CN112451679A - BCG complex combined with nano-drug carrier and preparation method thereof - Google Patents
BCG complex combined with nano-drug carrier and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- 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
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- 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
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61P35/00—Antineoplastic agents
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Abstract
The invention discloses a BCG complex combined with a nano-drug carrier, which is a complex structure formed by mutually combining a nano-drug carrier and BCG thalli by depending on the acting force between avidin and biotin protein; the nano-drug carrier is nano-particles which are coated by polylactic acid-glycolic acid copolymer and subjected to surface modification by avidin protein and have the particle size of 120nm-180 nm; each BCG thallus surface is combined with single to a plurality of nano particles. The BCG has good adhesion and absorption effects on bladder epithelium, the drug particles are loaded on the surface of BCG thallus, the BCG is used as a carrier, the uptake and tumor targeting of a nano drug carrier in the bladder are enhanced, and the tumor killing effect of the drug can be obviously improved. Meanwhile, the BCG loaded with the medicine can stimulate strong immune response locally and systemically in the bladder, thereby realizing a combined treatment mode combining immunotherapy and chemotherapy.
Description
Technical Field
The invention belongs to a nano drug carrier technology, and particularly relates to a BCG complex combined with a nano drug carrier and a preparation method thereof.
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 BCG has significant immunotherapeutic effects, large doses of BCG in bladder instillation can cause side effects such as bladder inflammation, infection, and the like, and there still remains a fraction of patients who are not sensitive to BCG treatment. Therefore, BCG perfusion-based combination therapies are gaining increasing attention. Clinical research results show that the combination of BCG and chemotherapeutic drugs (such as gemcitabine) and immunomodulators (such as PD-1/PD-L1 monoclonal antibody drugs) can further improve the prognosis of patients and 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 the BCG vaccine complex combined with the nano-drug carrier, the BCG vaccine complex has good adhesion and absorption effects on the bladder epithelium, the nano-drug carrier coated with different drug particles is loaded on the surface of BCG thallus, the BCG is used as the carrier, the uptake and the tumor targeting of the nano-drug carrier in the bladder are enhanced, and the tumor killing effect of the drug is obviously improved. Meanwhile, the BCG loaded with the medicine can stimulate strong immune response locally and systemically in the bladder, thereby realizing a combined treatment mode combining immunotherapy and chemotherapy.
In order to solve the technical problems, the BCG complex combined with the nano-drug carrier provided by the invention is a complex structure which is formed by mutually combining the nano-drug carrier and BCG thalli by depending on the acting force between avidin and biotin protein; wherein, the nano-drug carrier is nano-particles which are coated by polylactic acid-glycolic acid copolymer and subjected to surface modification by avidin protein and have particle size of 120nm-180 nm; in the complex, each BCG thallus surface has single to ten nanometer particle combination. The preparation method of the complex mainly comprises the following steps: synthesizing a nano-drug carrier by using a polymeric material water/oil/water double-emulsification method, preparing the avidin-labeled nano-drug carrier, premixing the BCG and the biotin-labeled polyclonal antibody, and combining the BCG and the nano-drug carrier to obtain the BCG complex combined with the nano-drug carrier.
According to the fact that the anti-cancer drug is soluble in an organic solvent or not soluble in the organic solvent and has good water solubility, the specific process of synthesizing the nano-drug carrier by using the polymer material water/oil/water double-emulsification method comprises the following two modes:
the first method is as follows: the anticancer drug is dissolved in an organic solvent, and the process of synthesizing 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 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; placing the obtained double emulsion in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, placing the solution at 12000rpm, and centrifuging for 10min to obtain precipitate; washing the precipitate with double distilled water for 2 times, and re-suspending the precipitate to obtain the nanometer anticancer medicine particle suspension coated with the polylactic acid-glycolic acid copolymer as the nanometer medicine carrier.
The second method comprises the following steps: the anticancer drug is not easy to dissolve in organic solvent, but has good water solubility, and the process of synthesizing 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 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; placing the obtained double emulsion in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, placing the solution at 12000rpm, and centrifuging for 10min to obtain precipitate; washing the precipitate with double distilled water for 2 times, and re-suspending the precipitate to obtain the nanometer anticancer medicine particle suspension coated with the polylactic acid-glycolic acid copolymer as the nanometer medicine carrier.
The specific contents for preparing the avidin labeled nano-drug carrier are as follows: diluting the nano-drug carrier with double distilled water to 4.5 × 1010～5.5×1010A nanoparticle suspension per ml, which was vortexed for 30min in a water bath at 37 ℃; slowly dropwise adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the mass volume concentration of 20mg/ml into the treated nanoparticle suspension, wherein the volume ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution to the nanoparticle suspension is 1:20, vortex for 5min in a 37 ℃ water bath; slowly dropwise adding an N-hydroxysuccinimide solution with the mass volume concentration of 58mg/ml, wherein the volume ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution to the N-hydroxysuccinimide solution is 1:1, and swirling for 1H in a water bath at 37 ℃ to obtain a solution which is marked as a mixed solution D; and (3) dropwise adding streptavidin with the mass volume concentration of 10ug/ml into the mixed solution D, wherein the volume ratio of the streptavidin to the nanoparticle suspension is 1:20, continuing to vortex for 4H in a water bath at 37 ℃, and collecting the solution; centrifuging the collected solution at 12000rpm for 10min, removing unbound avidin and activating reagent, obtaining precipitate obtained by centrifugation, washing the precipitate with double distilled water for 2 times, and resuspending the precipitate to obtain the avidin-labeled nano-drug carrier suspension.
Premixing BCG vaccine and biotin-labeled polyclonal antibody, wherein the biotin-labeled polyclonal antibody is a biotin-labeled Mycobacterium tuberculosis polyclonal antibody (biotin @ Anti-MT), and the method comprises the following specific steps: taking bacillus calmette-guerin bacterial liquid in logarithmic growth phase, and adjusting the concentration of the bacterial liquid to be 108Centrifuging at 5000rpm for 10min at CFU/ml, discarding the culture medium, washing the precipitate obtained by centrifuging with phosphate buffer solution for three times, and isovolumetrically resuspending the BCG precipitate; adding biotin-labeled mycobacterium tuberculosis polyclonal antibody (biotin @ Anti-MT) with mass volume concentration of 0.1mg/ml into the resuspended bacterial liquid, wherein the volume ratio of the biotin-labeled mycobacterium tuberculosis polyclonal antibody to the resuspended bacterial liquid is 1: 20; shaking the bacterial solution 1H on a shaking table at 37 ℃, centrifuging at 5000rpm for 10min, and removing the part without the antibody; and (3) washing the precipitate twice by using PBS buffer solution, and performing isometric resuspension on the precipitate to complete premixing of the BCG and the biotin-labeled polyclonal antibody so as to obtain the BCG bacterial liquid combined with the biotin-labeled Mycobacterium tuberculosis polyclonal antibody.
The combination of BCG vaccine and nano-drug carrier comprises the following steps: adding an avidin-labeled nano-drug carrier suspension into bacillus calmette-guerin bacterial liquid combined with a biotin-labeled mycobacterium tuberculosis polyclonal antibody, wherein the volume ratio of the avidin-labeled nano-drug carrier to the bacillus calmette-guerin bacterial liquid is 1: 10, shaking for 1H on a shaking table at 37 ℃, centrifuging for 10min at 5000rpm, and removing the part of the unbound nano-drug carrier; washing the precipitate obtained by centrifugation twice with PBS buffer solution to obtain the final product, namely the BCG complex combined with the 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 BCG complex combined with the nano drug carrier, namely BCG-nanoparticles (BCG-NPs). The most remarkable advantage of BCG-NPs is that the intratumoral transport and absorption of the medicine can be improved. The BCG has good adhesion and absorption effects on bladder epithelium, the drug particles are loaded on the surface of BCG thallus, the BCG is used as a carrier, the uptake and tumor targeting of a nano drug carrier in the bladder are enhanced, and the tumor killing effect of the drug can be obviously improved.
Drawings
FIG. 1 is a schematic diagram of the present invention for realizing the adsorption of the nano-drug carrier to BCG;
FIG. 2 is a transmission electron microscope image of the avidin-labeled nano-drug carrier obtained in the first embodiment of the present invention;
FIG. 3 is an electron microscope image of a BCG complex combined with Dox nano-drug carriers finally obtained according to an embodiment of the present invention;
FIG. 4 shows the killing effect of BCG complex combined with nano-drug carrier on bladder cancer cells, which is prepared in the first embodiment of 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 vaccine complex BCG-NPs combined with the nano-drug carrier is a complex structure which is formed by mutually combining the nano-drug carrier coated with drug molecules and BCG thalli by depending on the acting force between avidin and biotin protein. Wherein, the nano-drug is a nano-particle with the final particle diameter of 120nm-180, wherein the nano-drug is coated by a high molecular organic polymer polylactic-co-glycolic acid (PLGA) as a shell, and the avidin protein is used for surface modification of the nano-drug carrier. The combination of single to 10 nanoparticles can be seen on the surface of the BCG thallus.
The bcg complex combined with the nano-drug carrier is prepared by the process shown in figure 1, and mainly comprises the steps of coating PLGA (poly (lactic-co-glycolic acid)) with drug molecules to form nano-particles, and modifying avidin (SA) on the surface of the nano-particles to form the avidin-modified PLGA-coated nano-drug carrier (PLGA-NPs-SA). Meanwhile, biotin-labeled tubercle bacillus polyclonal antibody (B-Ab) is adhered to the surface of the BCG, so that biotinylated BCG (B-Ab-BCG) is formed. And finally, mixing the nano-drug carrier with the BCG, and realizing the preparation of the BCG complex combined with the nano-drug carrier by utilizing the binding force between biotin and avidin proteins. And is prepared according to the following steps:
step one, preparation of a nano-drug carrier: mainly utilizes a W/O/W emulsification method and an ultrasonic emulsification technology to realize the preparation of the polymer PLGA coated nano-drug carrier. And (3) premixing the medicament which is easy to dissolve in the organic solvent with a dichloromethane DCM solution of PLGA, mixing with water, and carrying out ultrasonic emulsification to finally obtain double emulsion of PLGA, thus obtaining the PLGA-coated nano medicament carrier particles. For drugs that are soluble in water but not in various organic solvents, the drugs are directly dissolved in the aqueous phase and then directly ultrasonically emulsified with the DCM organic phase of PLGA. The nano-drug carrier is spherical particles, and has the characteristics of high biological safety, small particle size, uniform distribution and the like. By utilizing the method, the preparation of nano-carriers of different drugs can be realized.
Step two, the combination of the nano-drug carrier and the avidin: the method is characterized in that 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activate carboxyl of PLGA on a nano-drug carrier by utilizing an amide reaction principle, and then are connected with avidin. Realize the labeling of the nano-drug carrier by the avidin. The shell of the nano-drug carrier marked with the avidin is made of a ring of avidin-marked PLGA polymeric material, and the thickness of the shell is 10nm-15 nm. The shell is internally provided with a drug molecule center with the diameter of 100-160 nm.
Step three, premixing BCG vaccine and biotin-labeled polyclonal antibody: by taking the Anti-mycobacterium tuberculosis polyclonal antibody (biotin @ Anti-MT) containing the biotin marker as an example, the binding rate of the biotin @ Anti-MT polyclonal antibody and BCG thallus can be more than 98 percent, so that the biotinylation of the BCG thallus is completed.
Step four, combining the BCG vaccine and the nano-drug carrier: and (3) combining the avidin-labeled nano-drug carrier synthesized in the second step with the biotinylated BCG bacteria synthesized in the third step by utilizing the strong binding capacity of biotin and avidin, thereby obtaining the BCG complex (BCG-NPs) combined with the nano-drug carrier.
Example 1: the preparation process of BCG complex combined with PLGA nano-drug carrier coated with Dox molecule is exemplified by clinical common chemotherapy drug adriamycin (Dox).
Step one, preparation of a Dox nano-drug carrier:
10mg of organic material PLGA was weighed out and dissolved in Dichloromethane (DCM) organic phase to prepare a 10mg/ml DCM solution of PLGA. 1mg of the chemotherapeutic drug Dox was weighed out and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10mg/ml DMSO solution of Dox. Mixing PLGA solution 0.9ml and Dox solution 0.1ml, and ultrasonic oscillating in water bath at 20 deg.C for 30min to mix thoroughly. And slowly adding the mixed solution into 1ml of polyvinyl alcohol (PVA) solution with the mass volume ratio of 0.5%, and performing ultrasonic treatment for 2min (power of 30W and frequency of 20 KHz) by using a probe ultrasonic homogenizer to obtain the primary emulsion. Mixing the obtained primary emulsion with 6ml of PVA solution with the mass percent of 0.5 percent again, and performing ultrasonic treatment for 10min (the power of 60W and the frequency of 20 KHz) by using a probe ultrasonic homogenizer to obtain the double emulsion. And then placing the obtained double emulsion in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, and collecting the solution after stirring. The solution was placed at 12000rpm and centrifuged for 10min to obtain a precipitate. And (3) washing the precipitate for 2 times by using double distilled water, and re-suspending the precipitate to obtain PLGA coated DOX nano-particle drug carrier suspension.
The preparation scheme of the nano-drug carrier for the drugs which are not dissolved in DMSO and are dissolved in other types of organic solvents 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 paclitaxel, and the organic solvent soluble correspondingly to paclitaxel can be acetone; the anti-cancer drug is mitomycin, and the corresponding soluble organic solvent of the mitomycin can be dimethylacetamide; the anticancer drug is cisplatin, and the corresponding soluble organic solvent of the cisplatin can be dimethylformamide; the particular anticancer drugs listed above and their corresponding soluble organic solvents are not limiting in the present invention.
In addition, particularly, for drugs (such as interferon preparation IFN) which are good in water solubility and not easy to dissolve in various organic solvents, the preparation scheme of the nano-drug carrier is as follows: PLGA was weighed and dissolved in the DCM organic phase to prepare a 10mg/ml solution of PLGA in DCM. Weighing water-soluble drug IFN, dissolving in double distilled water, and preparing drug solution with concentration of 1 mg/ml. 1ml of PLGA solution and 1ml of IFN solution were mixed in a 1:1, and performing ultrasonic treatment for 2min (30W power and 20KHz frequency) by using a probe ultrasonic homogenizer to obtain a primary emulsion. The obtained primary emulsion was mixed at a ratio of 1: 3 volume percent, and 6ml of PVA solution with the mass percent of 0.5 percent are mixed again, and a probe ultrasonic homogenizer is used for ultrasonic treatment for 10min (the power of 60W and the frequency of 20 KHz), thus obtaining the double emulsion. The double emulsion obtained was then placed in a ventilation unit, stirred at room temperature for 6H, and evaporated to remove the organic solvent component. After the stirring was completed, the solution was collected. The solution was placed at 12000rpm and centrifuged for 10min to obtain a precipitate. Washing the precipitate with double distilled water for 2 times, and re-suspending the precipitate to obtain PLGA coated IFN nano-drug carrier suspension.
Step two, binding the Dox nano-drug carrier and the avidin:
diluting the PLGA coated DOX nano-drug carrier suspension obtained in the last step with double distilled water to obtain the concentration of 4.5 multiplied by 1010～5.5×1010Per ml of nanoparticle suspension. The nanoparticle suspension was taken in a volume of 1ml, vortexed for 30min in a water bath at 37 ℃. 50ul of a 20mg/ml solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was slowly added dropwise to the nanoparticle suspension and vortexed for an additional 5min in a 37 ℃ water bath. Slowly adding 50ul of N-hydroxysuccinimide (NHS) solution with the concentration of 58mg/ml dropwise, and continuing to vortex for 1H in a water bath at 37 ℃; then 50ul of streptavidin (or other type of avidin) with a concentration of 10ug/ml is added dropwise to the solution, and the solution is collected after further vortexing for 4H in a water bath at 37 ℃. Centrifuging at 12000rpm for 10min to remove unbound avidin and activating reagent, collecting the precipitate, washing the precipitate with double distilled water for 2 times, and then resuspending the precipitate with 1ml of double distilled water. And obtaining the Dox nano-drug carrier suspension marked by the avidin. As shown in FIG. 2, the avidin-labeled nano-drug carrier obtained in this step is spherical particles with a particle size in the range of 120nm to 180 nm. The shell is made of a ring of avidin-labeled PLGA polymeric material, and the thickness of the shell is 10nm-15 nm. The shell is internally provided with a drug molecule center with the diameter of 100-160 nm.
Step three, premixing BCG and biotin labeled polyclonal antibody, wherein: mycobacterium tuberculosis with biotin-labeled polyclonal antibody as biotin labelPolyclonal antibodies (biotin @ Anti-MT); taking BCG vaccine BCG bacterial liquid in logarithmic growth phase, and adjusting the bacterial liquid concentration to 108CFU/ml. The bacterial solution was centrifuged at 5000rpm for 10min in a volume of 1 ml. The medium was discarded, washed three times with phosphate buffered saline PBS, and then 1ml of PBS buffer was used to resuspend BCG bacteria in equal volumes. 50ul of biotin-labeled Mycobacterium tuberculosis polyclonal antibody (biotin @ Anti-MT) was added to the bacterial solution at a concentration of 0.1 mg/ml. The cell suspension 1H was shaken on a shaker at 37 ℃ and centrifuged at 5000rpm for 10min to remove the unbound antibody. And (3) washing the obtained precipitate twice by using PBS buffer solution, and isovolumetrically resuspending the precipitate by using 1ml of PBS buffer solution to obtain the biotinylation BCG bacterial liquid combined with biotin @ Anti-MT.
Step four, the combination of the biotinylated BCG and the Dox nano-drug carrier:
and (3) adding 0.1ml of avidin-labeled drug nano-carrier suspension obtained in the third step into 1ml of biotinylated BCG bacterial solution combined with biotin @ Anti-MT obtained in the first step. Shaking the mixed solution for 1H on a shaking table at 37 deg.C to obtain mixed solution, centrifuging at 5000rpm for 10min, and removing unbound nanometer drug carrier. The precipitate obtained by centrifugation was washed twice with PBS wash buffer to obtain the final product, BCG complex (BCG-NPs) combined with Dox nano-drug carrier. As shown in figure 3, the nano-drug carriers can be successfully combined on the outer surface of the BCG thallus to form a complex structure with the BCG thallus, and each BCG thallus surface can be combined with from a single nano-drug carrier to 10 nano-drug carriers.
Example 2: killing effect of BCG-NPs on human bladder cancer T-24 cell strain in vitro:
the MTT detection method is used for detecting the killing capacity of the synthesized BCG-NPs compound on the T-24 cell strain of the human bladder cancer compared with the BCG alone. The experiment is divided into two groups, namely group A and group B, wherein the group A takes BCG bacterial liquid in logarithmic phase, and the group B obtains BCG-NPs suspension liquid by adopting the preparation method.
PBS was used to adjust the concentration of the bacterial liquid to 108CFU/ml. 10ul, 50ul and 100ul of each bacterial liquid (A and B) are respectively added into a 96-well cell culture plate paved with bladder cancer T-24 cells, and each group of experimentThree-hole repetition is set. The negative control group was supplemented with an equal volume of PBS. At 37 ℃, the cells were cultured for 24H in a 1640-cell complete medium in a cell culture chamber, and after the culture was completed, the supernatant was discarded, and each well was washed 2 times with PBS. Then 200ul of fresh 1640 cell complete medium containing 20ul of MTT with a concentration of 5mg/ml was added to each well of the well plate, the culture was continued for 4H, after the culture was completed, the supernatant was discarded, 150ul of DMSO was added to each well of the well plate, and the plate was gently shaken for 10 min. The absorbance of each well was measured at OD570 on a microplate reader. Cell viability ═ (mean OD value for each experimental group/mean OD value for control group).
Compared with BCG alone, BCG-NPs have the in vitro killing effect on human bladder cancer T-24 cells, as shown in figure 4. The concentration of three parts of bacterial liquid is 108CFU/ml, bacterial liquid volume 10ul, 50ul, 100ul respectively with T-24 cell for co-culture, culture 24H. As a result, the BCG-NPs have good cell killing effect compared with the BCG group. And the killing effect is enhanced with the increase of the dosage.
In conclusion, the invention realizes the combination between the nano-drug carrier and the BCG live bacteria by utilizing the binding force between biotin and avidin. The clinical common chemotherapeutic drug Dox is taken as an example for detailed explanation. The present invention is not limited to Dox chemotherapeutic agents. The invention can be used for preparing nano-drug carriers for various clinical therapeutic drugs, such as gemcitabine, mitomycin, immune preparation PDL1 monoclonal antibody drugs and the like, and finally realizes the BCG as a carrier-mediated delivery process. In addition, the method can also be applied to the combined collocation of different medicines to prepare the mixed medicine nano-carrier. Therefore, the invention has better clinical application value for individual bladder perfusion treatment of bladder cancer patients.
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 (8)
1. A BCG complex combined with a nano-drug carrier is characterized in that the complex is a complex structure which is formed by the mutual combination of the nano-drug carrier and BCG thalli by depending on the acting force between avidin and biotin protein; wherein, the nano-drug carrier is nano-particles which are coated by polylactic acid-glycolic acid copolymer and subjected to surface modification by avidin protein and have particle size of 120nm-180 nm; in the complex, each BCG thallus surface has single to ten nanometer particle combination.
2. The method of claim 1, wherein the method comprises the steps of: synthesizing a nano-drug carrier by using a polymeric material water/oil/water double-emulsification method, preparing the avidin-labeled nano-drug carrier, premixing the BCG and the biotin-labeled polyclonal antibody, and combining the BCG and the nano-drug carrier to obtain the BCG complex combined with the nano-drug carrier.
3. The method for preparing BCG complex combined with 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 emulsion 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 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;
placing the obtained double emulsion in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, placing the solution at 12000rpm, and centrifuging for 10min to obtain precipitate; washing the precipitate with double distilled water for 2 times, and re-suspending the precipitate to obtain the nanometer anticancer medicine particle suspension coated with the polylactic acid-glycolic acid copolymer as the nanometer medicine carrier.
4. The method for preparing BCG complex combined with nano-drug carrier according to 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 combined with 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 emulsion 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;
placing the obtained double emulsion in a ventilation device, stirring for 6H at room temperature, evaporating to remove organic solvent components, collecting the solution after stirring, placing the solution at 12000rpm, and centrifuging for 10min to obtain precipitate; washing the precipitate with double distilled water for 2 times, and re-suspending the precipitate to obtain the nanometer anticancer medicine particle suspension coated with the polylactic acid-glycolic acid copolymer as the nanometer medicine carrier.
6. The method for preparing BCG complex combined with nano-drug carrier according to any of claims 3 to 5, wherein the specific contents for preparing avidin-labeled nano-drug carrier are as follows:
2-1) diluting the nano-drug carrier with double distilled water to 4.5 × 1010～5.5×1010A nanoparticle suspension per ml, which was vortexed for 30min in a water bath at 37 ℃;
2-2) slowly dropwise adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the mass volume concentration of 20mg/ml into the nanoparticle suspension treated in the step 2-1), wherein the volume ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution to the nanoparticle suspension is 1:20, vortex for 5min in a 37 ℃ water bath; slowly dropwise adding an N-hydroxysuccinimide solution with the mass volume concentration of 58mg/ml, wherein the volume ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution to the N-hydroxysuccinimide solution is 1:1, and swirling for 1H in a water bath at 37 ℃ to obtain a solution which is marked as a mixed solution D; and (3) dropwise adding streptavidin with the mass volume concentration of 10ug/ml into the mixed solution D, wherein the volume ratio of the streptavidin to the nanoparticle suspension is 1:20, continuing to vortex for 4H in a water bath at 37 ℃, and collecting the solution;
2-3) centrifuging the solution collected in the step 2-2) at 12000rpm for 10min, removing unbound avidin and activating reagent, obtaining the precipitate obtained by centrifugation, washing the precipitate for 2 times by double distilled water, and re-suspending the precipitate to obtain the avidin-labeled nano-drug carrier suspension.
7. The method for preparing BCG complex combined with nano-drug carrier according to claim 6, characterized in that BCG is pre-mixed with biotin-labeled polyclonal antibody (biotin @ Anti-MT) of Mycobacterium tuberculosis, wherein the method comprises the following steps:
3-1) taking BCG vaccine 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 precipitate obtained by centrifuging with phosphate buffer solution for three times, and isovolumetrically resuspending the BCG precipitate;
3-2) adding biotin-labeled mycobacterium tuberculosis polyclonal antibody (biotin @ Anti-MT) with the mass volume concentration of 0.1mg/ml into the resuspended bacterial liquid in the step 3-1), wherein the volume ratio of the biotin-labeled mycobacterium tuberculosis polyclonal antibody to the resuspended bacterial liquid is 1: 20; shaking the bacterial solution 1H on a shaking table at 37 ℃, centrifuging at 5000rpm for 10min, and removing the part without the antibody; and (3) washing the precipitate twice by using PBS buffer solution, and performing isometric resuspension on the precipitate to complete premixing of the BCG and the biotin-labeled polyclonal antibody so as to obtain the BCG bacterial liquid combined with the biotin-labeled Mycobacterium tuberculosis polyclonal antibody.
8. The method for preparing BCG complex combined with nano-drug carrier as claimed in claim 7, wherein the combination of BCG and nano-drug carrier comprises the following steps:
adding an avidin-labeled nano-drug carrier suspension into bacillus calmette-guerin bacterial liquid combined with a biotin-labeled mycobacterium tuberculosis polyclonal antibody, wherein the volume ratio of the avidin-labeled nano-drug carrier to the bacillus calmette-guerin bacterial liquid is 1: 10, shaking for 1H on a shaking table at 37 ℃, centrifuging for 10min at 5000rpm, and removing the part of the unbound nano-drug carrier; washing the precipitate obtained by centrifugation twice with PBS buffer solution to obtain the final product, namely the BCG complex combined with the nano-drug carrier.
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