CN110559280A

CN110559280A - Paclitaxel-loaded liposome bacterium inhalation preparation for treating lung cancer

Info

Publication number: CN110559280A
Application number: CN201910982147.8A
Authority: CN
Inventors: 金义光; 张萌萌; 杜丽娜
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2019-10-16
Filing date: 2019-10-16
Publication date: 2019-12-13
Anticipated expiration: 2039-10-16
Also published as: CN110559280B

Abstract

The invention discloses a paclitaxel-loaded liposome bacterium inhalation preparation and finds that the paclitaxel-loaded liposome bacterium inhalation preparation has a special effect of treating lung cancer. The preparation is prepared by preparing paclitaxel liposome, introducing into bacteria such as Escherichia coli or Lactobacillus casei by electroporation, and directly administering to lung, wherein the bacteria carry drug and directly target to lung tumor, with high efficiency and safety.

Description

Paclitaxel-loaded liposome bacterium inhalation preparation for treating lung cancer

Technical Field

The invention relates to the field of medicines, in particular to a taxol-loaded liposome bacterium inhalation preparation for treating lung cancer.

background

Paclitaxel (PTX) is a complex compound extracted from the bark of the tree of the shortleaf yew, has a molecular weight of 853, is a white powder, and is soluble in various organic solvents, such as methanol, ethanol, acetone, dichloromethane, chloroform, etc.; is insoluble in water and petroleum ether.

paclitaxel is an anti-microtubule drug, and inhibits cell division and proliferation by inhibiting cell from forming spindle and spin bond during mitosis, so that cell can be arrested in G2 and M phase, and has anti-tumor effect. PTX is now widely used clinically, and paclitaxel is also widely used in clinical chemotherapy of hematological diseases and various solid tumors, in addition to advanced ovarian cancer, lung cancer, and emperor cancer. Clinically, paclitaxel is mostly used for administration after the recurrence of patients in the treatment process of small cell lung cancer, but has high price, takes in blood through intravenous absorption, reduces the amount of drugs reaching pathological changes, is distributed systemically, and has serious toxic and side effects such as anaphylaxis, bone marrow suppression, digestive tract reaction, neuromuscular toxicity, cardiotoxicity and the like.

The liposome (Liposomes) is a quasi-circular vesicle with a bilayer structure formed by phospholipid and cholesterol, can entrap hydrophilic and lipophilic drugs, can also entrap biological macromolecules, has the particle size of 10-1000 nm, and has a series of advantages of quasi-cell membrane structure, high biocompatibility, low immunogenicity, drug or active group protection, drug half-life extension, synergy and attenuation and the like.

gram-negative bacteria such as Escherichia coli, which is a representative of the genus Escherichia, are generally nonpathogenic, are common inhabitants of the intestinal tracts of humans and animals, and can cause parenteral infections under certain conditions. Some serotype strains are highly pathogenic, causing diarrhea, collectively referred to as pathogenic E.coli. The escherichia coli produces flagella all around, can move, has no spores, can ferment various saccharides to produce acid and gas, and is normal inhabitant bacteria in intestinal tracts of human beings and animals. Common gram-positive bacteria such as lactobacillus casei belong to lactobacillus, are common probiotics, do not produce spores, have flagella, do not move, facultative heterotypic fermented lactose, do not liquefy gelatin; the optimal growth temperature is 37 ℃; the thalli are different in length, and the two ends of the thalli are square and are often chained; the colony is coarse, grey white and sometimes yellowish, and can ferment various sugars.

Bacterial therapy is a novel technique for tumor treatment. The bacteria can target tumor, so that the bacteria can act on tumor cells with higher strength to play an anti-tumor role. Bacteria can aggregate in tumor tissues and exert antitumor activity, which may be related to the following factors. In tumor necrosis areas, the tumor cells are nutritious: the fast growing tumor tissue is easy to form low oxygen zone and tumor necrosis zone, and produces great amount of intermediate metabolite, so that the tumor tissue has different environment from normal tissue and can provide nutrients essential for bacteria propagation. The facultative anaerobic nature of the bacteria allows them to colonize both in small metastatic tumor cells with a certain oxygen content and in the deep hypoxic centers in larger tumor tissues. In tumor tissue, promoters of multiple genes of bacteria may all be preferentially activated. Tumor cells and stromal cells secrete tumor necrosis factor-P or other immunosuppressive factors to inhibit activation and infiltration of neutrophils, and simultaneously, the hypoxic environment in tumor tissues causes the bactericidal action of macrophages and neutrophils to be inhibited, so that the immunosuppressive action is generated in solid tumors, and bacteria are not easy to remove. The stimulation of the immune system by bacteria can induce inflammatory response, release inflammatory factors to kill tumor non-specifically, such as TNF-alpha, and directly affect the tumor vasculature. The bacteria inhibit tumor growth by competing with the nutrients required for growth of the tumor cells. Bacteria can continue to proliferate in tumor cells and cause nuclear fragmentation.

The pulmonary drug delivery is to deliver the drug to the lung through a special drug delivery device to perform systemic or local treatment of pulmonary diseases, and compared with the traditional oral administration or vein, the drug is mainly concentrated in the lung and is distributed less in other parts. Most of the medicine is deposited in the lung, so that the toxic and side effects of other tissues can be avoided or reduced. In contrast, the actual amount of drug reaching the lungs is typically lower by oral or injection, resulting in a lower ratio of drug in the lungs to drug in the plasma or directly in failure of the treatment. Common forms of pulmonary administration are aerosols, sprays and dusts. Clinically, pulmonary administration is mainly used for treating asthma, chronic obstructive pneumonia and the like.

The cancer seriously threatens the life and health of human beings, the lung cancer is the most common malignant tumor in China, and the morbidity and the mortality of the lung cancer are the first malignant tumors. According to statistics, the incidence of lung cancer accounts for 11.6 percent of the total number, the incidence accounts for the first cancer in the world, the mortality rate reaches 18.4 percent of all cancers, and the incidence accounts for the first cancer death in the world. Lung cancer refers to malignant epithelial tumors of the lung that originate in the bronchial epithelium, bronchial glands, bronchioles epithelium and alveolar epithelium. Because there are many types of normal airway epithelial cells, during the development of tumor, the pluripotent stem cells can differentiate in different directions, resulting in the heterogeneity of lung cancer in histology, so the lung cancer tissue morphology is complex. From a clinical perspective, lung cancer is currently divided into two main categories: small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (Non-Small Cell Lung Cancer, NSCLC), of which about 80% are Non-Small Cell Lung cancers. Clinically, surgery, chemotherapy and radiotherapy are the main means for treating non-small cell lung cancer, but the long-term curative effect of patients in middle and late stages is still poor, and the main death reasons are local control difficulty and distant metastasis. In recent years, traditional chemotherapy is mainly oral or intravenous administration, and generally distributes the drugs, so that the amount of the effective drugs reaching lung cancer parts is small, while the drugs reaching normal tissues possibly cause toxic and side effects, and the serious toxic and side effects and low treatment rate bring great pain to lung cancer patients.

Disclosure of Invention

The invention discloses a paclitaxel-loaded liposome bacterium inhalation preparation and finds that the paclitaxel-loaded liposome bacterium inhalation preparation has a special effect of treating lung cancer.

The taxol-loaded liposome bacterial inhalation preparation can be prepared by adopting the following steps:

(1) Preparing paclitaxel liposome;

(2) Selecting a suitable bacterium as a vector;

(3) The taxol liposome is loaded into bacteria through the action of electric pore-forming, and the taxol liposome-loaded bacteria inhalation preparation is obtained.

Further, the paclitaxel liposome-loaded bacterial inhalation preparation can also be prepared by adopting the following steps:

(1) preparing paclitaxel liposome;

(2) Preparing paclitaxel liposome into paclitaxel liposome solid;

(3) Selecting a suitable bacterium as a vector;

(4) Rehydrating the paclitaxel liposome solid in the step (2) to form liposome suspension, and loading the paclitaxel liposome into bacteria through electric pore-forming action to obtain the paclitaxel liposome-loaded bacteria inhalation preparation.

The paclitaxel liposome can be prepared by referring to relevant professional books and literature, and specifically, the preparation method is selected from techniques such as film dispersion method, multiple emulsion method, melting method, injection method, surfactant treatment method, centrifugation method, proliposome method, pressure extrusion method, calcium fusion method, etc., and preferably, the film dispersion method. The paclitaxel liposome of the invention has a particle size of 1-1000 nm. The amount of paclitaxel and the amount of the auxiliary materials in the paclitaxel liposome are not limited, and the preferred paclitaxel liposome contains 0.1-30% by weight of paclitaxel, 70-99.9% by weight of lipid and 0-50% by weight of other pharmaceutically acceptable auxiliary materials except water.

The paclitaxel liposome of the present invention, wherein the lipid for preparing the liposome is selected from lecithin, phosphatidylethanolamine, soybean phospholipid, cholesterol, cephalin, cholesterol acetyl ester, beta-sitosterol, sodium taurocholate, egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidic acid, phosphatidylserine, phosphatidylinositol, sphingomyelin, dicetyl phosphate, stearamide, preferably selected from lecithin, soybean phospholipid, cholesterol, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, more preferably selected from lecithin, soybean phospholipid, cholesterol, dipalmitoyl phosphatidylcholine, most preferably selected from soy phospholipids and cholesterol. When the paclitaxel liposome is prepared by adopting the soybean phospholipid and the cholesterol, the molar ratio of the soybean phospholipid and the cholesterol is selected from 50: 1-1: 1, preferably 20: 1-2: 1, and more preferably 10: 1-4: 1. The pharmaceutically acceptable adjuvants in the preparation step of paclitaxel liposome are selected from saccharides, alcohols, amino acids, pulmonary surfactant, cyclodextrin, polymer, glidant, antioxidant, citric acid and its salt, and phosphate.

The preparation method of the paclitaxel liposome solid in the preparation step of the paclitaxel-loaded liposome bacterial inhalation preparation is selected from spray drying method and freeze drying method, and preferably self-cooling freeze drying method. When the paclitaxel liposome solid is prepared by a freeze-drying method, the paclitaxel liposome solid is generally prepared by adding a freeze-drying protective agent into the paclitaxel liposome and freeze-drying the paclitaxel liposome. The freeze-drying protective agent can be added at the same time of preparing the paclitaxel liposome or after preparing the paclitaxel liposome. The lyoprotectant is selected from saccharides and alcohols, specifically sucrose, lactose, xylitol, dextran, mannitol, trehalose, preferably lactose and mannitol, and most preferably mannitol. The weight ratio of the liposome to the lyoprotectant in the paclitaxel liposome solid is not strictly defined, as long as the liposome obtained after hydration has a small and uniform particle size.

The bacterium in the above step of preparing the paclitaxel-loaded liposome-based bacterial inhalation formulation is not specifically limited, and may be selected from gram-negative bacteria and gram-positive bacteria in terms of genus; from the aspect of pathogenicity, the bacteria can be selected from pathogenic bacteria and probiotics. The gram-negative bacteria are selected from Escherichia coli, Salmonella, Proteus, Shigella, pneumobacillus, Brucella, Haemophilus influenzae, parainfluenza (Haemophilus), Moraxella, Acinetobacter, Yersinia, Legionella pneumophila, Shigella, Pasteurella, Parahemolytic bacilli, Shigella, preferably Escherichia coli, Salmonella. The gram-positive bacteria are selected from Staphylococcus, Streptococcus, Diplococcus pneumoniae, Bacillus anthracis, Bacillus diphtheriae, and Bacillus tetani. The pathogenic bacteria are selected from Streptococcus pneumoniae, Staphylococcus aureus, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Pseudomonas aeruginosa, anaerobe, Enterobacter, Klebsiella, Haemophilus influenzae, Serratia marcescens, Proteus proteus, Bacillus aneroides, Clostridium botulinum, Salmonella, and Vibrio parahaemolyticus. The probiotic bacteria are selected from Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus mansonii, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium ovorans, Bifidobacterium thermophilum, Streptococcus faecalis, lactococcus lactis, Streptococcus intermedius, yeast, Lactobacillus rhamnosus, and Lactobacillus paracasei, preferably Lactobacillus casei, Bifidobacterium thermophilum, lactococcus lactis, and Lactobacillus rhamnosus.

When the taxol liposome-loaded bacterial inhalation preparation is prepared, a chemical method and a physical method can be adopted as the preparation method. The chemical method can combine paclitaxel with bacteria via chemical bond, and can load drug after bacteria are prepared into protoplast carrier; bacteria can also be conjugated to drugs by recombinant plasmids; chemical methods often have a large impact on the activity of the bacteria themselves. The physical method mainly comprises a centrifugal method, an ultrasonic method and an electric pore-forming method. The taxol liposome-loaded bacterial inhalation preparation prepared by the electroporation method has high bacterial survival rate and high taxol liposome encapsulation rate.

The taxol-loaded liposome bacteria inhalation preparation is used for treating various lung cancers.

The bacteria have small grain size and rapid propagation, can selectively enter tumor tissues for rapid propagation and play a role in resisting tumors. The compound is used as an active carrier, so that the utilization rate of the medicine is improved, and the problems of poor water solubility, poor absorption, instability, great toxic and side effects and the like of a plurality of medicines can be solved. The medicine is wrapped in the medicine, so that the degradation of the medicine is reduced, and the stability is improved; meanwhile, the targeting property of the medicament is improved, the medicament is promoted to enter tumor cells, and the absorption is increased. The viable bacteria carrier has outstanding advantages when being used for lung inhalation administration of the antitumor drug. The taxol-loaded liposome bacterium inhalation preparation can enter the deep part of lung tissue after being inhaled into the lung, directly trend to lung tumor and rapidly propagate at the tumor part; the concentration of the carried medicine in lung tumor tissues is greatly increased, and the medicine effect can be quickly exerted; meanwhile, the medicine in normal cell tissues is less, and the toxic and side effects brought by the medicine are reduced. The lung inhalation mode of the paclitaxel liposome bacterium inhalation preparation can be inhalation after atomization, can also be directly injected by combining a respirator and a lung lavage device, and can also be inhalation in a powder inhalation mode after freeze drying.

Drawings

FIG. 1 photograph of paclitaxel liposome solid after redispersion

FIG. 2 shows the particle size distribution of paclitaxel liposome

FIG. 3 transmission electron micrograph of paclitaxel liposome

FIG. 4 Effect of electroporation on bacteria

FIG. 5 Effect of different concentrations of paclitaxel liposomes on bacterial survival

FIG. 6 Effect of electroporation on bacterial survival

Figure 7 toxicity of each formulation against a549 cells. PTX: paclitaxel; and (3) LP: a paclitaxel liposome; LP/E: physical mixture of paclitaxel liposome and E.coli (without electroporation); LP/L: physical mixture of paclitaxel liposome and lactobacillus casei (no electroporation); LPE: paclitaxel liposome-loaded E.coli (after electroporation); LPL: lactobacillus casei loaded with paclitaxel liposome (after electroporation). P < 0.05; p < 0.01.

FIG. 8 flow assay of each formulation for apoptosis of A549 cells. The significance of the symbols in the figure is illustrated in figure 7, where the percentages are percentages of apoptosis.

FIG. 9 is a confocal microscope of in vitro phagocytosis of cells from each formulation. LR: a rhodamine liposome; LR/E: physical mixture of rhodamine liposomes and E.coli (without electroporation); LR/L: physical mixture of rhodamine liposomes and lactobacillus casei (no electroporation); LRE: escherichia coli carrying rhodamine liposome (after electroporation); LRL: lactobacillus casei carrying rhodamine liposome (after electroporation).

FIG. 10 appearance of lung tissue in rats of each group. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 11 is a photograph of pathological sections of HE-stained lung tissues of rats in each group. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 12 is a photograph of pathological sections stained with Caspase-3 from rat lung tissues of each group. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 13.Tunel assay for apoptosis in lung tissue of various groups of rats. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 14 shows the distribution of homogenized colonies from rat organs. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 15 immunoblotting of lung tissue Bcl-2 and HIF-1. alpha. protein levels. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 16. white blood cell and neutrophil counts in rat blood. The meaning of the reference numbers in the figure is illustrated in figure 7.

FIG. 17 shows the change of the contents of VEGF, TNF- α, IL-4 and IFN- γ in rat lung tissues of various groups of rat lung tissues. The meaning of the reference numbers in the figure is illustrated in figure 7.

Detailed Description

Example 1 paclitaxel liposomes and lyophilized solids thereof

Preparing paclitaxel liposome by using a film dispersion method: weighing 0.553g of soybean phospholipid and 0.05g of cholesterol in a beaker, adding 5mL of ethanol for ultrasonic dissolution, precisely weighing 0.04g of paclitaxel, adding the paclitaxel into the ethanol solution for dissolution, transferring the solution to a rotary evaporator, carrying out rotary evaporation at 50 ℃ to obtain a uniform film, adding 20mL of water, and continuously carrying out rotary dispersion under the water bath condition to obtain the paclitaxel liposome.

Adding 1.2g mannitol into the above 20mL paclitaxel liposome, dissolving, lyophilizing in a lyophilizer to obtain loose solid, and sieving with 180 mesh sieve to obtain white powder as paclitaxel liposome solid.

A proper amount of paclitaxel liposome solid is dispersed in phosphate buffer solution with pH7.0 to obtain light blue opalescent liquid (figure 1), and no precipitate is generated after the liquid is placed at room temperature for 8 hours. The paclitaxel liposome solid is re-suspended to obtain paclitaxel liposome (figure 2), which has small particle size of about 70nm, and spherical vesicle under transmission microscope with particle size of about 100nm (figure 3).

Example 2 paclitaxel-loaded Liposome bacterial inhalation formulations

Thawing competent Escherichia coli on ice, adding paclitaxel liposome solid of example 1, and incubating on ice for about 5min to dissolve and disperse completely; setting parameters of the electric pore-forming instrument, wherein r is 550V, d is 60ms, s is 50, and i is 100 ms; and (3) carrying out electric pore-forming operation on the liquid for 20 times in total to prepare the taxol liposome-loaded bacterial inhalation preparation.

Replacing the escherichia coli in the preparation process with lactobacillus casei to obtain the taxol-loaded liposome bacteria inhalation preparation.

Experimental example 1 Properties of paclitaxel-loaded Liposome bacterial inhalation preparation

Sample preparation: paclitaxel liposomes prepared in example 1; the paclitaxel-loaded liposome bacterial inhalation formulation prepared in example 2; escherichia coli (e.coli); lactobacillus casei (l.casei).

Under a transmission electron microscope, the bacterial wall after the electroporation has obvious pore channels (figure 4), which can promote the medicine to enter the bacteria.

Through high performance liquid detection, the entrapment rate of the paclitaxel liposome-loaded bacteria after the electroporation effect is higher, and is more than 95%.

in an in vitro environment, the paclitaxel liposome has no obvious influence on the survival of bacteria, and the colony number has no obvious difference compared with a blank control group under the drug concentration from low concentration to high concentration (0.635-5 mmol/L paclitaxel), namely the paclitaxel liposome has no bacteriostatic action (figure 5).

For E.coli, the number of colonies after electroporation was 4.38X 10⁶CFU/mL, the number of colonies in the blank control group was 4.73X 10⁶CFU/mL. For Lactobacillus casei, the number of colonies after electroporation was 4.31X 10⁶CFU/mL, the number of colonies in the blank control group was 4.76X 10⁶CFU/mL. The survival rate of the escherichia coli is 92.6%, the survival rate of the escherichia coli is 90.5%, and the electroporation has no obvious influence on the growth of bacteria (figure 6).

Experimental example 2 cytological study of paclitaxel-loaded Liposome bacterial inhalation preparation

Sample preparation: human lung cancer cell line a 549; paclitaxel; the paclitaxel liposome of example 1; the paclitaxel-loaded liposome bacterial inhalation formulation of example 2; escherichia coli (e.coli); lactobacillus casei (l.casei). Physical mixtures of paclitaxel liposomes and E.coli and of Lactobacillus casei were prepared using the procedure of example 2, but without electroporation. The method comprises the following specific steps: the competent bacteria were frozen in a freezer at-80 ℃, the competent bacteria were thawed on ice, the paclitaxel liposome solid of example 1 was added and incubated on ice for about 5min to allow sufficient dissolution and dispersion to obtain a physical mixture of paclitaxel liposomes and bacteria.

The following English abbreviations represent the respective formulations. PTX: paclitaxel; and (3) LP: a paclitaxel liposome; LP/E: physical mixture of paclitaxel liposome and E.coli (without electroporation); LP/L: physical mixture of paclitaxel liposome and lactobacillus casei (no electroporation); LPE: paclitaxel liposome-loaded E.coli (after electroporation); LPL: lactobacillus casei loaded with paclitaxel liposome (after electroporation).

a549 cells were inoculated in 96-well plates, and the number of cells per well was 5X 10⁵And incubating the cells for 8 hours for adherence, adding PTX, LP, E.coli, LP/E, LPE, L.casei and LP/L, LPL (except pure bacterial preparations, the concentrations of PTX are 0.625-5 mu M respectively) with different concentrations into a Cell culture hole, incubating for 24 hours, adding a Cell Counting Kit-8(CCK-8) solution, continuously incubating for 2 hours, detecting the absorbance value at 450nm, and calculating the Cell survival rate of each group of medicines. A549 cells were seeded in 6-well plates (5X 10)⁵Perwell) was incubated at 37 ℃ for 8 h. After the formulation is added, the reaction lasts for 24h, and the apoptosis of A549 cells is detected according to the method of an Annexin V-FITC/PI kit operating manual. The effect of each formulation on apoptosis was examined by flow cytometry.

Phagocytosis of cells: liposomes and bacterial formulations of rhodamine (rhodamine) were prepared according to examples 1 and 2, and the methods reported in this experimental example, to give LR: a rhodamine liposome; LR/E: physical mixture of rhodamine liposomes and E.coli (without electroporation); LR/L: physical mixture of rhodamine liposomes and lactobacillus casei (no electroporation); LRE: escherichia coli carrying rhodamine liposome (after electroporation); LRL: lactobacillus casei carrying rhodamine liposome (after electroporation).

Measured by laser confocal microscopy, 5X 10⁵A549 cells were inoculated on a 2X 2mm slide glass, cultured at 37 ℃ for 8 hours, and the concentration of the cells was controlled to 10⁶CFU/mL and rhodamine concentration of 5 μ M, adding the above formula into cell culture solution, taking out the slide at 0.5, 2, 4h, washing with Phosphate Buffer Solution (PBS) for 3 times, fixing with 4% paraformaldehyde for 15min, washing with PBS for 3 times, staining with DAPI for 10min, sealing with immunofluorescent agent, and observing with laser confocal microscope.

As a result:

Coli and l.casei alone (10)⁶CFU/mL) has higher cytotoxicity to A549 cells, and the cell survival rates are 45.6% and 51.9% respectively. PTX vs. A549 finecells were somewhat cytotoxic, with LP toxicity at the same concentration greater than PTX effect and concentration dependent (figure 7). The liposome has better cell membrane permeability and is easy to be endocytosed by cells, and the medicine is promoted to enter the cells. The anti-A549 cell effect of the bacterial preparation containing the paclitaxel liposome is larger than that of the single paclitaxel and the paclitaxel liposome group, wherein the toxicity of the bacterial inhalation preparation (LPE, LPL) carrying the paclitaxel liposome to the A549 cell is larger than that of the corresponding physical mixed group (LP/E, LP/L).

Drug pair apoptosis status with flow assay (fig. 8), bacteria e.coli and l.casei alone (10)⁶CFU/mL) of the strain has apoptosis rates of 7.7% and 3.8% on A549 cells respectively, apoptosis rate of single LP on the A549 cells is 13.6%, the apoptosis rate of combined action of the bacteria and the LP is far higher than that of the single LP, and the apoptosis rate of the LPE and LPL groups on the A549 cells is the highest and far greater than that of the LP/E and the LP/L, which shows that the cytotoxic effect of the bacteria serving as a drug carrier is greatly increased.

the phagocytosis experiment shows that the amount of the drug entering cytoplasm of the rhodamine liposome-loaded bacterial preparation is increased along with the increase of time (figure 9), but the capacity of entering cells of the rhodamine liposome-loaded bacterial preparation (LRE and LRL) in each time period is obviously higher than that of the rhodamine liposome alone and a mixed group of the rhodamine liposome and the bacteria (LR/E, LR/L), and the surface bacterial carrier can promote the drug to enter the cells to exert the drug effect.

Experimental example 3 study of drug effect on treating rat Lung cancer

45 male rats (average weight 180 + -20 g/rat) were randomly divided into 9 groups of 5 rats, and after ether anesthesia, the other groups were intratracheally injected with 0.1mL of 10% trimethylxanthene iodized oil solution (m/v) at a constant rate except for the normal control group through the trachea. The primary lung cancer is spontaneously formed under the induction of a cancer inducer after being respectively bred for 45 days, and then is treated. Grouping was as follows (a) normal group control (normal), endotracheal intubation given saline, 0.2 mL; (b) lung cancer model group (model), saline, 0.2 mL; (c) lactobacillus casei, 10⁶CFU (l.casei); (d) escherichia coli, 10⁶CFU (e.coli); (e) paclitaxel liposome, paclitaxel 1mg (LP); (f) lactobacillus casei and paclitaxel liposome mixture, 10⁶CFU + paclitaxel 1mg (LP/L); (g) coli and paclitaxel liposome mixture, 10⁶CFU + paclitaxel 1mg (LP/E); (h) lactobacillus casei carrying paclitaxel liposome, 10⁶CFU/paclitaxel 1mg (LPL); (i) paclitaxel-loaded liposome Escherichia coli, 10⁶CFU + paclitaxel 1mg (LPE). The administration mode is realized by trachea spraying. The administration is carried out twice, once a week, the administration is carried out by injecting the rat into the trachea at the same time every day, 20 mu L of blood is taken through the tail vein before the rat is sacrificed 10 days after the final administration, and the content of the white blood cells and the content of the neutral granulocytes are measured by a blood cell counter after the dilution with a proper amount. Each group of rats was sacrificed by anesthesia with 1mL of 10% chloral hydrate, the trachea was dissected and exposed, the chest was opened to expose the main and bronchial tubes clearly to the operative field, and a small T-shaped opening was cut at the main trachea. The right bronchus was ligated with hemostats. The front end of the self-made catheter is embedded into the left main trachea. Extracting 2mL of 4 ℃ physiological saline, slowly injecting the physiological saline into the left lung through a catheter, observing the filling of the left lung, slightly withdrawing the perfusion liquid after the physiological saline stays in the lung for 30s, injecting the perfusion liquid into the lung, repeatedly withdrawing and injecting for 3 times, completely withdrawing the injected liquid, transferring the liquid into a 5mL centrifuge tube, and repeatedly irrigating for 1 time. The lavage solutions were pooled for 2 times, 4mL in total, centrifuged at 3500rmp for 10min, the supernatant aspirated, and stored at-80 ℃. Taking right lung tissue, fixing the upper lung leaves in 10% formaldehyde, conventionally dehydrating, embedding, staining by hematoxylin-eosin (HE), simultaneously marking Casepase-3 protein by immunohistochemistry, observing pathological morphological change of the lung tissue and expression condition of Casepase-3 under a light microscope, carrying out ELISA detection on tumor necrosis factor TNF-alpha, tumor necrosis factor VEGF, interleukin-4 IL-4 and hypoxia transcription factor IFN-gamma after tissue homogenization on the middle lung leaves, and analyzing apoptosis inhibiting protein Bcl-2 and hypoxia transcription factor HIF-1 alpha by immunoblotting on the lower lung leaves. Homogenating and culturing each tissue organ of the rat, counting the colony number, and observing the distribution in the bacteria-carried tissue.

As a result:

It can be seen from the appearance of the freshly taken lung tissue that the normal lung tissue is ruddy and glossy, there are a lot of white nodules and bleeding of lung cancer in the lung cancer group, the white nodules of lung tissue in each treatment group are obviously reduced, and the symptoms of the LPL and LPE groups are obviously alleviated (FIG. 10). After the healthy rats are fed with normal saline, the lung tissue structure is complete, and the alveolar spaces are clear. Lung cancer model group rats had destroyed pulmonary alveolar structure and a large amount of inflammatory tissue was proliferated. The pathological conditions were significantly improved in each treatment group. The paclitaxel-loaded liposome bacteria group had better improvement effect (figure 11).

Caspase-3 is the last loop of the apoptotic cascade and it is reported in the literature that paclitaxel can promote Caspase-3 expression. In this study, the model group had almost no effect of yellow staining, the yellow staining increased in each treatment group, and the effects of LPL and LPE groups were best (FIG. 12).

Apoptosis was detected in lung tissue by the Tunel method. Lung tissue apoptosis was less in the model group, apoptosis increased in the treated group, less in the l.casei and e.coli groups alone, significantly increased in the LP/L and LP/E groups than the LP and bacteria groups alone, and most in the LPL and LPE groups (fig. 13).

As can be seen from homogenate of each organ tissue, the number of colonies in lung tissue was very high in each treatment group, much higher than that in other organs (FIG. 14). Comparing the number of colonies in other organs, wherein a plurality of colonies exist in the central tissue, and the heart is probably closely related to the lung tissue; the presence of small amounts of bacteria in liver tissue is associated with metabolic functions of the liver; other tissues were substantially free of bacteria (fig. 14). The taxol liposome-carried bacterial inhalation preparation has obvious lung tissue deposition effect, and can bring chemotherapeutic drugs to tumor parts while treating tumors, so that the taxol liposome-carried bacterial inhalation preparation has an anti-tumor effect.

When lung cancer occurs, a large amount of lung cancer cells are proliferated, and the expression of anti-apoptosis protein is inhibited, so that apoptosis is reduced. Bcl-2 is an apoptosis inhibiting protein which can prevent cytochrome C in mitochondria from releasing outwards and inhibit apoptosis. HIF-1 α is a hypoxia regulated transcription factor that regulates the expression of a variety of hypoxia inducible genes. Expression of HIF-1 α protein levels is increased when tumors grow rapidly, leaving the tumor tissue relatively hypoxic. Thus, the oxygenation status of tumor tissue can be assessed by measuring the amount of HIF-1. alpha. protein. In addition, hypoxic conditions also induce expression of VEGF, thereby increasing tissue permeability and inducing angiogenesis. The protein levels of Bcl-2 and HIF-1 alpha are respectively detected by western blotting, and the results show that each formula promotes the reduction of the levels of Bal-2 and HIF-1 alpha, wherein the reduction of LPL and LPE groups is the most, which indicates that the taxol-loaded liposome bacterial inhalation preparation has obvious promotion effect on lung cancer cell apoptosis (figure 15).

Leukocytes and neutrophils are typical immune functional cells that clear foreign bacteria, viruses and mutated cells. After the bacterial preparation is used for treatment, the white blood cells and the neutrophils in blood are obviously increased, which indicates that the bacterial preparation can stimulate immunity after the action. The LP group was not significantly different from the normal group. After the action of each bacterial preparation group, the amount of leukocytes and neutrophils was significantly increased (fig. 16). Therefore, the bacteria stimulate an innate immune response in vivo and thus exert an antitumor effect.

TNF-alpha is a cellular immune mediator, can directly kill tumor cells, and does not harm normal cells. IL-4 is a marker cell for Th2 and plays an important role in humoral immune responses. After treatment with the bacterial preparation, the TNF-alpha and IL-4 factor levels are obviously increased, wherein the expression of LPL and LPE groups is higher than that of the L.casei and E.coli groups (figure 17) of single bacteria, thus obviously inducing the secretion of cell factors, generating immunoregulation action and enhancing the anti-tumor efficacy. Gamma interferon (IFN-gamma) is a kind of secretory protein with broad-spectrum antiviral, antitumor and immunoregulatory functions. In this study, IFN- γ expression was lower in the model group and increased in each bacterial preparation group. The group administered with bacteria alone was significantly higher than the group administered with paclitaxel liposomes. The paclitaxel-loaded liposome-containing bacterial inhalation formulation group was significantly increased compared to the bacterial administration group alone (fig. 17). The bacteria can remarkably promote CD⁸⁺The T cell secretes IFN-gamma to delay the growth of tumor. These results all indicate that the bacteria are primarily involved in CD in the tumor microenvironment⁸⁺t cell mediated anti-tumor immune responses. The bacteria can increase the CD in the tumor by increasing the immune response level in the tumor⁸⁺T cells and CD^11b+The proportion of the mononuclear-macrophage induces the mononuclear-macrophage to generate tumor necrosis factors TNF-alpha and IFN-gamma, destroys the immune escape environment in the tumor and generates the anti-tumor effect.

VEGF is a major regulator of induction of angiogenesis and is capable of promoting the growth and high density vascularization of a variety of tumor tissues. In this study, VEGF-expressing lung cancer model groups showed the highest expression, and each treatment group was significantly reduced compared to the model group, and the ratio of LPL to LP/L group, and the ratio of LPE to LP/E group, the former further reduced VEGF expression, with significant differences (fig. 17).

The chemotherapy drug paclitaxel is prepared into liposome and then combined with bacteria, the targeting property of the paclitaxel is improved by using the bacteria as a carrier, and the local administration mode is realized by a pulmonary inhalation administration technology, so that the toxic and side effects are greatly reduced. Through the exploration of a pharmacodynamic mechanism, the taxol-loaded liposome bacterium inhalation preparation can reduce the expression of Caspase-3 and Bcl-2, reduce the expression of VEGF, inhibit the proliferation of tumors, enhance the anti-tumor effect, promote the generation of factors such as TNF-alpha, IL-4, IFN-gamma and the like, enhance the anti-tumor effect through immune regulation, and is a novel method for treating lung cancer.

Claims

1. A paclitaxel-loaded liposome bacterial inhalation preparation.

2. The paclitaxel-loaded liposome bacterial inhalation formulation of claim 1, prepared by the following steps:

(1) Preparing paclitaxel liposome;

(2) Selecting a suitable bacterium as a vector;

3. The paclitaxel-loaded liposome bacterial inhalation formulation of claim 1, prepared by the following steps:

(1) Preparing paclitaxel liposome;

(2) Preparing paclitaxel liposome into paclitaxel liposome solid;

(3) Selecting a suitable bacterium as a vector;

4. the taxol liposome-loaded bacterial inhalation formulation as claimed in claims 2 and 3, wherein the taxol liposome is prepared by a membrane dispersion method.

5. The taxol-loaded liposome bacterial inhalation formulation of claim 3, wherein the taxol liposome solid is prepared by freeze-drying.

6. The paclitaxel-loaded liposome bacterial inhalation formulation of claims 2 and 3, wherein the bacteria are selected from the group consisting of gram-negative bacteria and gram-positive bacteria.

7. The paclitaxel-loaded liposome bacterial inhalation formulation of claims 2 and 3, wherein the bacteria are selected from the group consisting of E.coli and Lactobacillus casei.

8. The paclitaxel-loaded liposome bacterial inhalation formulation of claim 1, prepared by the following steps:

Weighing 0.553g of soybean phospholipid and 0.05g of cholesterol in a beaker, adding 5mL of ethanol for ultrasonic dissolution, precisely weighing 0.04g of paclitaxel, adding the paclitaxel into the ethanol solution for dissolution, transferring the solution to a rotary evaporator, carrying out rotary evaporation at 50 ℃ to obtain a uniform film, adding 20mL of water, and continuously carrying out rotary dispersion under the water bath condition to obtain paclitaxel liposome; adding 1.2g of mannitol into the 20mL of paclitaxel liposome, dissolving, freeze-drying in a freeze-drying machine to obtain loose solid, and sieving with a 180-mesh sieve to obtain white powder as paclitaxel liposome solid; thawing competent Escherichia coli on ice, adding paclitaxel liposome solid, and incubating on ice for about 5min to dissolve and disperse completely; setting parameters of the electric pore-forming instrument, wherein r is 550V, d is 60ms, s is 50, and i is 100 ms; and (3) carrying out electric pore-forming operation on the liquid for 20 times in total to prepare the taxol liposome-loaded bacterial inhalation preparation.

9. The paclitaxel-loaded liposome bacterial inhalation formulation of claim 1, which is used for treating various lung cancers.