CN109078183B - Bionic multifunctional nano preparation based on cancer cell membrane and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a bionic multifunctional nano preparation based on cancer cell membranes and a preparation method and application thereof, wherein the nano preparation is characterized in that an inner core is formed by chemotherapeutic drug desalted adriamycin and photothermal treatment drug indocyanine green, an outer layer is formed into a shell structure by human cervical cancer cell membranes, when the preparation is carried out, firstly, a nano particle inner core obtained by self-assembling two anti-cancer drugs (DOX, ICG) is prepared, and then, the outer layer is wrapped with the human cervical cancer cell membranes for bionic camouflage, and finally, the multifunctional nano preparation which has both chemotherapy and photothermal treatment and cancer cell targeting is obtained. The nano preparation has high biological safety and simple preparation, causes the disturbance of cell membranes under the stimulation of near infrared light, finally realizes the quick release of the medicine, and has very great application prospect.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a cancer cell membrane-based bionic multifunctional nano preparation with chemotherapy and photothermal treatment functions, and a preparation method and application thereof.
Background
Cancer causes millions of human deaths each year, and is a big killer of human health. Has important clinical significance for the research of cancer drug therapy. However, monotherapy has its inherent limitations that greatly limit its application in the clinical field. For example, chemotherapy is a common treatment for cancer, but its therapeutic effect is affected by dose-dependent toxicity. Therefore, many researches and clinical applications show that the combined treatment of a plurality of treatment methods can achieve the effect of synergistic complementation, show various advantages of high-efficiency tumor inhibition, low side effect, delayed drug resistance and the like, and have good development prospect.
Among the multiple synergistic treatment modes, the treatment mode of 'photothermal treatment + chemotherapy' can effectively kill cancer cells, and the synergistic treatment effect is achieved. Currently, a nano-drug delivery system is usually obtained by co-loading a chemotherapeutic drug and a photosensitizer into a synthetic nanocarrier. These nano-drug delivery vehicles generally have certain toxic and side effects in blood circulation, are easy to remove by reticuloendothelial, and are difficult to realize targeted therapy, and have poor therapeutic effect. In recent years, biomimetic camouflage nano-drug delivery systems have attracted attention. The drug is wrapped by the cell membrane, so that the biocompatibility is improved, the blood circulation time of the drug is prolonged, and the aggregation of a drug delivery system near cancer cells is improved. The cell membrane surface is provided with specific protein molecules which can be recognized by cancer cells, thereby achieving the aim of targeted therapy. However, achieving rapid release of cell membrane-loaded drugs remains a challenge.
Disclosure of Invention
The invention aims to provide a bionic multifunctional nano preparation based on a cancer cell membrane and a preparation method and application thereof, aiming at the technical defects in the prior art.
The technical scheme adopted for realizing the purpose of the invention is as follows:
a bionic multifunctional nanometer preparation based on cancer cell membrane comprises nanometer drug particle inner core and outer cancer cell membrane, wherein: the nano-drug particle inner core is composed of chemotherapeutic drugs and photo-thermal treatment drugs coated outside the chemotherapeutic drugs, wherein: the chemotherapy drug is adriamycin with hydrophobicity, the photothermal therapy drug is indocyanine green with amphipathy, and the cancer cell membrane is cell membrane fragments of cervical cancer cells growing in logarithmic phase.
Preferably, the nano preparation has regular spherical appearance and average particle size of 220-240 nm.
Preferably, the adriamycin is doxorubicin dihydrochloride.
In another aspect of the present invention, a method for preparing the nano-formulation is further included, which comprises the following steps:
(1) slowly dripping chemotherapeutic drug into magnetically-stirred pure water dropwise by using a micro-injector under the condition of keeping out of the sun to form nanoparticles of the chemotherapeutic drug, and stirring for 20-40 min;
(2) dissolving photo-thermal treatment drugs in water, dropwise adding the photo-thermal treatment drugs into the nanoparticle solution obtained in the step (1) by using a micro-injector under the condition of keeping out of the sun, stirring for 10-15h, and coating the photo-thermal treatment drugs on the exterior of the chemotherapy drugs to obtain nanoparticle cores, wherein the molar ratio of the photo-thermal treatment drugs to the chemotherapy drugs is 1 (2-3);
(3) extruding the nanoparticle inner core obtained in the step (2) and the cancer cell membrane fragments in a grading manner, wherein the mass ratio of the nanoparticle inner core to the cancer cell membrane fragments is 1: (3-4) mixing;
wherein the steps (1) to (3) are all carried out in a dark environment.
Preferably, the chemotherapeutic agent in step (1) is desalted hydrophobic adriamycin, the photothermal therapeutic agent in step (2) is indocyanine green, and the cancer cell membrane in step (3) is a cell membrane of human cervical cancer cells growing in log phase.
Preferably, the step (3) of fractional extrusion comprises the following steps: mixing the nanoparticle inner core and the cancer cell membrane fragments according to the mass ratio of 1: 3, uniformly mixing by vortex, standing for 30-40 minutes, and respectively filtering for 3-5 times by microporous filter membranes of 1 μm, 0.85 μm and 0.45 μm to obtain the bionic multifunctional nanoparticle preparation wrapped by the cancer cell membrane.
Preferably, the method for preparing the cancer cell membrane fragments in step (3) comprises the steps of: breaking cell membrane by hypotonic extraction method to remove inclusion to obtain cell membrane fragment, ultrasonic processing to obtain uniform nanometer cancer cell membrane fragment, and placing in ice bath.
Preferably, the cancer cell membrane in step (3) is extracted by hypotonic method, which comprises the following steps: taking human cervical carcinoma cells growing in a logarithmic phase, scraping the cells by using a cell scraper, centrifuging 700g to collect the cells, washing by using isotonic precooled PBS, centrifuging, removing supernate, adding hypotonic solution into the cell solution for ice bath for 15-20 minutes, repeatedly freezing and thawing the cell solution subjected to ice bath in liquid nitrogen at room temperature until the cells are completely crushed, centrifuging 700g to obtain supernate, centrifuging 14000g to obtain sediment which is cell membrane fragments, and freeze-drying and storing the cell membrane fragments at-80-90 ℃.
Preferably, the preparation method of the bionic multifunctional nano preparation comprises the following steps:
adding 5mg of doxorubicin hydrochloride into a 10mL sample bottle, carrying out light-shielding treatment, adding 4.5mL of dimethyl sulfoxide solution to fully dissolve the doxorubicin hydrochloride, adding 0.5mL of triethylamine under the condition of magnetic stirring, adjusting the rotation speed of magnetons to 600rpm, and reacting overnight. The product obtained was desalted doxorubicin.
mu.L of the desalted Doxorubicin (DOX) was slowly added dropwise into 5mL of pure water magnetically stirred with a micro-syringe in the dark to form doxorubicin nanoparticles. After 30 minutes, 200. mu.L of indocyanine green solution (1mg/mL) (ICG) dissolved in water was added dropwise to the above nanoparticle solution by the same method overnight with stirring to obtain nanoparticle cores (DINPs).
The hypotonic extraction method of cancer cell membrane comprises the following steps: cell membrane protein and cytoplasm protein extraction kit are adopted to extract cell membrane. Taking human cervical carcinoma cells growing in the logarithmic phase, scraping the cells by using a cell scraper, centrifuging 700g for 5 minutes, collecting the cells, washing by using isotonic precooled PBS (pH 7.4), centrifuging, removing supernatant, and adding hypotonic solution to stand for 15 minutes under the condition of ice bath. The cell solution was repeatedly frozen and thawed in liquid nitrogen at room temperature until the cells were completely disrupted. Subsequently, the supernatant was carefully obtained by centrifugation at 700g for 10 minutes, and the pellet was obtained by centrifugation at 14000g for 30 minutes, i.e., cell membrane fragments (CMs). The cell membrane fragments were lyophilized at-80 deg.C for storage.
The encapsulation fusion of the nanoparticle and the cancer cell membrane fragment comprises the following steps: dissolving the extracted cancer cell membrane fragments in pure water, and carrying out ice-bath ultrasonic treatment by an ultrasonic cell disruptor to obtain uniformly dispersed nano-scale cell membrane fragments. And then, mixing the nanoparticles with the cancer cell membrane fragments according to the mass ratio of 1: 3, evenly mixing by vortex, standing for 30 minutes, and then respectively filtering for 3 times by microporous filter membranes with the diameters of 1 micron, 0.85 micron and 0.45 micron to obtain the cancer cell membrane coated bionic multifunctional nanoparticles (DICNPs).
On the other hand, the invention also comprises the application of the bionic multifunctional nano preparation in preparing anti-cancer drugs.
In the above scheme, the drop-by-drop addition of DOX to water to form nanoparticles is driven by hydrophobic forces. The ICG is coated on the outer layer of the DOX nano particle because the ICG is an amphiphilic material, the hydrophobic layer is coated inside, and the hydrophilic layer is coated outside.
Compared with the prior art, the invention has the beneficial effects that:
1. the multifunctional nano preparation for simulating the cancer cell membrane has the effect of the synergistic treatment of chemotherapy and photothermal therapy, has good biocompatibility and degradability due to the fact that the cancer cell membrane is wrapped by the outer layer, can effectively avoid the phagocytosis of a reticuloendothelial system, and has a high targeting effect on the same kind of cancer cells.
2. The inner layer is a compound of two drugs, DOX is a chemotherapeutic drug, ICG is a photo-thermal therapeutic drug, so that the synergistic therapeutic effect of chemotherapy and photo-thermal therapy can be realized, and meanwhile, the photo-thermal responsiveness of ICG can accelerate the release speed of the DOX drug.
3. The nano preparation has good biocompatibility and the capability of actively targeting cancer cells, realizes the capability of chemotherapy and photothermal therapy cooperative treatment, stimulates the drug release process by near infrared light (NIR), ensures that the drug is exploded at the cancer cell part, improves the treatment effect to the maximum extent, and has important significance in the cancer treatment process.
Drawings
FIG. 1 TEM image of cancer cell membrane-based biomimetic multifunctional nano-formulation DICNPs of the present invention.
FIG. 2 shows the particle size of DICNPs based on the multifunctional nano-preparations for cancer cell membrane simulation.
FIG. 3 shows the targeting analysis (scale: 25 μm) of the cancer cell membrane-based biomimetic multifunctional nano-preparation DICNPs of the present invention.
FIG. 4 is the in vitro drug release diagram of the cancer cell membrane-based biomimetic multifunctional nano-preparation DICNPs of the present invention.
FIG. 5 shows the analysis of the cell dark toxicity of the cancer cell membrane-based biomimetic multifunctional nano-preparation DICNPs.
FIG. 6 is a cytophototoxicity analysis of cancer cell membrane-based biomimetic multifunctional nano-formulation DICNPs.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Doxorubicin hcl and indocyanine green were purchased from gangrenum biotechnology limited, and cell membrane protein and cytosolic protein extraction kits were purchased from piceatian biotechnology limited.
(1) Hydrochloric acid removal treatment of doxorubicin hydrochloride: 5mg of doxorubicin hydrochloride was weighed into a 10mL sample bottle and treated with light, 4.5mL of dimethyl sulfoxide (DMSO) solution was added to dissolve doxorubicin hydrochloride sufficiently, and 0.5mL of triethylamine was added under magnetic stirring (600rpm, 37 ℃ C.) for overnight reaction. The product obtained was desalted doxorubicin.
(2) Preparing a nanoparticle core: mu.L of the desalted Doxorubicin (DOX) was slowly added dropwise into 5mL of pure water magnetically stirred with a micro-syringe under protection from light to form doxorubicin nanoparticles (DOX NPs). After 30 minutes, 200. mu.L of indocyanine green solution (1mg/mL) (ICG) dissolved in water was dropped into the above nanoparticle solution with a micro-syringe under protection from light overnight with stirring to obtain nanoparticle cores (DINPs).
(3) Extracting cancer cell membranes: extracting by hypotonic method, and extracting cell membrane by cell membrane protein and cytoplasm protein extraction kit. In order to prevent the change of the membrane protein caused by the protease contained or adsorbed on the membrane, the following operation should be performed at a low temperature of 4 ℃. Taking human cervical carcinoma cells growing in the logarithmic phase, scraping the cells by a cell scraper, centrifuging for 5min at 700g, collecting cell precipitates, adding isotonic precooled PBS (pH 7.4) for resuspension, centrifuging for 5min at 600g, removing supernatant, centrifuging for 1min at 600g, precipitating residual liquid on the wall of the centrifuge tube and further precipitating the cells, and completely sucking up the residues as far as possible. Adding hypotonic solution, and standing for 15min under ice bath condition. The cell solution was repeatedly frozen and thawed in liquid nitrogen several times at room temperature until the cells were completely disrupted. Subsequently, the supernatant was carefully obtained by centrifugation at 700g for 10 minutes to remove nuclei, organelles and unbroken cells. Finally, 14000g was centrifuged for 30 minutes to obtain a pellet, which was cell membrane debris (CMs). The cell membrane fragments were lyophilized at-80 deg.C for storage.
(4) Preparing the cancer cell membrane-based bionic multifunctional nano preparation DICNPs: dissolving the extracted cancer cell membrane fragments in pure water, and carrying out ice-bath ultrasonic treatment by an ultrasonic cell disruptor to obtain uniformly dispersed nano-scale cell membrane fragments. And then, mixing the nanoparticles with the cancer cell membrane fragments according to the mass ratio of 1: 3, evenly mixing by vortex, standing for 30 minutes, and then respectively filtering for 3 times by microporous filter membranes with the diameters of 1 micron, 0.85 micron and 0.45 micron to obtain the cancer cell membrane coated bionic multifunctional nanoparticles (DICNPs). The morphology is characterized by TEM as in figure 1. The bionic carrier can be observed to have a regular spherical structure, uniform size and a particle size of about 220 nm. The results of measuring the particle diameter and Zeta potential of the nanoparticles by a laser nanometer particle size analyzer are shown in FIG. 2.
Based on the targeting characterization of the cancer cell membrane bionic multifunctional nano preparation DICNPs, monkey-derived renal cell COS 7 cells and smooth muscle cell L929 cells are used as controls, and the operation steps are as follows: HeLa cells, COS 7 cells and L929 cells were seeded in confocal dishes at 1X 10 cells/dish51mL of medium per dish. Adding DICNPs after 24h of cell culture and adherence, continuing to culture for 2h, then sucking off liquid and washing cells with PBS, staining nuclei with Hochest 33342 dye for 15min, washing with PBS, then adding paraformaldehyde for fixing for 30min, washing, and finally adding 1mL of PBS solution. The confocal culture dish is placed under laser confocal observation and photographs are taken.
The tumor targeting result is shown in fig. 3, blue represents fluorescence of cell nucleus Hochest 33342, red represents fluorescence of DOX, and green represents fluorescence of ICG, because DICNPs are coated by the shell of the cancer cell membrane, the nanoparticles can effectively improve targeting of the same tumor cells, the specific binding (strong fluorescence intensity) of the DICNPs and HeLa cells can be seen, the binding (weak fluorescence intensity) of the DICNPs with COS 7 cells and L929 cells is poor, in addition, the fluorescence intensity of an experimental group (DICNPs + NIR) of the DICNPs under near infrared light is stronger than that of an experimental group (DICNPs) which does not provide near infrared light, and the near infrared light (NIR) has the effect of stimulating drug release.
The in-vitro drug release behavior of the cancer cell membrane-based bionic multifunctional nano preparation DICNPs is as follows: the in-vitro drug release behavior of the nano system is evaluated by a dialysis method, and the release medium is PBS phosphate buffer solution with pH 7.4. In a 37 ℃ constant temperature water bath kettle, oscillating at 160rpm to carry out the medicament release behaviors of DINPs, DINPs + NIR, DICNPs and DICNPs + NIR, respectively replacing equal amount of PBS solution at time points of 0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60 and 72h, measuring the taken out medium by a fluorescence spectrophotometer (EX:480nm, EM:560nm) and calculating the cumulative release rate. As can be seen from FIG. 4, the drug release of the nanoparticles wrapping the cancer cell membrane is significantly slowed, while the drug release wrapping the cell membrane and applying near-infrared illumination is significantly enhanced.
Cytotoxicity analysis based on cancer cell membrane bionic multifunctional nano preparation DICNPs: cytotoxicity was determined by MTT method. HeLa cells at 6X 103The density of each cell per well was seeded in 96-well plates. Before adding the material, the medium was cultured in 100. mu.L of DMEM medium containing 10% FBS for 24 hours. Then DOX, ICG, DINPs and DICNPs are added in different concentrations. One plate was illuminated and the other plate was protected from light, and after co-culturing with the cells for 24 hours, the original medium was replaced with 200. mu.L of fresh DMEM medium containing 10% FBS, 20. mu.L of MTT (5mg/mL in PBS buffer) solution was added to each well, culturing was continued in an incubator at 37 ℃ for 4 hours, the medium was discarded, 150. mu.L of DMSO was added to each well, and the absorbance intensity of the solution at 570nm was measured on a microplate reader. The relative survival rate of the cells was calculated as follows: cell viability (%) (OD570(sample)/OD570(Control)) x 100%, where OD570(Control) and OD570(sample) represent absorbance of the solution without and with sample treatment, respectively. The values obtained are the mean of the values obtained in three replicates, the values being shown as mean ± Standard Deviation (SD). Compared with the images shown in fig. 5-6, the DICNPs have smaller cytotoxicity when the NIR is not added, and the cell survival rate of the DICNPs group is obviously reduced after the NIR illumination, which shows that the cell membranes have better coating effect on the DINPs, and the drugs are obviously released and the cytotoxicity is obviously increased under the stimulation of the near-infrared illumination.
The invention has been described in an illustrative manner, and it is to be understood that the invention is not limited to the precise details set forth herein. It should be noted that any simple variation, modification or other equivalent substitution by a person skilled in the art without any inventive step shall fall within the scope of protection of the present invention, without departing from the inventive concept. Therefore, the protection scope of the patent of the invention should be subject to the attached claims.
Claims (9)
1. A bionic multifunctional nanometer preparation based on cancer cell membranes is characterized by consisting of an inner core of nanometer drug particles and an outer cancer cell membrane, wherein: the nano-drug particle inner core is composed of chemotherapeutic drugs and photo-thermal treatment drugs coated outside the chemotherapeutic drugs, wherein: the chemotherapy drug is adriamycin with hydrophobicity, the photothermal therapy drug is indocyanine green with amphipathy, and the cancer cell membrane is cell membrane fragments of cervical cancer cells growing in logarithmic phase;
the bionic multifunctional nano preparation is prepared by the following method:
(1) slowly dripping chemotherapeutic drug into magnetically-stirred pure water dropwise by using a micro-injector under the condition of keeping out of the sun to form nanoparticles of the chemotherapeutic drug, and stirring for 20-40 min;
(2) dissolving photo-thermal treatment drugs in water, dropwise adding the photo-thermal treatment drugs into the nanoparticle solution obtained in the step (1) by using a micro-injector under the condition of keeping out of the sun, stirring for 10-15h, and coating the photo-thermal treatment drugs on the exterior of the chemotherapy drugs to obtain nanoparticle cores, wherein the molar ratio of the photo-thermal treatment drugs to the chemotherapy drugs is 1 (2-3);
(3) extruding the nanoparticle inner core obtained in the step (2) and the cancer cell membrane fragments in a grading manner, wherein the mass ratio of the nanoparticle inner core to the cancer cell membrane fragments is 1: (3-4) mixing;
wherein the steps (1) to (3) are all carried out in a dark environment.
2. The multifunctional biomimetic nano preparation based on cancer cell membranes as recited in claim 1, wherein the nano preparation has a regular spherical appearance with an average particle size of 220-240 nm.
3. The biomimetic multifunctional nano preparation based on cancer cell membranes according to claim 1, wherein the doxorubicin is doxorubicin dihydrochloride.
4. The biomimetic multifunctional cancer cell membrane-based nano-formulation according to claim 1, wherein the chemotherapeutic agent in step (1) is desalted hydrophobic adriamycin, the photothermal therapeutic agent in step (2) is indocyanine green, and the cancer cell membrane in step (3) is a cell membrane of a human cervical cancer cell grown in a logarithmic phase.
5. The biomimetic multifunctional nano preparation based on cancer cell membranes as claimed in claim 1, wherein the step (3) of step-extrusion comprises the steps of: mixing the nanoparticle inner core and the cancer cell membrane fragments according to the mass ratio of 1: 3, uniformly mixing by vortex, standing for 30-40 minutes, and respectively filtering for 3-5 times by microporous filter membranes of 1 μm, 0.85 μm and 0.45 μm to obtain the bionic multifunctional nanoparticle preparation wrapped by the cancer cell membrane.
6. The biomimetic multifunctional nano preparation based on cancer cell membranes as claimed in claim 1, wherein the preparation method of the cancer cell membrane fragments in the step (3) comprises the following steps: breaking cell membrane by hypotonic extraction method to remove inclusion to obtain cell membrane fragment, ultrasonic processing to obtain uniform nanometer cancer cell membrane fragment, and placing in ice bath.
7. The biomimetic multifunctional nano preparation based on cancer cell membranes as claimed in claim 1, wherein the cancer cell membranes in the step (3) are extracted by hypotonic method, comprising the following steps: taking human cervical carcinoma cells growing in a logarithmic phase, scraping the cells by using a cell scraper, centrifuging 700g to collect the cells, washing by using isotonic precooled PBS, centrifuging, removing supernate, adding hypotonic solution into the cell solution for ice bath for 15-20 minutes, repeatedly freezing and thawing the cell solution subjected to ice bath in liquid nitrogen at room temperature until the cells are completely crushed, centrifuging 700g to obtain supernate, centrifuging 14000g to obtain sediment which is cell membrane fragments, and freeze-drying and storing the cell membrane fragments at-80-90 ℃.
8. The biomimetic multifunctional nano preparation based on cancer cell membranes according to claim 1, wherein the preparation method of the biomimetic multifunctional nano preparation comprises the following steps:
adding 5mg of doxorubicin hydrochloride into a 10mL sample bottle, carrying out light-shielding treatment, adding 4.5mL of dimethyl sulfoxide solution to fully dissolve the doxorubicin hydrochloride, adding 0.5mL of triethylamine under the condition of magnetic stirring, adjusting the rotation speed of magnetons to 600rpm, and carrying out overnight reaction to obtain a product, namely desalted doxorubicin;
dropwise and slowly adding 100 mu L of the desalted adriamycin into 5mL of pure water subjected to magnetic stirring by using a micro-syringe under the condition of keeping out of the sun to form adriamycin nanoparticles, and after 30 minutes, dropwise adding 200 mu L of indocyanine green solution dissolved in water into the nanoparticle solution by using the same method for overnight stirring, wherein the concentration of the indocyanine green solution is 1mg/mL, so as to obtain nanoparticle cores;
the hypotonic extraction method of cancer cell membrane comprises the following steps: extracting cell membranes by using a cell membrane protein and cytoplasm protein extraction kit, taking human cervical cancer cells growing in a logarithmic phase, scraping the cells by using a cell scraper, centrifuging 700g for 5 minutes to collect the cells, washing by using isotonic precooled PBS (PBS), wherein the pH of the PBS is 7.4, centrifuging, discarding supernatant, adding hypotonic solution, standing for 15 minutes under the condition of ice bath, repeatedly freezing and thawing the cell solution in liquid nitrogen at room temperature until the cells are completely crushed, centrifuging 700g for 10 minutes to obtain supernatant, centrifuging 14000g for 30 minutes to obtain precipitate, namely cell membrane fragments, and freeze-drying and storing the cell membrane fragments at-80 ℃;
the encapsulation fusion of the nanoparticle and the cancer cell membrane fragment comprises the following steps: dissolving the extracted cancer cell membrane fragments in pure water, performing ice-bath ultrasonic treatment by using an ultrasonic cell disruptor to obtain uniformly dispersed nano-scale cell membrane fragments, and then mixing the nano-particles and the cancer cell membrane fragments according to a mass ratio of 1: 3, uniformly mixing by vortex, standing for 30 minutes, and then respectively filtering for 3 times by microporous filter membranes with the diameters of 1 micron, 0.85 micron and 0.45 micron to obtain the bionic multifunctional nano-particles wrapped by the cancer cell membranes.
9. The use of the biomimetic multifunctional nano-formulation based on cancer cell membrane according to claim 1 in the preparation of anticancer drugs.
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