sections of mice heart, liver, spleen, lung, kidney on day 24 of treatment.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Dopamine hydrochloride is purchased from Shanghai Michelia Biotechnology Co., ltd, heat shock protein inhibitor is purchased from Guangzhou Zuoke Biotechnology Co., and cell membrane protein and cytoplasmic protein extraction kit is purchased from Biyun Tian Biotechnology Co., ltd.
Example 1 Synthesis of Polydopamine nanospheres (PDAs)
50mg of dopamine hydrochloride and 250 mu L of concentrated ammonia water with the concentration of 25% (v/v) are added into 25mL of deionized water, the mixture is reacted for 20 to 28 hours at the room temperature under the rotating speed of 500rpm, and then the mixture is centrifuged for 10 minutes at 12000rpm, the precipitate is collected, the precipitate is repeatedly washed and centrifuged by the deionized water, and the precipitate is collected until the supernatant is clear and transparent, and the precipitate product is the polydopamine nanospheres.
Example 2 preparation of polydopamine nanodrug System (PDA/HI) loaded with Heat shock protein inhibitor
6mg of the heat shock protein inhibitor was dissolved in 1mL of DMSO as an organic solution to obtain a heat shock protein inhibitor solution. 7mg of the polydopamine nanospheres obtained in example 1 were dispersed in 9mL of deionized water to obtain polydopamine nanosphere solution. Mixing the two solutions, wherein the volume ratio of the solutions is 9:1, and the mass ratio of the solutes is 7:6. stirring at 25deg.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)
Extracting cell membrane by using cell membrane protein and cell plasma protein extraction kit. In order to avoid membrane protein changes by proteases contained or adsorbed on the membrane, the following operations should be performed under ice bath conditions.
To obtain cell membrane debris, mouse breast cancer cells 4T1 were cultured in a cell culture dish having a diameter of 25cm, then the cells were collected with a cell scraper, centrifuged at 700g for 5min to obtain a cell pellet, resuspended in pre-chilled 1×pbs (0.01 m, ph=7.4), and centrifuged at 700g for 5min, and the obtained cell pellet was resuspended in hypotonic cell lysate (containing membrane protein and plasma protein extraction reagent a and 1mM PMSF in a cell plasma protein extraction kit) for 10 to 15min with 2000 to 5000 tens of thousands of cells lysed with 990 μl of cell membrane extraction reagent+10 μl PMSF) and placed in an ice-water mixture. Then, the cells were broken by freeze thawing, and centrifuged at 700g for 10min at 4℃to carefully collect the supernatant, and centrifuged at 14,000g for 30min to obtain a precipitate as cell membrane fragments. Freeze-drying, weighing, and storing in-80deg.C ultra-low temperature refrigerator. The lyophilized film material was dissolved and dispersed in ultrapure water or 1 XPBS before use.
Example 4 preparation of Heat shock protein inhibitor-loaded nanomedicine System (PDA/HI@CCM) with cancer cell membrane encapsulation
The cell membrane fragments extracted in example 3 were dissolved in pure water and subjected to ultrasonic treatment in an ice bath by an ultrasonic cell disrupter to obtain uniformly dispersed nanoscale cell membrane fragments (ultrasonic power 97.5W, working time 30 min). Then the polydopamine nanospheres prepared in example 2 and cancer cell membrane fragments are mixed according to a mass ratio of 1:1.2, and then sequentially filtering the mixture for 10 times through polycarbonate microporous filter membranes with the thickness of 1 mu m,0.85 mu m and 0.45 mu m respectively, thus obtaining the polydopamine drug (PDA/HI@CCM) coated with cancer cell membranes and loaded with heat shock protein inhibitors. The particle size of the nanoparticle is measured by a Markov nanosize meter as shown in FIG. 1. The morphology was characterized by Transmission Electron Microscopy (TEM) as shown in figure 2. The medicine has regular spherical structure and particle size of 150-200 nm.
Example 5 changes in surface potential of cancer cell membranes, polydopamine nanospheres, gold nanostars and lignin at different pH conditions.
Four materials (polydopamine nanospheres with concentration of 1mg/mL are synthesized in example 1, cancer cell membranes are obtained by extracting broken cell membranes in example 3, lignin with photothermal conversion is purchased from German Sigma company, a photothermal material gold nanostar is synthesized according to literature steps (Zhu et al 2018. Research the Potential Interacting Area of Nanomedicine Enhances Its Homotypic Cancer Targeting Efficacy [ J ]. ACS NANO.2020,14 (3): 2557-2560 ])) and is respectively and ultrasonically dispersed in 1.5mL of deionized water, pH of the solution is regulated to 2, 3, 4, 5, 6 and 7 by 0.1M hydrochloric acid (HCl), and the change of surface potential is measured by a Markov nanoscaler, so that the pH value when the surface potential is 0 is obtained.
Example 6 Targeted analysis of the encapsulation of Heat shock protein inhibitor loaded Polydopamine drug (PDA/HI@CCM) by cancer cell membranes
Experiments were performed with murine breast cancer cells 4T1, with murine melanoma B16F-10 cells and smooth muscle cells L929 as controls. The operation steps are as follows: 4T1 cells, B16F-10 cells and L929 cells were inoculated into copolymer Jiao Min, respectively, 1X 10 per dish 5 Cells were added to each dish in 1.5mL of complete medium. After 24h of Cell culture adherence 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 and the excess material was washed away with fresh 1 XPBS, after which the nuclei and Cell Mask were stained with the host 33342 dye TM Green Plasma Membrane Stain dyeing cell membrane of living cells at 37deg.C for 10min, washing off excessive dye and flooding with fresh 1 XPBS, adding paraformaldehyde for fixing for 40min, washing off fixing solution with fresh 1 XPBS after fixing, and adding 1mL 1 XPBS solution to prevent cell adhesion. The confocal petri dishes were placed under laser confocal observation and photographs were taken. As shown in FIG. 4, the tumor targeting result is shown in blue for fluorescence of the Cell nucleus host 33342, red for fluorescence of PDA/HI@CCM, and green for Cell Mask of living Cell membrane TM Green Plasma Membrane Stain. As PDA/HI@CCM has the outer shell coating of the cell membrane of the cancer cell 4T1, the targeting of the same tumor cell can be effectively improved, and as can be seen from FIG. 4, the PDA/HI@CCM has specific binding with the 4T1 cell (stronger fluorescence intensity), and the PDA/HI@CCM has poor binding with B16F-10 cells and L929 cells (weak fluorescence intensity).
Example 7 in vitro drug release assay of Heat shock protein inhibitor loaded polydopamine (PDA/HI@CCM) encapsulated by cancer cell membranes
The in vitro drug release behavior of the nanosystems was evaluated by dialysis methods with phosphate buffer (1 x PBS) at ph=7.4 and acetate buffer (1 x ABS) at ph=5.2. The PDA/HI@CCM obtained in example 4 was filled into dialysis bags (molecular weight cut-off 1000 kDa), then added to phosphate buffer and acetate buffer, respectively, and dialyzed in a thermostatic water bath at 37℃and shaken at 160rpm to investigate the drug release behavior of the PDA/HI@CCM. The buffer was aspirated at time points 0,1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96h, respectively, 5mL of buffer was then added, the volume of buffer was kept unchanged, and the buffer removed was measured with an ultraviolet spectrophotometer and the cumulative release rate was calculated. As can be seen from fig. 5, the cumulative release of PDA/hi@ccm in acetate buffer environment at pH 5.0 is significantly higher than in 1 x PBS environment at pH 7.4. Therefore, PDA/HI@CCM can release heat shock protein inhibitor rapidly and effectively in the environment of tumor cells with acidity, and when the tumor cells are heated by illumination, the tumor cells are prevented from expressing heat shock protein, so that the killing effect of photo-thermal treatment on the tumor cells is improved.
Example 8 cytotoxicity assay of Heat shock protein inhibitor loaded polydopamine nanomaterial (PDA/HI@CCM) encapsulated by cancer cell membranes
(1) The in vitro dark toxicity of PDA/HI@CCM obtained in example 4 on 4T1 cells and L929 cells was evaluated by using the method of detecting cell activity by CCK8 method. The specific operation steps are as follows: first, 4T1 cells and L929 cells were respectively treated as 1X 10 cells 4 The density of each/well was inoculated into a 96-well plate, and then placed in a carbon dioxide incubator to culture the cells for adherence overnight (4T 1 cells were cultured in RPMI1640 medium, and L929 cells were cultured in DMEM high-sugar medium). Subsequently, the original medium was aspirated and replaced with fresh complete medium containing PDA/HI@CCM at different concentrations. No laser irradiation was used throughout the experiment. The concentration of the selected PDA/HI@CCM is based on the concentration of HI, the HI concentration ranges from 5 to 80 mug/mL (gradient arrangement: 5 mug/mL, 10 mug/mL, 20 mug/mL, 40 mug/mL, 60 mug/mL, 80 mug/mL), 3 in parallel each concentration; the pure cell group was set as a positive control group. Cells were incubated with the material in a cell incubator for 24h, after which time the medium was removed, the cells were washed twice with 1 XPBS and 100. Mu.L of fresh medium (containing 10% by volume CCK-8) was added to each well. Incubation in incubator for a period of time, and finally detection and recording of absorbance at 450nm wavelength using a microplate reader, cell viability was calculated by the following formula:
(％)＝(OD450sample-OD450background)/(OD450control-OD450background)×100％；
where OD450sample is the absorbance of the cells added to PDA/HI@CCM, OD450 control is the absorbance of the cells without material, and OD450 background is the absorbance of the blank background without material and cells.
Data are expressed as mean ± Standard Deviation (SD) based on three independent replicates.
(2) The in vitro phototoxicity of PDA/HI@CCM on 4T1 cells and L929 cells was evaluated by using the method of detecting cell activity by the CCK-8 method. The specific operation steps are as follows: first, 4T1 cells and L929 cells were treated with 1.10 4 The density of each/well was inoculated into a 96-well plate, which was then placed in a carbon dioxide incubator for culture adherence overnight. Subsequently, the original medium was aspirated and replaced with fresh complete medium containing PDA/HI@CCM at different concentrations. After 4h incubation of cells with PDA/HI@CCM, they were placed at 808nm, 1.0W/cm 2 Irradiating with near infrared light for 5min. After the irradiation was completed, the cells were returned to the cell incubator and cultured for 24 hours. After the end of the incubation, the medium containing the material was aspirated, the cells were washed twice with 1 XPBS and 100. Mu.L of fresh medium (containing 10% CCK-8) was added to each well. Incubation in incubator for 20-40min, and detection and recording of absorbance at 450nm wavelength using a microplate reader. The concentration of PDA/HI@CCM selected was based on the HI concentration, which ranged from 5-80 μg/mL (gradient set: 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL). 3 replicates were set for each concentration; wherein the pure cell group was set as a positive control group. Cell viability was calculated by the following formula:
(％)＝(OD450sample-OD450background)/(OD450control-OD450background)×100％；
where OD450sample is the absorbance of the cells added to PDA/HI@CCM, OD450 control is the absorbance of the cells without material, and OD450 background is the absorbance of the blank background without material and cells.
Data are expressed as mean ± Standard Deviation (SD) based on three independent replicates.
As shown in FIG. 6, PDA/HI@CCM had lower in vitro cytotoxicity against 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 excellent cell selectivity of PDA/hi@ccm towards (homology targeting) homotypic cancer cells 4T1 cells.
Comparing FIGS. 6 and 7, the heat shock protein inhibitor is significantly released and cytotoxicity is significantly increased under the stimulation of near infrared light.
EXAMPLE 9 in vivo anti-tumor Activity study of Heat shock protein inhibitor-loaded polydopamine nanomaterial (PDA/HI@CCM) encapsulated by cancer cell membranes
The mouse 4T1 tumor model building steps are specifically as follows: pre-cultured breast cancer cells (4T 1) were digested with pancreatin, centrifuged, resuspended in an appropriate amount of 1 XPBS, and assayed at 5X 10 5 Cell density of 100. Mu.L was inoculated with 100. Mu.L to the right hind limb of BALB/C mice (purchased from Peking Violet laboratory animal technologies Co., ltd.) and tumor size was observed daily until the tumor volume of the mice reached 100mm 3 And when the test is left and right, carrying out the next experiment.
The 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 represents the wavelength at 808nm, 1.0W/cm 2 Irradiating with near infrared light for 5min. The tail vein injection of the experiment group is based on the injection amount of 10mg/kg mice of the heat shock protein inhibitor HI. Dosing was started on the first day, every other day and light was applied once for a total of 3 treatments. At the same time, tumor volumes were recorded every two days. Tumor volume is defined as: v=w 2 L/2, wherein W and L refer to the longest and shortest diameters of the tumor, respectively.
After the treatment was completed, the observation was continued for 19 days, and then all mice were euthanized, and tumors were 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, sectioned, and then the sections were analyzed by hematoxylin-eosin (H & E) staining and photographed using an optical microscope. Tumor volume changes for each group of mice throughout the treatment period are shown in figure 8. Tumors of each group of mice isolated after the end of the whole treatment period are shown in fig. 9. As can be seen intuitively from fig. 8, both the pda+nir group and the pda@ccm+nir group show a degree of tumor suppression capacity compared to the PBS group, mainly due to EPR effect and PTT effect. As expected, the PDA/HI@CCM+NIR treatment group had the best treatment effect, even completely eliminating part of the mice' tumors, demonstrating that the PDA/HI@CCM of the invention achieves good tumor inhibition effect under laser irradiation. As shown in fig. 9, the in vitro tumor of each experimental group mice after 24 days met the expected results, confirming that PDA/hi@ccm+nir group had excellent antitumor efficacy. The results of H & E staining of the major organs (heart, liver, spleen, lung, kidney) of each group of mice are shown in fig. 10, with no pathological changes of the major organs observed in the PDA/hi@ccm+nir group compared to the control group. It was further demonstrated that the systemic toxicity of PDA/HI@CCM was negligible and that HI was greatly ameliorated by hepatotoxicity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. A nanometer photothermal therapeutic drug is characterized by comprising cancer cell membranes at the inner and outer layers of nanometer drug particles;
the nano-drug particles are nano-drug systems consisting of heat shock protein inhibitors and polydopamine nanospheres loaded with the heat shock protein inhibitors;
the cancer cell membrane is the cell membrane of a homologous tumor cell of the cancer to be treated in the logarithmic phase, and the cancer cell membrane is the breast cancer cell membrane;
the heat shock protein inhibitor is geldanamycin;
the polydopamine nanospheres are obtained by stirring dopamine hydrochloride and ammonia water in deionized water for reaction;
the polydopamine nanospheres and the heat shock protein inhibitor are respectively dissolved and dispersed by deionized water and an organic solvent to obtain polydopamine nanosphere solution and heat shock protein inhibitor solution;
the isoelectric point of polydopamine and the isoelectric point of cancer cell membranes are both between pH4 and 5;
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 1mg: 5. Mu.L;
the volume ratio of the polydopamine nanosphere solution to the heat shock protein inhibitor solution is 9:1, the mass ratio of the solute is 7:6, preparing a base material;
the concentration of the ammonia water is 25%;
the dosage of the deionized water is 1mL according to the proportion of the deionized water and the dopamine hydrochloride: 2mg calculation;
the organic solvent is dimethyl sulfoxide.
2. The method for preparing the nano photothermal therapeutic drug according to claim 1, comprising the steps of:
(1) Synthesis of polydopamine nanospheres: mixing dopamine hydrochloride and ammonia water with a solvent, stirring for reaction, centrifuging, collecting precipitate, and washing to obtain polydopamine nanospheres;
the dosage of the dopamine hydrochloride and the ammonia water is 1mg: 5. Mu.L;
the solvent is deionized water; the dosage of the solvent is 1mL according to the proportion of the solvent and the dopamine hydrochloride: 2mg calculation;
the concentration of the ammonia water is 25%;
(2) Preparation of heat shock protein inhibitor-loaded nano-drug system: mixing the heat shock protein inhibitor and the polydopamine nanospheres prepared in the step (1), stirring for reaction, and centrifuging to obtain a nano-drug system;
before mixing the heat shock protein inhibitor and the polydopamine nanospheres, respectively adopting an organic solvent and deionized water for dissolution and dispersion 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 9:1, the mass ratio of the solute is 7:6, preparing a base material;
the heat shock protein inhibitor is geldanamycin;
the organic solvent is dimethyl sulfoxide;
(3) Preparation of a heat shock protein inhibitor-loaded polydopamine drug wrapped by cancer cell membranes: blending and extruding the nano-drug system obtained in the step (2) with cancer cell membrane fragments to obtain a nano-photothermal therapeutic drug;
the dosage of the nano-drug system and the cancer cell membrane fragments is 1:1.2;
the cancer cell membrane fragments are obtained by treating the cell membranes of homologous tumor cells of cancers which grow in log phase and need to be treated.
3. The method for preparing the nanometer photothermal therapeutic agent according to claim 2, wherein,
the stirring reaction in the step (1) is carried out at the room temperature of 250 rpm-1000 rpm for 12 h-48 h;
the centrifugation in the step (1) is 8000-16000 rpm for 5-20 min;
the washing in the step (1) is to wash the precipitate to clear and transparent supernatant;
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 8000-16000 rpm for 5-20 min;
both steps (2) and (3) are carried out under the light-shielding condition.
4. The method for preparing the nanometer photothermal therapeutic agent according to claim 2, wherein,
the stirring reaction in the step (1) is carried out at 400-600 rpm for 20-28 h at 20-25 ℃;
the centrifugation in the step (1) is 10000 rpm-12000 rpm for 8 min-12 min;
stirring reaction in the step (2) is carried out at the temperature of 25 ℃ at 300-500 rpm for 20-28 h;
the centrifugation in step (2) was 12000rpm for 10min.
5. The method for preparing the nanometer photothermal therapeutic agent according to claim 2, wherein,
the extrusion in step (3) comprises the steps of: uniformly mixing the nano-drug system and cancer cell membrane fragments in proportion, and then filtering by adopting a polycarbonate microporous filter membrane to obtain the nano-photothermal therapeutic drug;
the uniform mixing is realized by vortex mixing;
the filtration by using the polycarbonate microporous filter membrane is carried out by adopting the polycarbonate microporous filter membranes with the specification of 1 mu m,0.85 mu m and 0.45 mu m in sequence, and the filtration is carried out for 8 to 15 times respectively;
the preparation method of the cancer cell membrane fragments 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 conditions.
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