CN113398275B - Bacterial vector for photodynamic therapy and preparation method and application thereof - Google Patents
Bacterial vector for photodynamic therapy and preparation method and application thereof Download PDFInfo
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- CN113398275B CN113398275B CN202010188454.1A CN202010188454A CN113398275B CN 113398275 B CN113398275 B CN 113398275B CN 202010188454 A CN202010188454 A CN 202010188454A CN 113398275 B CN113398275 B CN 113398275B
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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
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a bacterial carrier for photodynamic therapy and a preparation method and application thereof, wherein non-pathogenic escherichia coli is used as a carrier, a photosensitizer is loaded, photodynamic therapy is combined, photodynamic therapy on tumors is realized, delivery of the photosensitizer is realized, uptake of the photosensitizer by tumor cells is obviously improved, sensitivity of focuses to photodynamic therapy is increased, treatment effect can be obviously improved, and biological safety is good.
Description
Technical Field
The invention relates to the field of bacterial drug loading, in particular to a bacterial carrier for photodynamic therapy and a preparation method and application thereof.
Background
Bacterial treatment of tumors has become a hotspot of recent research and has made breakthrough progress since Coley discovered that bacteria were able to treat tumors over 100 years ago. The hypothesis of the bacterial mechanism of tumor chemotaxis can be summarized as three points: (1) The immune escape mechanism caused by the tumor site provides a shelter for bacteria; (2) The environment of the tumor and surrounding tissues with acidic property is suitable for bacterial growth; (3) The hypoxic environment of the tumor site, as well as the necrotic area of the core, all provide an ideal living environment for the bacteria. Most of the current researches on bacterial therapies are in the aspect of gene modification, the bacterial gene modification is simple to operate, the effect is remarkable in the preclinical researches, but the bacterial gene modification has the defect that the retention time of bacteria at a tumor site is not longer than 15 days generally, and the amount of protein expressed by engineering bacteria at the tumor site cannot effectively inhibit the growth of tumors. Thus, bacterial therapies are often used as a supplement to gene therapy oncology methods.
There are many bacterial species that can accumulate inside tumors, and more than ten bacteria have been found to be effective in mouse tumor models. Most of the bacteria are pathogenic bacteria, and the structure of Lipopolysaccharide (LPS) is changed by mutating the musB gene, so that pathogenicity of the Lipopolysaccharide (LPS) is eliminated or weakened, septicemia caused by the bacteria is avoided, and a good anti-tumor effect is achieved in a preclinical experiment, but the number of the bacteria to be administered is still low. The non-pathogenic escherichia coli DH5 alpha has good biological safety, and can be used as a drug carrier because the non-pathogenic escherichia coli DH5 alpha can be injected into mice intravenously in large dose without death.
Photodynamic therapy (PDT) has the characteristics of being rapid, efficient, low-toxic in tumor treatment, and has been approved by the United states food and drug administration for use in specific tumor treatments. The phthalocyanine type photosensitizer is widely used in anti-tumor research, and the phthalocyanine type compound can generate singlet oxygen and related Reactive Oxygen Species (ROS) to kill surrounding tissues and cells under the irradiation of near infrared band light, so that good anti-tumor effect is achieved in preclinical research. At present, the research on phthalocyanine is mostly carried out on the transformation of phthalocyanine molecules, and a modification targeting group targets a specific tumor, but the targeting photosensitizers often do not play a good role in treatment, and the main reason is that the drug cannot enter the inside of the tumor due to the rejection of the tumor to the drug, so that the effect of the drug is greatly reduced.
Disclosure of Invention
The invention aims to provide a bacterial vector, a preparation method and application thereof.
In order to achieve the purpose of the invention, the following technical scheme is adopted:
the invention provides the application of non-pathogenic escherichia coli as a photosensitizer carrier for photodynamic therapy.
According to an embodiment of the present invention, the photosensitizer is a photodynamic therapy photosensitizer known in the art, such as a phthalocyanine type photosensitizer, a benzylidene cycloalkanone photosensitizer, rose bengal (rose bengal), tin ethyl purplish, hematoporphyrin or 5-aminolevulinic acid hydrochloride, etc.; phthalocyanine type photosensitizers are preferred.
The invention also provides a bacterial carrier, which comprises escherichia coli and a photosensitizer, wherein the photosensitizer is loaded on the escherichia coli.
According to an embodiment of the invention, the E.coli is a non-pathogenic E.coli, for example E.coli DH 5. Alpha.
According to an embodiment of the present invention, the loading ratio of the E.coli and the photosensitizer is not particularly limited, and one skilled in the art can determine the concentration of E.coli, for example, 10, according to an effective amount of photosensitizer required for photodynamic therapy 9 The saturation concentration of the photosensitizer loaded by the CFU/ml escherichia coli is 100 mu M. In one embodiment, every 10 5 The concentration of the CFU/ml of the Escherichia coli-loaded photosensitizer is 1-25. Mu.M.
According to an embodiment of the present invention, the photosensitizer is a photodynamic therapy photosensitizer known in the art, such as a phthalocyanine type photosensitizer, a benzylidene cycloalkanone photosensitizer, rose bengal (rose bengal), tin ethyl purplish, hematoporphyrin or 5-aminolevulinic acid hydrochloride, and the like; phthalocyanine type photosensitizers are preferred. In a specific embodiment, the phthalocyanine-type photosensitizer is a compound of formula I:
the invention further provides a preparation method of the bacterial vector, which comprises the following steps:
(1) Activating escherichia coli to prepare escherichia coli suspension;
(2) And adding a photosensitizer into the activated escherichia coli suspension, and incubating in a dark place to obtain a photosensitizer-loaded bacterial carrier.
According to an embodiment of the present invention, step (1) may be performed using the known E.coli activation method of the present invention, for example, inoculating into LB medium for activation.
According to an embodiment of the invention, the OD of the E.coli suspension according to step (1) 600 The value is 0.6-0.8.
According to an embodiment of the present invention, the photosensitizer in step (2) is a phthalocyanine photosensitizer, for example, the photosensitizer is a compound represented by formula (I).
According to an embodiment of the invention, the final concentration of the photosensitizer added in step (2) is 1-20. Mu.M, e.g. 3-15. Mu.M, and further e.g. 1. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M.
According to embodiments of the invention, the incubation time in the absence of light may be from 0.1 to 1h, for example from 20 to 30min; the light-shielding incubation temperature is 25-37 ℃.
The invention further provides application of the bacterial vector in preparation of photodynamic therapy drugs.
According to an embodiment of the present invention, the photodynamic therapy drug is for treating an individual suffering from a tumor, for example, breast cancer, colon adenoma, colorectal cancer, rectal cancer, anal cancer, small intestine cancer, breast cancer, lung cancer, stomach cancer, liver cancer, lung cancer, lymphocytic lymphoma, pancreatic cancer, skin melanoma, eye melanoma, uterine sarcoma, ovarian cancer, endometrial cancer, cervical cancer, endocrine cancer, thyroid cancer, parathyroid cancer, renal cancer, soft tissue tumor, prostate cancer, bronchial cancer, renal cancer, more specifically, breast cancer or lung cancer, but is not limited thereto.
According to embodiments of the invention, the subject includes humans or non-human primates, as well as other mammals, such as pigs, cows, horses, sheep, goats, dogs, and the like.
The invention also provides a pharmaceutical composition for photodynamic therapy comprising a bacterial vector of the invention and optionally other pharmaceutically acceptable components.
According to embodiments of the present invention, the other pharmaceutically acceptable component may be an antineoplastic agent. The antineoplastic agent may be an antineoplastic agent known in the art.
Photodynamic therapy (PDT) is a method of treating medical conditions using light activated drugs (photosensitizers) in the present invention. Aggregation of photosensitizers in target tissue capable of direct illumination makes PDT a selective treatment. When the photosensitizer is activated by light, singlet oxygen and other free radicals are generated in the tissue where the drug is retained, interactions between these reactive oxygen species and biological macromolecules can cause changes in cellular metabolism and can cause cell death at high doses of drug and/or light, resulting in therapeutic effects.
In the present invention, a "therapeutically effective amount" refers to the amount of a photosensitizer that produces a desired effect, particularly a killing effect on tumor cells, in a patient or subject. The amount will depend on a number of factors including the nature of the photosensitizer, the physical characteristics of the subject and the type of tumor cells. The range of effective amounts required to achieve the desired result of the treatment can be readily determined by one skilled in the art.
Advantageous effects
According to the invention, the nonpathogenic escherichia coli is adopted as a photosensitizer carrier, the photodynamics therapy is combined with the photodynamic therapy by loading an effective amount of photosensitizer on the escherichia coli, so that the photodynamic therapy on tumors is realized, the delivery of the photosensitizer is realized, the uptake of the photosensitizer by tumor cells is obviously improved, the sensitivity of focus on the photodynamic therapy is increased, the treatment effect can be obviously improved, the biological safety is good, and the method has a wide clinical application prospect.
Drawings
Fig. 1: standard curves of emitted light intensities at 680nm for different concentrations of ZnPc-IR710 photosensitizer solutions.
Fig. 2: standard curve of autofluorescence intensity for E.coli DH 5. Alpha. Suspensions at different colony concentrations.
Fig. 3: e.coli DH 5. Alpha. ZnPc-IR710 photosensitizer-carrying capacity curve.
Fig. 4: the in-vitro photodynamic anti-tumor activity of the bacterial vector is schematically shown in the specification, and the bacterial vector is the killing effect of ZnPc-IR710 and E.coli@ZnPc-IR710 with different concentrations in application example 1 on human breast cancer MCF-7 cells.
Fig. 5: the results of the in vitro tumor cell localization imaging experiment of the bacterial vector of the invention are confocal microscopic photographs of MCF-7 cell distribution and ZnPc-IR710 and E.coli@ZnPc-IR710 co-culture in application example 2.
Fig. 6: the experimental result of the in-vitro photodynamic anti-tumor action mechanism of the bacterial vector is the detection result of the MCF-7 cells and E.coli@ZnPc-IR710 in application example 3 after co-culture by a flow cytometer.
Fig. 7: graph of E.coli DH 5. Alpha. Concentration versus mouse body weight.
Fig. 8: the in vivo experimental results of the inhibition effect of the bacterial vector on the mouse tumor show that the tumor weight change condition (A) and the mouse weight change condition (B) of the 4T1 tumor-bearing mouse model in the application example 5 comprise E.coli@ZnPc-IR710 experimental group (2×10) 8 CFU per mouse), e.coli experimental group (2×10 8 CFU per mouse), znPc-IR710 experimental group (400 ng per mouse), znPc-IR710 experimental group (4. Mu.g per mouse), physiological saline negative control group.
Fig. 9: the in vivo imaging result of the inhibition effect of the bacterial vector on the mouse tumor is the in vivo tumor imaging condition (A) and the time change (B) of the concentration of the drug in the mouse body of the 4T1 tumor-bearing mouse model in the application example 6, comprising the experimental group (2X 10 8 CFU per mouse), znPc-IR710 experimental group (4. Mu.g/per mouse, 200. Mu.g/kg).
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1:
1. the activation of the escherichia coli DH5 alpha is carried out by the following culture method:
(1) Preparing an LB culture medium: 1g Trypton,0.5g Yeast Extract,1g NaCl dissolved in 100ml water, pH 7.0, and sterilized at 121deg.C for 20min.
(2) The frozen glycerol bacteria were removed and inoculated into 2ml of LB medium for activation, and cultured overnight at 37℃in a shaker (220 rpm). 100. Mu.l of the activated bacterial liquid was inoculated into 10ml of LB medium (1% inoculation), and the mixture was shaken at 37℃for 4 hours (220 rpm). 200 μl of bacterial liquid is taken, and the OD600 absorption value is read by an enzyme-labeled instrument, and is about 0.6-0.8 for standby.
2. The preparation method of the bacterial vector comprises the following specific steps:
phthalocyanine ZnPc-IR710 (compound of formula I) with four positive charges was selected and prepared as a 2mM solution with DMSO for use.
Samples (1. Mu.M, 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M) of varying gradient concentrations were prepared by diluting ZnPc-IR710 solution with 1% SDS containing 0.1M NaOH, and then excited at 610nm with a microplate reader, the emitted light intensity at 680nm was measured, and a standard curve was prepared as shown in FIG. 1.
And (3) taking 200 mu l of the bacterial liquid prepared in the step one, and reading an OD600 absorption value by using an enzyme-labeled instrument, wherein the value is about 0.6-0.8. By counting, the coliform bacteria liquid is prepared into samples (10) 9 CFU/ml、10 8 CFU/ml、10 7 CFU/ml、10 6 CFU/ml、10 5 CFU/ml、10 4 CFU/ml and 10 3 CFU/ml) and then detected for biological autofluorescence using an enzyme-labeled instrument and a standard curve was prepared as shown in fig. 2.
And 200 mu l of bacterial liquid is taken, and the OD600 absorption value is read by an enzyme-labeled instrument, wherein the value is about 0.6-0.8 for standby. The culture medium was discarded after centrifugation at 4000rpm for 6min at 1ml per tube of the above-mentioned bacterial liquid, resuspended in PBS, and washed again by centrifugation 3 times, with a concentration of E.coli of about 10 9 CFU/ml, znPc-IR710 (final concentration of ZnPc-IR710 1. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M, 1)0. Mu.M, 15. Mu.M, 20. Mu.M), shaking table (150 rpm) at 37℃for 30 minutes in the absence of light, to obtain bacterial vectors loaded with photosensitizer ZnPc-IR710, 3 replicates per group.
The bacterial liquid after the incubation was centrifuged at 4000rpm for 6min, the supernatant was discarded, resuspended in PBS and washed by centrifugation again 3 times.
The above-mentioned centrifugally washed bacteria were lysed with 1% SDS containing 0.1M NaOH, and then excited at 610nm by an enzyme-labeled instrument to measure the light emitted at 680nm, and the resulting product was substituted into the standard curve of fluorescence intensity of the above-mentioned photosensitizer (the standard curve shown in FIG. 1) to calculate the concentration of the photosensitizer. Meanwhile, before bacteria are lysed, the autofluorescence value of the bacteria is measured and substituted into the standard curve of the bacterial count and fluorescence (the standard curve shown in fig. 2) to calculate the bacterial count. Thus, a bacterial drug loading capacity curve as shown in FIG. 3 was obtained.
As can be seen from FIG. 3, it was found that the adsorption capacity of Escherichia coli to a photosensitizer tends to saturate when the photosensitizer concentration reaches 10. Mu.M.
Application example 1
The in vitro photodynamic anti-tumor activity of the bacterial vector is studied. The in vitro photodynamic antitumor activity of the bacterial vectors was accomplished by cytotoxicity assays using the MTT method. The specific implementation steps are as follows:
the human breast cancer MCF-7 cells are subjected to subculture after resuscitating. Cells passed through the 2 passages were observed for cell status under an inverted microscope. The adherent cells should be paved with about 90% of the area of the bottom of the T-25 cell culture flask, so that the number of cells required by the experiment is ensured. Observing cells should be in a good adherence state, eliminating cells in a bad state, and avoiding influencing experimental results. The cell status was observed using an inverted microscope, and then the cells were digested for counting. According to the cell count result, adding a certain amount of fresh 37 ℃ cell culture solution (DMEM culture medium containing 10wt% of fetal calf serum) into the cell suspension, diluting the cell suspension to obtain the required experimental cell concentration (100000/ml), blowing and uniformly mixing, inoculating into 24-hole cell culture plates, adding 1ml of cell suspension into each hole, and adding CO at 37 DEG C 2 The incubator was incubated overnight.
Comparing the pair of ZnPc-IR710 and E.colli@ZnPc-IR 710Phototoxicity of MCF-7 cells. The culture medium in the cell suspension in the 24-well cell culture plate was aspirated, the culture medium containing ZnPc-IR710 and E.coli@ZnPc-IR710 was added, the concentrations of the added ZnPc-IR710 were 0, 20, 50, 100, 200, 400nM, and the cells were incubated in the drug for 2 hours. After 2 hours, the 24-well cell culture plate was aspirated to contain the drug culture solution, and then 1ml of fresh 37℃cell culture solution was added for washing once. The cell culture solution was aspirated for washing, and 1ml of fresh 37℃cell culture solution was added again. After mixing, according to the experimental scheme of phototoxicity, a light source with the wavelength of 670nm is used for irradiating to the light dose of 7.5J/cm 2 . After the illumination is finished, the mixture is placed at 37 ℃ and CO 2 Culturing overnight in an incubator. After 24 hours, the viability of the cells was checked using the MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide) method. The old medium was first aspirated, then 1ml of MTT medium mixed with 0.5mg/ml was added, and after four hours of incubation, blue-violet crystalline Formazan (Formazan) compounds appeared in the cells, and then 500. Mu.l of dimethyl sulfoxide (DMSO) was used to dissolve these Formazan, the light absorption value was measured at 490nm wavelength with an enzyme-labeled instrument, and the survival rate of the cells of the administration group was calculated by colorimetry with the control group. The data obtained were processed by GraphPad Prism statistical analysis software after three parallel experiments and the results are expressed as mean±sem. From the experimental results, the E.coli@ZnPc-IR710 of the MCF-7 cell shows stronger killing effect under the illumination condition. The research shows that the bacterial vector has stronger photodynamic anti-tumor activity in vitro than pure phthalocyanine, as shown in figure 4.
Application example 2
The invention researches the localization imaging in the isolated tumor cells of the bacterial vector, and provides basis for the mechanism analysis of the bacterial vector for treating tumors. In vitro intracellular localization imaging of bacterial vectors is by confocal laser imaging. The specific implementation steps are as follows.
Photosensitizers, by virtue of their own fluorescent nature, fluoresce under specific excitation light. The phthalocyanine type photosensitizer ZnPc-IR710 of the embodiment has absorption in a red light wave band, and can emit characteristic red fluorescence by using excitation light in the red light wave band; the E.coli used in the present invention is a modified luminescent E.coli (modification of E.coli is carried out with reference to Zhang Y, zheng K, chen Z, et al Rapid killing of bacteria by a new type of photosensizer. Applied microbiology and biotechnology,2017,101:4691-4700;Chen Z,Zhou S,Chen J,et al.An effective zinc phthalocyanine derivative for photodynamic antimicrobial chemotherapy.Journal of Luminescence,2014,152:103-107), and the modified E.coli has the property of emitting green fluorescence under 488nm excitation light for positioning of bacteria in tumor cells.
Cells were cultured in the same manner as in example 1, and then MCF-7 cells (5X 10 4 Each ml,2 ml) was inoculated on a specific confocal dish and cultured in an incubator at 37℃for 18 hours to allow the cells to adhere. 1ml of a medium containing ZnPc-IR710 and E.coli@ZnPc-IR710 was added to the cell culture dish at a concentration of 100nM. After incubation for 2h, the floating E.coli was removed by rinsing gently with 1ml of PBS buffer for 6 times, then 1ml of medium was added and observed using a confocal microscope (detection pathway: bacterial fluorescence: excitation light 488nm, emission light 500-550nm; phthalocyanine fluorescence: excitation light 641nm, emission light 650-1000 nm). From the experimental results, as shown in fig. 5, the tumor cells showed stronger uptake of e.coll@znpc-IR 710 than the simple phthalocyanine type photosensitizer. The research shows that the bacterial vector has stronger tumor targeting property than a pure phthalocyanine photosensitizer in vitro.
Application example 3
The in-vitro anti-tumor mechanism of the bacterial vector is studied in depth, and the tumor killing way is analyzed, so that a basis is provided for the mechanism analysis of the bacterial vector for treating tumors in vivo. The in vitro tumor killing pathway of bacterial vectors was analyzed by flow cytometry. The method comprises the following specific steps:
reactive oxygen species can promote apoptosis in cells, which die through apoptotic pathways after treatment of the cells with PDT. Therefore, we used apoptosis and necrosis detection kit (Apoptosis and Necrosis Assay Kit, from bi-cloudy days) to detect apoptosis. Double staining method using Hoechst 33342 and Propidium Iodide (PI). Chromatin can shrink as cells undergo apoptosis. Hoechst 33342 can penetrate cell membranes, and the fluorescence of the apoptosis cells after staining can be obviously enhanced compared with that of normal cells. PI cannot penetrate cell membranes and cannot stain normal or apoptotic cells with intact cell membranes. Whereas for necrotic cells, the integrity of their cell membranes is lost, PI can stain necrotic cells.
Cells were cultured in the same manner as in application example 1, and the cell state was observed using an inverted microscope and plated (six-well plate). After digestion of MCF-7 cells, the cells were resuspended and inoculated 5X 10 5 The cells were cultured overnight in six well plates. The next day, the experimental group was added with E.coli@ZnPc-IR710 (100 nM), incubated for 30min, and the non-ingested drug was washed off, and then irradiated with 670nM LED flat-panel light source at a light dose of 3.0J/cm 2 . The irradiated MCF-7 cells are washed by PBS and then digested by pancreatin, and are collected in a culture medium, and the apoptosis condition is detected by a flow cytometer. ZnPc-IR710 (100 nM) was added to the control. Both control and experimental groups collected about 30 ten thousand cells in a 1.5ml EP tube, centrifuged first and the supernatant was aspirated. The precipitated cells were resuspended in 1ml of cell staining buffer. Mu.l Hoechst and 5. Mu.l PI staining solution were added. Incubation is carried out for 20 minutes at 4 ℃, and the incubation is carried out for a plurality of times. And then detected by a flow cytometer (detection path: PI fluorescence: excitation light 638nm, emission light 675-725nm; hoechst fluorescence: excitation light 405nm, emission light 450-510 nm). The experimental results are shown in FIG. 6, and E.coli@ZnPc-IR710 kills tumor cells through the apoptosis pathway of the cells. Research shows that the killing mechanism of the bacterial vector to tumor cells in vitro is apoptosis of the tumor cells caused by active oxygen.
Application example 4
The biological safety of the escherichia coli DH5 alpha is researched, and further safety support is provided for bacteria serving as a drug carrier. The biosafety of bacteria in mice is achieved by changes in weight and behavior of mice. The method comprises the following specific steps:
kunming mice (4-6 weeks old, purchased from SLAC laboratory animals Co., shanghai, china) were maintained and treated as recommended by the animal protection and use Committee (IACUC) of the university of Fujian, china. Small sizeMice were acclimated in light (12:12 h light and dark cycle), humidity (50-55%) and temperature controlled chambers. Throughout the course of the experiment, mice were able to obtain food and water ad libitum. Mice (4-6 weeks, -23 g) were randomly divided into 4 groups (6 per group). The initial body weights of the groups were identical. Shaking, and taking 200 μl of bacterial liquid, and reading OD600 absorption value with a Bio-Tek microplate reader, wherein the value is about 0.6-0.8. Mice were injected with 100. Mu.l of E.coli by tail vein, diluted with PBS at a concentration of 10 7 、10 8 、10 9 、10 10 CFU/ml. Changes in body weight and behavior (diet and activity) of mice were detected daily for one month after injection of non-photosensitizer-loaded E.coli DH 5. Alpha. The experimental results are shown in FIG. 7, and it can be seen that each mouse was injected 10 times 8 The CFU escherichia coli can not cause obvious behavior change and weight change of the mice in a continuous observation period of one month, the weight can be quickly restored to normal weight within a few days after administration, and the escherichia coli injection has no obvious toxicity to the mice and has good biological safety.
Application example 5
The anti-tumor activity of the bacterial vector in the mouse body is subjected to preliminary research, and experimental basis is provided for popularization of the bacterial vector to clinical application. The antitumor activity of bacterial vectors in mice was accomplished by a 4T1 tumor implantation model. The specific experimental procedure is as follows.
The back of female Kunming mice was inoculated subcutaneously with a 4T1 murine breast cancer cell suspension, creating a stable 4T1 mouse tumor model: taking pre-frozen 4T1 cell suspension, resuscitating the cells, performing expansion culture, then digesting by using trypsin to obtain the cell suspension, centrifuging at 1000rpm for 5 minutes, and discarding the supernatant. The obtained 4T1 cells were then counted and diluted to 1X 10 with physiological saline 7 200 μl/ml of the strain was inoculated on the right side of the back of the mouse, and after inoculation, a tumor bubble of about soybean size was observed to be located at the inoculation site, and disappeared about 1 day, i.e., the inoculated cells were gradually absorbed and permeated by subcutaneous tissue. After inoculation, the tumor condition is observed every day, the weight of the mice is weighed, and after about 3 to 5 days of inoculation, the tumor volume is as long as 50mm 3 Proved to be successful in establishing a tumor model of the 4T1 mouse. Tumor volumeThe calculation method of (a) is v=w 2 X L x 0.5, w represents the shortest diameter of the tumor and L represents the longest diameter of the tumor.
Randomly dividing the established 4T1 tumor-bearing mouse model into 5 groups of 8 mice each, wherein the groups are respectively as follows: E.coli@ZnPc-IR710 experimental group (2X 10) 8 CFU per mouse), e.coli experimental group (2×10 8 CFU per mouse), znPc-IR710 experimental group (400 ng per mouse), znPc-IR710 experimental group (4. Mu.g per mouse, 200. Mu.g/kg), physiological saline negative control group. Wherein, the E.coli@ZnPc-IR710 experimental group and the ZnPc-IR710 experimental group (400 ng of ZnPc-IR710 per mouse) have the same dosage of the photosensitizer. The back tumor of the mice reaches 50mm 3 The drug was administered by tail vein injection at the indicated dose (all drugs were dissolved in 200 μl physiological saline). To avoid interfering with photodynamic therapy, each group of mice was dehaired with a depilatory agent at the tumor inoculation site of each group of mice. After 24 hours of administration, the light source with the wavelength of 640-720nm is utilized, and the light dose is 30J/cm 2 Photodynamic therapy is performed on the 4T1 solid tumor site. Tumor volumes were then measured daily using vernier calipers and mouse weights were measured using balances. The experimental results are shown in fig. 8, and the experimental results show better photodynamic treatment effect on 4T1 mice transplanted tumor e.coll@znpc-IR 710 under the illumination condition, and the weights of all groups of mice are not changed obviously. The research shows that the bacterial vector has stronger anti-tumor activity than the pure phthalocyanine photosensitizer and bacteria in mice, and the no obvious change of the weight of the mice indicates that the biological safety of the bacterial vector is better.
Application example 6
The bacterial vector provided by the invention is subjected to preliminary research on tumor imaging effects in mice, and experimental basis is provided for popularization of the bacterial vector to clinical application. Bacterial vectors were imaged in mice against tumors by FMT mouse imager. The specific experimental steps are as follows:
by virtue of FMT (Fluorescence Molecular Tomography ) technology, living animal fluorescence tomography of small animals is developed, so that the information of the metabolism process related to the phthalocyanine photosensitizer can be obtained at the living animal level. In this study, in order to monitor the photosensitizer ZnPc-IR710 at the tumor site of miceWe utilized the FMT small animal in vivo fluorescence tomography system (PerkinElmer VisEn FMT 2500) TM LX, waltham, MA) to monitor the distribution and metabolism of photosensitizers in mice. All scans are performed under the same conditions, namely, the mode of 670nm laser bottom penetration scanning and ultrasonic probe depth positioning is used for obtaining 10 ten thousand-level quantity of fluorescence information with different fault depths, and three-dimensional fault signal scanning and reconstruction are realized by combining 3D reconstruction and analysis software, so that three-dimensional quantitative information such as the form, concentration, volume and the like of target fluorescence is obtained.
Randomly dividing the established 4T1 tumor-bearing mouse models into 5 groups, wherein the groups are as follows: E.coli@ZnPc-IR710 experimental group (2X 10) 8 CFU per mouse), znPc-IR710 experimental group (4. Mu.g/per mouse, 200. Mu.g/kg). The back tumor of the mice reaches 75mm 3 The drug was administered by tail vein injection at the indicated dose (all drugs were dissolved in 200 μl physiological saline). After administration, to avoid interference of hair with the imaging results, each group of mice was dehaired at the tumor inoculation site with a depilatory agent. Calibration was performed using 1. Mu.M ZnPc-IR710 and physiological saline, respectively, at the time of quantification. The mice are subjected to gas anesthesia treatment before entering the living body imager, so that fluorescence errors caused by the movement of the mice are avoided. We imaged mice at 5 time points (6 h, 12h, 24h, 48h, 96 h) after intravenous drug administration at the tail of the mice, respectively. And scanning analysis is carried out by using a small animal living body fluorescence tomography system. The experimental results are shown in fig. 9A, and the 4T1 mouse engraftment tumor e.colli@znpc-IR 710 showed better imaging effect at all time points. Research shows that the bacterial vector has stronger imaging effect in mice than a pure phthalocyanine photosensitizer, and shows that bacteria can more efficiently carry the phthalocyanine photosensitizer to tumor sites to exert photodynamic effect; as can be seen from fig. 9B, the drug concentration in the mice reached the maximum 24 hours after injection administration, and the drug concentration of the e.coll@znpc-IR 710 experimental group was significantly higher than that of the ZnPc-IR710 experimental group without e.coll load, indicating that the delivery amount of the photosensitizer was effectively increased by using e.coll as a carrier, and the effective delivery thereof was realized.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. Application of escherichia coli in preparation of photosensitizer carrier for photodynamic therapy, wherein escherichia coli is non-pathogenic escherichia coli DH5 alpha, and the photosensitizer is adsorbed on the surface of escherichia coli every 10 5 The concentration of the CFU/ml escherichia coli loaded photosensitizer is 1-25 mu M, and the photosensitizer is a compound shown in a formula (I):
the preparation method of the photosensitizer carrier comprises the following steps:
(1) Activating escherichia coli to prepare escherichia coli suspension;
(2) And adding a photosensitizer into the activated escherichia coli suspension, and incubating in a dark place to obtain a photosensitizer-loaded bacterial carrier.
2. A bacterial carrier comprises Escherichia coli and a photosensitizer, wherein the photosensitizer is loaded on the Escherichia coli, the Escherichia coli is non-pathogenic Escherichia coli DH5 alpha, and the photosensitizer is adsorbed on the surface of the Escherichia coli every 10 5 The concentration of the CFU/ml escherichia coli loaded photosensitizer is 1-25 mu M, and the photosensitizer is a compound shown in a formula (I):
the preparation method of the bacterial vector comprises the following steps:
(1) Activating escherichia coli to prepare escherichia coli suspension;
(2) And adding a photosensitizer into the activated escherichia coli suspension, and incubating in a dark place to obtain a photosensitizer-loaded bacterial carrier.
3. The method for preparing a bacterial vector according to claim 2, comprising the steps of:
(1) Activating escherichia coli to prepare escherichia coli suspension;
(2) And adding a photosensitizer into the activated escherichia coli suspension, and incubating in a dark place to obtain a photosensitizer-loaded bacterial carrier.
4. The method according to claim 3, wherein the activation in the step (1) is performed by inoculating Escherichia coli into LB medium.
5. The method according to claim 3 or 4, wherein the E.coli suspension in step (1) has an OD 600 The value is 0.6-0.8.
6. The method according to claim 3 or 4, wherein the incubation in the dark in step (2) is performed for a period of 0.1 to 1 hour.
7. The preparation method according to claim 6, wherein the light-shielding incubation time in the step (2) is 20-30min.
8. The method according to claim 3 or 4, wherein the light-shielding incubation temperature in step (2) is 25-37 ℃.
9. Use of the bacterial vector of claim 2 for the preparation of a medicament for photodynamic therapy.
10. The use according to claim 9, wherein the photodynamic therapy medicament is for treating an individual suffering from a tumour.
11. The use according to claim 10, wherein the tumor is breast cancer, colon adenoma, colorectal cancer, rectal cancer, anal cancer, small intestine cancer, lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, eye melanoma, uterine sarcoma, ovarian cancer, endometrial cancer, cervical cancer, endocrine cancer, thyroid cancer, parathyroid cancer, soft tissue tumor, prostate cancer, bronchial cancer or renal cancer.
12. A pharmaceutical composition for photodynamic therapy comprising the bacterial vector of claim 2 and an antineoplastic agent.
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