CN111001014B

CN111001014B - Anti-tumor drug based on immobilized bacteria as carrier and application thereof

Info

Publication number: CN111001014B
Application number: CN201911278243.0A
Authority: CN
Inventors: 杨寒朔; 姬改利; 李琪琪
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2022-04-22
Anticipated expiration: 2039-12-12
Also published as: CN111001014A

Abstract

The invention belongs to the field of biological medicines, and particularly relates to an anti-tumor medicine based on immobilized bacteria as a carrier, and application of the immobilized bacteria in the field of anti-tumor treatment. The invention provides a medicament with an anti-tumor effect, which comprises fixed bacteria and a chemotherapeutic medicament, wherein the fixed bacteria is a carrier of the chemotherapeutic medicament. The immobilized bacteria include gram-positive and gram-negative bacteria. Compared with other nano material carriers, the fixed bacteria provided by the invention as a chemotherapeutic drug carrier has the advantages of simple preparation, low cost, stable process and good anti-tumor effect.

Description

Anti-tumor drug based on immobilized bacteria as carrier and application thereof

This application will likely serve as a priority basis for subsequent patent applications (including, but not limited to, chinese invention patent applications, chinese utility model applications, PCT applications, foreign applications based on the paris convention).

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an anti-tumor medicine based on immobilized bacteria as a carrier, and application of the immobilized bacteria in the field of anti-tumor treatment.

Background

In recent years, the treatment of malignant tumors remains a major problem for mankind. Currently, chemotherapy is the primary method of clinical treatment of tumors. The chemotherapy drug has the characteristics of strong tumor cell killing power, quick response and the like, can effectively control the state of an illness within a period of time, but can also damage normal cells and influence the body health of a patient because the chemotherapy drug has no obvious targeting property. In 2014, researchers have counted and analyzed the information of 90 tumor chemotherapy patients in a certain traditional Chinese medicine, and found that the adverse reaction rate of platinum drugs (cisplatin and oxaliplatin) is 56% by taking the adverse reaction of a digestive system as an example; the adverse reaction rate of the anti-microtubule drug (paclitaxel) is 55 percent; the adverse reaction rate of antimetabolites (tegafur and fluorouracil) is 65 percent; in addition, doxorubicin and epidoxorubicin inflicted damage to the heart. Therefore, the development of highly specific drug delivery systems, combining traditional chemotherapy with novel drug delivery systems, is the hot topic of current tumor research.

Bacteria, especially certain anaerobes and facultative anaerobes can infiltrate into and preferentially multiply and accumulate in the tumor, so that the bacteria can be used as a treatment carrier, and a new direction for treating the tumor is opened. The protocols used are live bacteria, and ideally live bacterial vectors should have the following characteristics: 1) is non-toxic to the host and is suitable for various administration modes; 2) has high targeting performance; 3) can be genetically modified to express exogenous gene with high efficiency; 4) is effective for various tumors; 5) can not be fixed in normal tissues and can be removed by organisms after treatment; 6) has the function of dissolving tumor. At present, no living bacterium can satisfy all the conditions at the same time. In addition, live bacterial vectors face other problems, such as significant differences in efficacy in treating different tumors; the living bacteria have higher requirements on preparation, transportation and storage conditions; inherent toxicity limitations of the live bacteria themselves, and the like. More importantly, the immunity of tumor patients is relatively low and live bacterial preparations can lead to the risk of infection. Moreover, live bacteria are difficult to be carriers of chemotherapeutic drugs.

Based on the above, the invention provides a fixed bacterium capable of carrying a drug and an application thereof in antitumor treatment.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a drug having an antitumor effect.

In order to achieve the purpose, the technical scheme of the invention is as follows.

A medicine with anti-tumor effect comprises fixed bacteria and chemotherapeutic medicine, wherein the fixed bacteria is a carrier of the chemotherapeutic medicine.

Further, the immobilized bacteria include gram-positive bacteria and gram-negative bacteria.

Further, the gram-positive bacteria include one or more of streptococcus thermophilus, staphylococcus aureus, bacillus, and genetically engineered strains of the foregoing bacteria.

Further, the gram-negative bacteria include one or more of pseudomonas aeruginosa, salmonella, escherichia coli, and genetically engineered strains of the foregoing.

Preferably, the salmonella is the RE88 strain.

Further, the fixing agent for fixing the bacteria comprises one or more of 4% paraformaldehyde, methanol, ethanol, acetone, glutaraldehyde and pentanediol.

Further, the chemotherapeutic drug comprises one or more of doxorubicin, 5-fluorouracil, paclitaxel, docetaxel, vinblastine, oxaliplatin and irinotecan.

Preferably, the chemotherapeutic agent comprises one or more of doxorubicin, 5-fluorouracil and paclitaxel.

Further, the medicine also comprises other pharmaceutically acceptable carriers and/or auxiliary agents.

Further, the pharmaceutical dosage form is an injection.

The chemotherapeutic drug has the capability of naturally combining DNA and tubulin, and the immobilized bacteria retain the DNA and tubulin of the bacteria, so the immobilized bacteria can be used as a natural carrier of the chemotherapeutic drug.

It is another object of the present invention to provide the use of immobilized bacteria.

The use of immobilized bacteria in the preparation of anti-tumor drug carriers.

Further, the drug is bound on the immobilized bacteria in an incubation mode, and the amount of the drug bound on the immobilized bacteria is positively correlated with the incubation amount.

Further, the drug is a chemotherapeutic drug comprising one or more of doxorubicin, 5-fluorouracil, paclitaxel, docetaxel, vinblastine, oxaliplatin and irinotecan.

Further, the immobilized bacteria can increase the concentration of the chemotherapeutic drug in the tumor and inhibit the growth of the tumor.

Further, the immobilized bacteria can increase the concentration of the chemotherapeutic drug in the tumor and reduce the volume of the tumor.

Further, the tumor is a solid tumor.

Further, the tumor also comprises a volume of more than 1000mm³Large tumors.

Bacteria can be phagocytized by phagocytic cells (neutrophils, macrophages), and the immobilized bacteria retain their phagocytic capacity. A large number of phagocytes such as central granulocytes are arranged in an organism, and after the phagocytes bacteria, the fixed bacteria carrying the medicine can be brought into the tumor, so that the chemotherapeutic medicine is accumulated in the tumor, and the traditional chemotherapeutic medicine cannot effectively reach the inside of the tumor.

It is another object of the present invention to provide a method for increasing the permeability of a drug within a tumor.

A method for improving the permeability of medicine in tumor is to combine the medicine on the fixed bacteria by incubation.

Further, the method comprises the steps of: 1) culturing bacteria; 2) preparing immobilized bacteria; 3) preparing drug-loaded bacteria.

Further, the bacteria in step 1) include gram-positive bacteria and gram-negative bacteria.

Preferably, the salmonella is the RE88 strain.

Further, taking the bacterial liquid growing to the logarithmic phase in the step 2), centrifuging to remove the liquid culture medium, washing by using a buffer solution, taking a proper amount of bacteria to be resuspended in the stationary liquid, treating for 0.5-24h, centrifuging to remove the stationary liquid, and washing the bacteria by using the buffer solution to obtain the stationary bacteria.

Further, the fixative comprises one or more of 4% paraformaldehyde, methanol, ethanol, acetone, glutaraldehyde and pentanediol.

Further, the obtained fixed bacteria had a final concentration of 5X 10⁶-5×10¹⁰Bacteria/ml.

Further, the obtained fixed bacteria had a final concentration of 5X 10⁶Bacteria/ml, 5X 10⁷Bacteria/ml, 5X 10⁸Bacteria/ml, 5X 10⁹Bacteria/ml, 5X 10¹⁰Bacteria/ml.

Further, uniformly mixing the medicine obtained in the step 3) with the fixed bacteria obtained in the step 2), wherein the medicine accounts for 50-200% of the fixed bacteria by weight; incubating for 0.5-24h at 0-50 ℃, then washing the bacteria by using buffer solution, and resuspending in the buffer solution to obtain the drug-loaded immobilized bacteria.

Further, the drug is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% by weight of the immobilized bacteria.

Further, the medicine-carrying fixed bacteria obtained in the step 3) are taken to prepare an injection during treatment.

The invention has the beneficial effects

The fixed bacteria provided by the invention can be used as a chemotherapeutic drug carrier to deliver chemotherapeutic drugs to the interior of a tumor, so that the accumulation of the chemotherapeutic drugs in the tumor is increased. The fixed bacteria can destroy tumor blood vessels and inhibit tumor growth, but tumor cells living around necrotic tumors can grow again to cause tumor recurrence, the fixed bacteria is used as a chemotherapeutic drug carrier to bring chemotherapeutic drugs to tumor parts, and the tumor cells living around necrotic tissues are killed and killed by combining the antitumor effect of the fixed bacteria, so that the tumor recurrence is effectively inhibited, and the antitumor effect of the fixed bacteria and the chemotherapeutic drugs is enhanced. In addition, compared with other nano material carriers, the fixed bacteria serving as the chemotherapeutic drug carrier has the advantages of simple preparation, low cost, stable process and good anti-tumor effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.

FIG. 1 shows the amount of adsorbed immobilized bacteria detected by HPLC.

FIG. 2 is a flow cytometry analysis of the permeability of doxorubicin DOX in tumors.

FIG. 3 is a picture of tumors after tumor treatment with immobilized bacteria carrying doxorubicin, immobilized bacteria alone, doxorubicin combined with immobilized bacteria, and doxorubicin alone.

FIG. 4 is a line graph showing the effect of doxorubicin-loaded immobilized bacteria on tumors treated with doxorubicin alone, immobilized bacteria in combination with doxorubicin, and doxorubicin alone.

FIG. 5 is a graph showing the survival of mice treated with immobilized bacteria such as doxorubicin, tumor-associated immobilized bacteria, and doxorubicin alone.

FIG. 6 is a line graph showing the effect of doxorubicin-loaded immobilized bacteria RE88 on tumor treatment with doxorubicin alone, immobilized bacteria in combination with doxorubicin, and doxorubicin alone.

FIG. 7 is a line graph showing the effect of immobilized RE88 carrying 5-FU fixed bacteria in combination with adriamycin or adriamycin alone on tumor treatment. (wherein, the solid line represents the necrotic area of the tumor after treatment, and the dotted line represents the tumor tissue that recurs around the tumor after treatment with immobilized bacteria alone or in combination with doxorubicin)

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Unless otherwise specified, the terms "comprise" and "comprise," as well as grammatical variations thereof, are used to denote "open" or "including" language such that they include the recited features but also allow for the inclusion of additional, non-recited features.

As used in this specification, the term "about" (e.g., the weight percent of the drug to the immobilized bacteria), typically represents +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range (e.g., the weight percent of the drug to the immobilized bacteria). For example, the range

The description should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

Example 1

1. Bacterial culture

Coli (TOP10) was cultured in LB liquid medium (10 g of tryptone, 5g of yeast extract and 10g of NaCl per liter of LB liquid medium) under normoxic conditions at 37 ℃.

2. Cell culture

CT26 cells (mouse colon cancer cells) were maintained in the national emphasis laboratory of biotherapy, Sichuan university, using 1640 medium containing 10% calf serum at 37 deg.C and 5% CO₂Culturing under the condition of normal oxygen.

3. Preparation of immobilized bacteria

Growth to logarithmic phase (based on OD)₆₀₀，1OD＝5×10⁸Bacteria/ml), 13000rpm, 5min centrifugation to remove liquid medium, using PBS washing bacteria 3 times. Take 5X 10⁸The bacteria were resuspended in 1ml of fixative (4% paraformaldehyde)The solution was treated for 0.5-24 hours, then centrifuged at 13000rpm for 5min to remove the fixative and the bacteria were washed 3 times by resuspension in PBS to a final concentration of 5X 10⁹Bacteria/ml.

4. Preparation of drug-loaded bacteria

100ul of chemotherapeutic drug (Dox (adriamycin): 2 mg/ml; 5-FU:10mg/ml) was mixed with 100ul of fixed bacteria, incubated at 0-50 ℃ for 0.5-24 hours, and then the bacteria were washed 3 times with PBS and resuspended in 200ml of PBS, and 100ul of drug-loaded bacteria were injected into each mouse through the tail vein at the time of treatment.

In other embodiments, the chemotherapeutic agent comprises 50% -200% by weight of the immobilized bacteria, including 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. Within the range of the weight percentage of the chemotherapeutic drug in the fixed bacteria, the drug-loaded bacteria with better effect can be prepared.

5. Determination of drug-loading Performance of bacteria

Immobilized bacterial suspension (5X 10)⁸100ul) were incubated with different concentrations of chemotherapeutic (DOX (doxorubicin): 0.5, 1, 2, 4, 6, 8 mg/ml; 5-FU: 2. 4, 8, 12, 16, 24, 32, 48, 64, 72, 80 mg/ml; tax (paclitaxel): 1, 2, 4, 6, 8, 16mg/ml), incubated at 37 ℃ for 2 hours, and centrifuged to collect the supernatant (about 200 ul). The drug-loaded bacteria were further washed twice with PBS (400 ul each), and the supernatants were collected together (about 1ml) for drug content detection using high performance liquid chromatography.

6. Drug-loaded bacterial drug delivery capacity assay

CT26 Colon cancer tumor-bearing mice were injected with 150. mu.g DOX or drug-loaded bacteria (containing 150. mu.g DOX), respectively, via the tail vein. Mice were sacrificed 48 hours post-surgery, tumors were excised, cells were isolated, washed with pre-cooled PBS and resuspended, and DOX tumor excision analyzed by flow cytometry, weighed, and subdivided by homogenization. After 40 μ filtration, cells were collected and washed ice with PBS. Doxorubicin fluorescence was then analyzed by flow cytometry.

7. Animal experiments

7.1. Animal feeding

Female BALB/c mice of 6-8 weeks old were selected for the experiment and were provided by the Experimental animal center of Sichuan university. All experimental mice were housed in standard SPF-rated animal houses, maintained at 25 ℃ at room temperature, and were free to eat and drink water during the experiment.

7.2. Mouse subcutaneous transplantation tumor model modeling

The tumor cell suspension cultured by step 2 was washed 2 times with PBS, and the cells were resuspended to a final concentration of 10 using serum-free medium⁷And/ml. Take 1x10⁶100ul of tumor cell suspension was inoculated subcutaneously into the right flank of the mouse.

7.3. Mice for treating subcutaneous transplantation tumor by drug-loaded bacteria

When the average volume of the subcutaneous transplanted tumor of the mouse reaches 400mm³Mice were randomly divided into five groups, namely (i) a control group (i.v. saline) and (ii) a simple fixed bacteria treatment group, (iii) a simple chemotherapy drug treatment group, (iv) a fixed bacteria-combined chemotherapy drug group (tail vein chemotherapy drug administration was performed after 8 hours of fixed bacteria treatment), and (v) a fixed bacteria-loaded drug group.

7.4. Treatment effect monitoring

After treatment, tumors were measured every two days with a caliper using the formula 0.52 × (tumor length × tumor width)²) Tumor volume was determined and tumor growth curves were plotted.

Example 2

1. The immobilized bacteria can be used as carrier of chemotherapeutic drugs

The chemotherapeutic drugs adriamycin, 5-FU and taxol can be combined on the fixed bacteria in a mode of incubation with the fixed bacteria, and the chemotherapeutic drugs combined on the fixed bacteria are increased along with the increase of the incubation dosage (figure 1), and the experiment is measured by a bacterial drug loading performance measuring method in steps 1-5.

2. The immobilized bacteria can deliver chemotherapeutic drugs to tumors

After 48 hours of tail vein injection of pure adriamycin and injection of adriamycin-loaded bacteria to treat CT26 tumor-loaded mice, cells in tumor tissues are separated and flow analysis is carried out (the experiment is measured by the step 1-6. the delivery capacity of the drug-loaded bacteria drug is measured), the permeation rate of the adriamycin in the pure adriamycin treatment group is detected to be 2.15%, the permeation rate of the adriamycin in the drug-loaded bacteria treatment group is detected to be 22.90%, the chemotherapeutic drug is loaded on the fixed bacteria, and the amount of the chemotherapeutic drug entering the tumor is obviously improved (figure 2).

3. Immobilized bacteria-carried chemotherapeutic drug with anti-tumor effect

The fixed bacteria loaded chemotherapeutic drug adriamycin has obvious anti-tumor effect, can inhibit tumor growth and reduce tumor volume after fixed bacteria treatment or fixed bacteria combined chemotherapeutic drug treatment (figure 3), has obviously better treatment effect than the single fixed bacteria, fixed bacteria combined chemotherapeutic drug and single chemotherapeutic drug treatment (figure 4), and prolongs the life cycle of mice (figure 5).

4. Fixing different bacteria carrying chemotherapeutic drugs also has antitumor effect on large tumor

Fixed bacteria RE88 chemotherapy drug-loaded adriamycin pair with volume of 1000mm³The large tumor has obvious anti-tumor effect, can inhibit the growth of the large tumor, prolongs the life cycle of mice, and has better treatment effect than the treatment of pure fixed bacteria, the combination of the fixed bacteria and chemotherapeutic drugs and the pure chemotherapeutic drugs (figure 6).

5. Fixing different bacteria carrying different chemotherapeutics also has anti-tumor effect on large tumor

The fixed bacteria RE88 carries chemotherapeutic drug 5-FU with the volume of 1000mm³The large tumor has obvious anti-tumor effect, can inhibit the growth of the large tumor, reduce the tumor volume and prolong the life of mice, and the treatment effect is better than that of the treatment of pure fixed bacteria, the combination of the fixed bacteria and chemotherapeutic drugs and the pure chemotherapeutic drugs (figure 7).

The results of the experiments demonstrate that chemotherapeutic agents can bind to the immobilized bacteria and be delivered to the tumor with the immobilized bacteria, enhancing the anti-tumor effect of the immobilized bacteria and chemotherapeutic agents. In the embodiment, the fixed bacteria have the drug carrying capacity for different chemotherapeutic drugs, and the use of different bacteria for carrying different chemotherapeutic drugs has the anti-tumor effect, so that the tumor bleeding can be caused and the tumor growth can be inhibited for the large-size refractory subcutaneous transplantation tumor.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A drug having an antitumor effect, comprising an immobilized bacterium which is a carrier for a chemotherapeutic drug, the immobilized bacterium comprising salmonella, and a chemotherapeutic drug having an ability to naturally bind to DNA or tubulin, the immobilized bacterium retaining the DNA and tubulin.

2. The medicament of claim 1, wherein the salmonella is strain RE 88.

3. The medicament of claim 1, wherein the bacteria are immobilized using a fixative comprising one or more of 4% paraformaldehyde, methanol, ethanol, acetone, glutaraldehyde, and pentanediol.

4. The medicament of claim 1, wherein the chemotherapeutic agent comprises one or more of doxorubicin, 5-fluorouracil, and paclitaxel.

5. The medicament of any one of claims 1 to 4, further comprising other pharmaceutically acceptable carriers and/or adjuvants.

6. The medicament of claim 1, wherein the pharmaceutical dosage form is an injectable formulation.

7. Use of immobilized bacteria, including salmonella, in the preparation of a carrier for an anti-tumor drug, the anti-tumor drug being a chemotherapeutic agent having the ability to naturally bind DNA or tubulin, the immobilized bacteria retaining the DNA and tubulin.

8. The use of claim 7, wherein the anti-tumor agent is bound to the immobilized bacteria in an incubated form, and the amount of the agent bound to the immobilized bacteria is positively correlated to the amount of the incubated agent.

9. The use of claim 7, wherein the chemotherapeutic agent comprises one or more of doxorubicin, 5-fluorouracil, and paclitaxel.

10. The use of claim 7, wherein said immobilized bacteria is capable of increasing the concentration of said chemotherapeutic agent in a tumor and inhibiting tumor growth.

11. The use of claim 7, wherein said immobilized bacteria is capable of increasing the concentration of said chemotherapeutic agent in the tumor and decreasing the volume of the tumor.

12. The use according to any one of claims 8 to 11, wherein the tumour is a solid tumour.

13. The use of claim 12, wherein the tumor further comprises a volume greater than 1000mm³Large tumors.

14. A method for preparing drug-loaded immobilized bacteria, comprising the steps of:

culturing bacteria, including salmonella;

preparing immobilized bacteria: taking a bacterial liquid growing to a logarithmic phase, centrifuging to remove a liquid culture medium, washing with a buffer solution, taking a proper amount of bacteria, re-suspending in a fixing agent, centrifuging to remove the fixing agent, and washing the bacteria with the buffer solution to obtain the fixed bacteria, wherein the fixed bacteria retain DNA and tubulin;

preparing medicine-carrying immobilized bacteria: uniformly mixing a chemotherapeutic drug and the fixed bacteria, wherein the chemotherapeutic drug accounts for 50-200% of the fixed bacteria by weight percent and has the capacity of naturally combining DNA or tubulin; the chemotherapeutic drug is combined on the fixed bacteria in an incubation mode, and the incubation temperature is 0-50 ℃; the immobilized bacteria were then washed with buffer and resuspended in buffer to give the drug-loaded immobilized bacteria.

15. The method of claim 14, wherein the salmonella is strain RE 88.

16. The method of claim 14, wherein the fixative comprises one or more of 4% paraformaldehyde, methanol, ethanol, acetone, glutaraldehyde, and pentanediol.

17. The method of claim 14, wherein the resulting fixed bacteria has a final concentration of 5 x10⁶-5×10¹⁰Bacteria/ml.

18. The method of claim 17, wherein the resulting fixed bacteria has a final concentration of 5 x10⁶Bacteria/ml, 5X 10⁷Bacteria/ml, 5X 10⁸Bacteria/ml, 5X 10⁹Bacteria/ml, 5X 10¹⁰Bacteria/ml.

19. The method of claim 14, wherein the drug-loaded immobilized bacteria is an injection.