CN112402394A - BCG complex adsorbing nano-drug carrier by utilizing positive and negative charge characteristics and preparation method thereof - Google Patents
BCG complex adsorbing nano-drug carrier by utilizing positive and negative charge characteristics and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a BCG vaccine complex which adsorbs a nano-drug carrier by utilizing the characteristics of positive and negative charges, and the complex is a complex structure which realizes the mutual combination between the nano-drug carrier with positive charges and BCG thalli by the mutual attraction of the positive and negative charges; wherein, the nano-drug carrier is a nano-particle which is coated by polylactic acid-glycolic acid copolymer, surface modification is carried out on cationic polymer polyethyleneimine, and the particle size is 80nm-100 nm; the average number of the nanoparticles is 10-20 per BCG thallus surface. The preparation method comprises the following steps: the cationic nano-drug carrier is synthesized by a polymeric material water/oil/water double-emulsification method, and the BCG is combined with the cationic nano-drug carrier. The method has good adsorption and absorption effects on the bladder epithelium by the BCG, loads the drug particles on the surface of the BCG thallus, utilizes the BCG as a carrier, enhances the uptake and tumor targeting of the nano drug carrier in the bladder, and can obviously improve the tumor killing effect of the drug.
Description
Technical Field
The invention belongs to the field of nano-drug carriers and tumor immunity, and particularly relates to preparation of a BCG complex adsorbed with the nano-drug carriers.
Background
Bladder cancer is the second most common malignancy of the urinary system, with non-muscle-layer-infiltrating patients (NMIBC) accounting for over 70% of total bladder cancer patients at first diagnosis. The high recurrence rate and progression rate of tumors are the most important factors affecting the prognosis of patients with bladder cancer. Intravesical BCG Vaccine (BCG Vaccine) perfusion is a treatment mode recommended after the operation of clinical high-risk NMIBC patients and is widely applied clinically. BCG perfusion in the bladder can kill operation residual or tiny tumor focus by inducing body local to generate strong immune response, thereby obviously reducing the recurrence and the progression of cancer and improving the prognosis of patients. Although the immunotherapeutic effect of BCG was significant, there still existed a portion of patients who were not sensitive to BCG treatment. Therefore, BCG perfusion-based combination therapies are gaining increasing attention. Clinical research results show that the combined intravesical perfusion of BCG and chemotherapeutic drugs (such as gemcitabine and the like) and immunomodulators (such as PD-1/PD-L1 monoclonal antibody drugs and the like) can further improve the prognosis of patients and improve the disease-free survival rate of the patients.
The nano-drug carrier is used for loading different types of drugs into nano-grade particles, so that the absorption and circulation time of the drugs in vivo can be improved, and the drug effect can be improved. Different from intravenous administration, the intravesical perfusion of the medicine can directly reach the local part of the tumor, and the serious side effect of systemic administration is greatly weakened. However, the biggest problem of the perfusion administration to the bladder is that the retention time of the drug in the bladder is short and the absorption of the drug is insufficient. Therefore, how to increase the retention time of the nano-drug carrier in the bladder and enhance the intratumoral targeting effect of the drug is a problem to be solved at present.
Disclosure of Invention
Aiming at the prior art, the invention provides a preparation method of BCG adsorption nano-drug carrier, which is characterized in that BCG has good adhesion and absorption effects on bladder epithelium, drug particles are loaded on the surface of BCG thallus, the BCG is used as a carrier, the uptake and tumor targeting of the nano-drug carrier in the bladder are enhanced, and the tumor killing effect of the drug can be obviously improved. Meanwhile, the adhesion of the BCG loaded with the medicine in the bladder can stimulate the local and systemic immunoreaction of the bladder to kill tumor cells, thereby realizing the combined treatment mode of local immunotherapy and chemotherapy.
In order to solve the technical problems, the invention provides a BCG complex which utilizes the characteristics of positive and negative charges to adsorb a positive nano-drug carrier, and the complex is a complex structure which realizes mutual combination by the mutual attraction of the positive and negative charges between the nano-drug carrier with the positive charges and the BCG thallus; wherein, the nano-drug carrier is a nano-particle which is coated by polylactic acid-glycolic acid copolymer, surface modification is carried out on cationic polymer polyethyleneimine, and the particle size is 80nm-100 nm; in the complex, each BCG bacterial surface is combined with 10-20 nano particles.
The preparation method of the BCG complex mainly comprises the following steps: synthesizing a cationic nano-drug carrier by using a polymeric material water/oil/water double-emulsification method, and combining the BCG with the cationic nano-drug carrier to obtain the BCG complex combined with the nano-drug carrier.
The invention relates to a preparation method of a BCG complex adsorbing a positive nano-drug carrier by utilizing the characteristics of positive and negative charges, which comprises the following steps:
when the cationic nano-drug carrier is synthesized by a polymeric material water/oil/water double-emulsification method, when the anticancer drug is dissolved in a corresponding soluble organic solvent, the synthesis method comprises the following specific steps:
dissolving a proper amount of polylactic acid-glycolic acid copolymer in dichloromethane to prepare a dichloromethane solution of the polylactic acid-glycolic acid copolymer with the mass volume concentration of 10mg/ml, and marking as a solution A; dissolving a proper amount of anticancer drug in a corresponding soluble organic solvent to prepare an organic solvent solution of the anticancer drug with the mass volume concentration of 10mg/ml, and marking as a solution B; according to the volume ratio of 9: 1, mixing the solution A and the solution B, and carrying out ultrasonic oscillation for 30min at normal temperature in an ultrasonic water bath to fully mix the solution A and the solution B to obtain a mixed solution C; according to the volume ratio of 1: 1, slowly adding the mixed solution C into a polyvinyl alcohol solution with the mass percent of 0.5%, and carrying out ultrasonic treatment for 2min by using a probe ultrasonic homogenizer with the power of 10-30W and the frequency of 20KHz to obtain a primary emulsion; according to the volume ratio of 1: 3, mixing the primary emulsion with 0.5 percent polyvinyl alcohol solution again, and performing ultrasonic treatment for 10min by using a probe ultrasonic homogenizer at the power of 50-80W and the frequency of 20KHz to obtain double emulsion; immediately, a 10% polyethyleneimine solution in percentage by mass is dropwise added into the double emulsion, and the obtained mixed solution is marked as a mixed solution D, wherein the volume ratio of the polyethyleneimine solution to the solution C is 1: 10; placing the mixed solution D in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, and centrifuging at 12000rpm for 10min to obtain a precipitate; washing the precipitate with double distilled water for 2 times to obtain positively charged adriamycin nanoparticles coated with polylactic acid-glycolic acid copolymer; finally, the mixture was diluted to a concentration of 4.5X 10 with phosphate buffer10～5.5×1010Per ml of nanoparticle suspension.
When the cationic nano-drug carrier is synthesized by a polymeric material water/oil/water double-emulsification method, the anticancer drug is an anticancer drug which is not easy to dissolve in an organic solvent but has good water solubility, and the synthesis method comprises the following specific steps:
dissolving a proper amount of polylactic acid-glycolic acid copolymer in dichloromethane to prepare the polylactic acid-glycolic acid copolymer with the mass volume concentration of10mg/ml of a dichloromethane solution of polylactic acid-glycolic acid copolymer is marked as solution A; dissolving a proper amount of anticancer drug in double distilled water, wherein the anticancer drug is not easy to dissolve in an organic solvent but has good water solubility, and preparing an aqueous solution of the anticancer drug with the mass volume concentration of 10mg/ml, and marking the aqueous solution as a solution B; according to the volume ratio of 1: 1, mixing the solution A and the solution B, and carrying out ultrasonic treatment for 2min by using a probe ultrasonic homogenizer at the power of 10-30W and the frequency of 20KHz to obtain a primary emulsion; according to the volume ratio of 1: 3, mixing the primary emulsion with 0.5 mass percent polyvinyl alcohol solution, and performing ultrasonic treatment for 10min by using a probe ultrasonic homogenizer at the power of 50-80W and the frequency of 20KHz to obtain double emulsion; immediately, a 10% polyethyleneimine solution in percentage by mass is dropwise added into the double emulsion, and the obtained mixed solution is marked as a mixed solution D, wherein the volume ratio of the polyethyleneimine solution to the solution a is 1: 10; placing the mixed solution D in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, and centrifuging at 12000rpm for 10min to obtain a precipitate; washing the precipitate with double distilled water for 2 times to obtain positively charged adriamycin nanoparticles coated with polylactic acid-glycolic acid copolymer; finally, the mixture was diluted to a concentration of 4.5X 10 with phosphate buffer10～5.5×1010Per ml of nanoparticle suspension.
The combination of BCG vaccine and cationic nano-drug carrier comprises the following specific contents:
taking BCG vaccine bacterial liquid in logarithmic growth phase, and adjusting the concentration of the bacterial liquid to 108CFU/ml, centrifuging at 5000rpm for 10min, discarding the culture medium, washing the precipitate obtained by centrifugation with phosphate buffer solution for three times, and isovolumetrically resuspending the BCG precipitate; and adding the nanoparticle suspension prepared in the step into the resuspended BCG bacterial liquid, wherein the volume ratio of the nanoparticle suspension to the BCG bacterial liquid is 1:5, shaking the mixed liquid for 1H on a shaking table at 37 ℃, centrifuging for 10min at 5000rpm, removing the unbound particle part, and washing the obtained precipitate twice by using a phosphate buffer solution to obtain the product, namely the BCG complex combined with the positive nano-drug carrier.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method can successfully prepare various different drug molecules into the nano drug carrier, and realizes the direct combination with BCG bacteria to form a complex of the nano drug carrier and BCG, namely BCG-nanoparticles (BCG-NPs). The most remarkable advantages of BCG-NPs are that the intratumoral transport and absorption of the medicine can be improved, and the effect of single BCG perfusion treatment is enhanced. The BCG-based nano drug carrier has good adsorption and absorption effects on bladder epithelium, drug particles are loaded on the surface of BCG thallus, and BCG is used as a carrier to form a complex of BCG and the nano drug carrier, so that the uptake and tumor targeting of the nano drug carrier in the bladder are enhanced.
Drawings
FIG. 1 is a schematic diagram of the present invention for achieving adsorption of nano-drug carriers with BCG by positive and negative charge attraction;
FIG. 2 is a low power transmission electron micrograph of positively charged PLGA coated DOX nanoparticles made according to the present invention;
FIG. 3 is a high power transmission electron micrograph of positively charged PLGA coated DOX nanoparticles made according to the present invention;
FIG. 4 is the scanning electron microscope image of BCG complex adsorbed with nano-drug carrier prepared by the present invention.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, which are not intended to limit the invention in any way.
The BCG complex which utilizes the characteristics of positive and negative charges and adsorbs a positive nano-drug carrier is designed according to the idea that the complex is a complex structure which realizes mutual combination by the mutual attraction of the positive and negative charges between the nano-drug carrier with the positive charges and the BCG thallus; wherein, the nano-drug carrier is a spherical nano-particle which is coated by polylactic acid-glycolic acid copolymer (PLGA), surface modification is carried out on cationic polymer Polyethyleneimine (PEI), and the particle size is 80nm-100 nm; in the complex, the surface of each BCG thallus has 10-20 nanoparticles combined on average. The preparation of the composite mainly comprises the following steps: synthesizing a cationic nano-drug carrier by using a polymeric material water/oil/water double-emulsification method, and combining BCG with the cationic nano-drug carrier to obtain the BCG complex adsorbed with the nano-drug carrier.
Fig. 1 shows the synthesis route of BCG compound with adsorbed positive nano-drug carrier of the present invention, first using PLGA as the outer shell, coating the various drug molecules into spherical nano-particles. And then carrying out surface polymerization on the cationic polymer PEI on the particle surface, so that the whole nano-drug carrier has positive charges. Because the normal BCG bacteria surface has certain negative charges, the positive nano-drug carrier can be successfully adsorbed to the BCG bacteria surface by utilizing the characteristic that the positive charges and the negative charges are mutually attracted.
Preparing the BCG complex absorbed with the nano-drug carrier according to the following steps:
1) synthesizing a cationic nano-drug carrier by using a polymeric material water/oil/water double-emulsification method:
dissolving a proper amount of polylactic acid-glycolic acid copolymer in dichloromethane to prepare a dichloromethane solution of the polylactic acid-glycolic acid copolymer with the mass volume concentration of 10mg/ml, and marking as a solution A; dissolving a proper amount of anticancer drug in a corresponding soluble organic solvent to prepare an organic solvent solution of the anticancer drug with the mass volume concentration of 10mg/ml, and marking as a solution B; according to the volume ratio of 9: 1, mixing the solution A and the solution B, and carrying out ultrasonic oscillation for 30min at normal temperature in an ultrasonic water bath to fully mix the solution A and the solution B to obtain a mixed solution C; according to the volume ratio of 1: 1, slowly adding the mixed solution C into a polyvinyl alcohol solution with the mass percent of 0.5%, and carrying out ultrasonic treatment for 2min by using a probe ultrasonic homogenizer with the power of 10-30W and the frequency of 20KHz to obtain a primary emulsion; according to the volume ratio of 1: 3, mixing the primary emulsion with 0.5 percent polyvinyl alcohol solution again, and performing ultrasonic treatment for 10min by using a probe ultrasonic homogenizer at the power of 50-80W and the frequency of 20KHz to obtain double emulsion; immediately, a 10% polyethyleneimine solution in percentage by mass is added dropwise to the double emulsion, and the obtained mixed solution is marked as a mixed solution D, wherein the mixed solution is prepared from the polyethyleneimine solution and the solution CThe volume ratio is 1: 10; placing the mixed solution D in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, and centrifuging at 12000rpm for 10min to obtain a precipitate; washing the precipitate with double distilled water for 2 times to obtain positively charged adriamycin nanoparticles coated with polylactic acid-glycolic acid copolymer; finally, the mixture was diluted to a concentration of 4.5X 10 with phosphate buffer10～5.5×1010Per ml of nanoparticle suspension.
In the invention, the positive charge nanoparticles of the cationic polymer PEI are modified on the surface, obtained in the step 1), and the potential is increased along with the increase of the added PEI amount. In addition, for the drugs which are insoluble in DMSO and soluble in other types of organic solvents, the preparation scheme of the nano drug carrier is that DMSO in the step is replaced by the corresponding soluble organic solvent, and other steps are the same. For example: the anticancer drug is adriamycin, and the organic solvent which is correspondingly soluble to the adriamycin is dimethyl sulfoxide. The anti-cancer drug is paclitaxel, and the organic solvent which is soluble correspondingly to the paclitaxel is acetone; the anti-cancer drug is mitomycin, and the corresponding soluble organic solvent of mitomycin is dimethylacetamide; the anticancer drug is cisplatin, and the corresponding soluble organic solvent of the cisplatin is dimethylformamide. Although specific classes of the above-mentioned drugs and the corresponding soluble organic solvents are listed, the present invention is not limited to the above.
Particularly, for drugs which are good in water solubility but not easy to dissolve in various common organic solvents, the preparation method of the nano drug carrier is as follows: dissolving a proper amount of polylactic acid-glycolic acid copolymer in dichloromethane to prepare a dichloromethane solution of the polylactic acid-glycolic acid copolymer with the mass volume concentration of 10mg/ml, and marking as a solution A; dissolving a proper amount of anticancer drug in double distilled water, wherein the anticancer drug is not easy to dissolve in an organic solvent but has good water solubility, and preparing an aqueous solution of the anticancer drug with the mass volume concentration of 10mg/ml, and marking the aqueous solution as a solution B; according to the volume ratio of 1: 1, mixing the solution A and the solution B, and carrying out ultrasonic treatment for 2min by using a probe ultrasonic homogenizer at the power of 10-30W and the frequency of 20KHz to obtain a primary emulsion; according to volume ratioIs 1: 3, mixing the primary emulsion with 0.5 mass percent polyvinyl alcohol solution, and performing ultrasonic treatment for 10min by using a probe ultrasonic homogenizer at the power of 50-80W and the frequency of 20KHz to obtain double emulsion; immediately, a 10% polyethyleneimine solution in percentage by mass is dropwise added into the double emulsion, and the obtained mixed solution is marked as a mixed solution D, wherein the volume ratio of the polyethyleneimine solution to the solution a is 1: 10; placing the mixed solution D in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, and centrifuging at 12000rpm for 10min to obtain a precipitate; washing the precipitate with double distilled water for 2 times to obtain positively charged adriamycin nanoparticles coated with polylactic acid-glycolic acid copolymer; finally, the mixture was diluted to a concentration of 4.5X 10 with phosphate buffer10～5.5×1010Per ml of nanoparticle suspension.
2) The specific steps of the combination of BCG and cationic nano-drug carrier are as follows: taking BCG bacterial liquid in logarithmic growth phase, and adjusting the concentration of the bacterial liquid to 108Centrifuging at 5000rpm for 10min at CFU/ml, discarding the culture medium, washing the centrifuged bacteria precipitate with PBS for three times, and isovolumetrically resuspending the BCG bacteria precipitate with PBS; adding the nanoparticle suspension prepared in the step 1) into the resuspended BCG bacterial liquid, wherein the volume ratio of the nanoparticle suspension to the BCG bacterial liquid is 1:5, shaking the mixed liquid for 1H on a shaking table at 37 ℃, collecting the bacterial liquid, centrifuging for 10min at 5000rpm, removing the part of the unbound nano-drug carrier, washing the obtained precipitate twice with PBS buffer solution, and obtaining the final product, namely the BCG complex bound with the positive nano-drug carrier.
Example (b): taking clinical common chemotherapy drug adriamycin (Dox) as an example, the BCG compound adsorbing the positive Dox nano drug carrier is prepared by the following method:
1) preparation of positively charged PLGA-coated DOX nanoparticles:
weighing 10mg of organic material polylactic-co-glycolic acid (PLGA) and dissolving the PLGA in 1ml of Dichloromethane (DCM) organic phase to prepare PLGA-DCM solution; Dox-DMSO solutions were prepared by weighing 1mg of doxorubicin (Dox) and dissolving in 0.1ml of dimethyl sulfoxide (DMSO). 0.1ml of Dox-DMSO solution is added into 0.9ml of PLGA-DCM solution, and ultrasonic oscillation is carried out for 30min at normal temperature under ultrasonic water bath to ensure that the mixture is fully mixed. And slowly adding the mixed solution into 1ml of 0.5% polyvinyl alcohol (PVA) solution in mass percent, and performing ultrasonic treatment for 2min (power 30W and frequency 20KHz) by using a probe ultrasonic homogenizer to obtain a primary emulsion.
Mixing the obtained primary emulsion with 6ml of PVA solution with the mass percent of 0.5%, and performing ultrasonic treatment for 10min (power 60W and frequency 20KHz) by using a probe ultrasonic homogenizer to obtain the double emulsion.
Immediately dripping 0.1ml of 10 percent PEI solution in percentage by mass into the double emulsion; the mixture was placed in a ventilation device, stirred at room temperature for 6H, and the organic solvent component was removed. After the stirring was completed, the solution was collected, and the solution was centrifuged at 12000rpm for 10min to obtain a precipitate. And (3) washing the precipitate for 2 times by using double distilled water to obtain the PLGA coated DOX nano-drug carrier with positive charges. Diluted to a concentration of about 5X 10 with PBS buffer10Per ml of nanoparticle suspension.
FIG. 2 is a low power electron microscope image of positively charged PLGA coated DOX nano-drug carrier particles synthesized in step 1) of the example, wherein the nano-drug carrier is spherical particles with a particle size of 80nm-100 nm. The nano-drug carrier synthesized by the method has the characteristics of small particle size, uniform particle size, high safety and the like.
FIG. 3 is a high power electron microscope image of the positively charged PLGA coated DOX nano-drug carrier particle synthesized in the step 1) of the example, which shows that the outer layer of the nano-drug carrier particle is a circle of outer shell composed of polymer PLGA and PEI, the diameter is 10-20nm, the inner part is a Dox drug molecule core, and the inner particle size is 50-80 nm.
2) Combination of BCG with cationic nano-drug carriers: taking BCG vaccine BCG bacterial liquid in logarithmic growth phase, and adjusting the bacterial liquid concentration to 108CFU/ml, 1ml of the bacterial liquid was centrifuged at 5000rpm for 10 min. The medium was discarded, washed three times with PBS buffer, and then the pellet was resuspended with 1ml of PBS buffer, and then 200. mu.l of the positive-charged nanoparticle suspension synthesized in step 1) was added thereto, and shaken on a shaker at 37 ℃ for 1H. Collecting bacterial liquid toCentrifuging at 5000rpm for 10min to remove the part of nanoparticles (i.e. nano drug carrier) not bound to BCG. And washing the precipitate twice with PBS buffer solution to obtain the BCG complex adsorbing the positive Dox nano-drug carrier.
FIG. 4 is a scanning electron microscope image of BCG compound adsorbed with positive Dox nano-drug carrier finally prepared in example. As can be seen, the surface of each BCG thallus can be adsorbed by 10-20 nano drug carriers, and the positive Dox nano drug carriers are firmly combined on the surface of the BCG thallus to form a composite structure together with the BCG.
The invention realizes the mutual adsorption between the nano-drug carrier and the BCG thalli by utilizing the characteristic of mutual attraction of positive and negative charges. The nano-drug carrier is nano-particles which are coated by a high-molecular organic polymer PLGA and surface-modified by a cationic polymer PEI, and have the particle size of about 80-100 nm. The surface of each BCG thallus can be combined with 10-20 nano-particles. Therefore, the invention can successfully prepare various different drug molecules into the nano drug carrier and realize the mutual adsorption with BCG bacteria to form a composite structure of the BCG and the nano drug carrier, namely a BCG-NPs composite.
While the present invention has been described with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments, which are illustrative only and not restrictive, and various modifications which do not depart from the spirit of the present invention and which are intended to be covered by the claims of the present invention may be made by those skilled in the art.
Claims (6)
1. A BCG complex which utilizes the characteristics of positive and negative charges to adsorb a positive nano-drug carrier is characterized in that the complex is a complex structure which realizes mutual combination by the mutual attraction of the positive and negative charges between the nano-drug carrier with the positive charges and BCG thalli; wherein, the nano-drug carrier is a nano-particle which is coated by polylactic acid-glycolic acid copolymer, surface modification is carried out on cationic polymer polyethyleneimine, and the particle size is 80nm-100 nm; in the complex, each BCG bacterial surface is combined with 10-20 nano particles.
2. The method for preparing BCG complex with positive and negative charges adsorbed on nano-drug carrier according to claim 1, wherein the method comprises the steps of: synthesizing a cationic nano-drug carrier by using a polymeric material water/oil/water double-emulsification method, and combining the BCG with the cationic nano-drug carrier to obtain the BCG complex combined with the nano-drug carrier.
3. The method for preparing BCG complex with positive and negative charges characteristic adsorbed positive nano-drug carrier according to claim 2, wherein the cationic nano-drug carrier is synthesized by using polymeric material water/oil/water double-emulsification method as follows:
dissolving a proper amount of polylactic acid-glycolic acid copolymer in dichloromethane to prepare a dichloromethane solution of the polylactic acid-glycolic acid copolymer with the mass volume concentration of 10mg/ml, and marking as a solution A;
dissolving a proper amount of anticancer drug in a corresponding soluble organic solvent to prepare an organic solvent solution of the anticancer drug with the mass volume concentration of 10mg/ml, and marking as a solution B;
according to the volume ratio of 9: 1, mixing the solution A and the solution B, and carrying out ultrasonic oscillation for 30min at normal temperature in an ultrasonic water bath to fully mix the solution A and the solution B to obtain a mixed solution C;
according to the volume ratio of 1: 1, slowly adding the mixed solution C into a polyvinyl alcohol solution with the mass percent of 0.5%, and carrying out ultrasonic treatment for 2min by using a probe ultrasonic homogenizer with the power of 10-30W and the frequency of 20KHz to obtain a primary emulsion;
according to the volume ratio of 1: 3, mixing the primary emulsion with 0.5 percent polyvinyl alcohol solution again, and performing ultrasonic treatment for 10min by using a probe ultrasonic homogenizer at the power of 50-80W and the frequency of 20KHz to obtain double emulsion;
immediately, a 10% polyethyleneimine solution in percentage by mass is dropwise added into the double emulsion, and the obtained mixed solution is marked as a mixed solution D, wherein the volume ratio of the polyethyleneimine solution to the solution C is 1: 10;
placing the mixed solution D in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, and centrifuging at 12000rpm for 10min to obtain a precipitate; washing the precipitate with double distilled water for 2 times to obtain positively charged adriamycin nanoparticles coated with polylactic acid-glycolic acid copolymer;
finally, the mixture was diluted to a concentration of 4.5X 10 with phosphate buffer10～5.5×1010Per ml of nanoparticle suspension.
4. The method for preparing BCG complex with adsorbed positive nanometer drug carrier based on positive and negative charge characteristics as claimed in claim 3,
the anticancer drug is adriamycin, and the organic solvent which is correspondingly soluble to the adriamycin is dimethyl sulfoxide.
The anti-cancer drug is paclitaxel, and the organic solvent which is soluble correspondingly to the paclitaxel is acetone;
the anti-cancer drug is mitomycin, and the corresponding soluble organic solvent of mitomycin is dimethylacetamide;
the anticancer drug is cisplatin, and the corresponding soluble organic solvent of the cisplatin is dimethylformamide.
5. The method for preparing BCG complex with positive and negative charges characteristic adsorbing positive nano-drug carrier according to claim 2, wherein the specific content of the nano-drug carrier synthesized by the polymeric material water/oil/water double-emulsification method is as follows:
dissolving a proper amount of polylactic acid-glycolic acid copolymer in dichloromethane to prepare a dichloromethane solution of the polylactic acid-glycolic acid copolymer with the mass volume concentration of 10mg/ml, and marking as a solution A;
dissolving a proper amount of anticancer drug in double distilled water, wherein the anticancer drug is not easy to dissolve in an organic solvent but has good water solubility, and preparing an aqueous solution of the anticancer drug with the mass volume concentration of 10mg/ml, and marking the aqueous solution as a solution B;
according to the volume ratio of 1: 1, mixing the solution A and the solution B, and carrying out ultrasonic treatment for 2min by using a probe ultrasonic homogenizer at the power of 10-30W and the frequency of 20KHz to obtain a primary emulsion;
according to the volume ratio of 1: 3, mixing the primary emulsion with 0.5 mass percent polyvinyl alcohol solution, and performing ultrasonic treatment for 10min by using a probe ultrasonic homogenizer at the power of 50-80W and the frequency of 20KHz to obtain double emulsion;
immediately, a 10% polyethyleneimine solution in percentage by mass is dropwise added into the double emulsion, and the obtained mixed solution is marked as a mixed solution D, wherein the volume ratio of the polyethyleneimine solution to the solution a is 1: 10;
placing the mixed solution D in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, and centrifuging at 12000rpm for 10min to obtain a precipitate; washing the precipitate with double distilled water for 2 times to obtain positively charged adriamycin nanoparticles coated with polylactic acid-glycolic acid copolymer;
finally, the mixture was diluted to a concentration of 4.5X 10 with phosphate buffer10～5.5×1010Per ml of nanoparticle suspension.
6. The method for preparing BCG complex with positive and negative charges adsorbed on positive nanometer drug carrier according to claim 2, wherein the combination of BCG and cationic nanometer drug carrier comprises the following specific contents:
taking BCG vaccine bacterial liquid in logarithmic growth phase, and adjusting the concentration of the bacterial liquid to 108CFU/ml, centrifuging at 5000rpm for 10min, discarding the culture medium, washing the precipitate obtained by centrifugation with phosphate buffer solution for three times, and isovolumetrically resuspending the BCG precipitate;
and adding the nanoparticle suspension prepared in the step into the resuspended BCG bacterial liquid, wherein the volume ratio of the nanoparticle suspension to the BCG bacterial liquid is 1:5, shaking the mixed liquid for 1H on a shaking table at 37 ℃, centrifuging for 10min at 5000rpm, removing the unbound particle part, and washing the obtained precipitate twice by using a phosphate buffer solution to obtain the product, namely the BCG complex combined with the positive nano-drug carrier.
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