CN114887059A - Preparation method and application of nano copper sulfide with bacterial outer membrane vesicle as ligand - Google Patents

Preparation method and application of nano copper sulfide with bacterial outer membrane vesicle as ligand Download PDF

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CN114887059A
CN114887059A CN202210522818.4A CN202210522818A CN114887059A CN 114887059 A CN114887059 A CN 114887059A CN 202210522818 A CN202210522818 A CN 202210522818A CN 114887059 A CN114887059 A CN 114887059A
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甘璐
杨祥良
秦佳琪
雍土莹
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Huazhong University of Science and Technology
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Abstract

The invention belongs to the technical field of nano material preparation, and discloses a preparation method and application of nano copper sulfide with bacterial outer membrane vesicles as ligands. The nano copper sulfide with the bacterial outer membrane vesicle as the ligand has an immune activation effect, has a strong photo-thermal conversion effect under the irradiation of near infrared light, and can realize immune/photo-thermal synergistic treatment of tumors.

Description

Preparation method and application of nano copper sulfide with bacterial outer membrane vesicle as ligand
Technical Field
The invention belongs to the technical field of nano material preparation, and particularly relates to a preparation method and application of nano copper sulfide with a bacterial outer membrane vesicle as a ligand.
Background
The tumor seriously threatens the health of human beings, and photothermal therapy (PTT) is a novel tumor treatment method which is widely researched due to the unique advantages of high selectivity, small invasiveness and the like. PTT is a photo-thermal material which converts light energy into heat under the irradiation of near infrared light (wave band: 780-1100 nanometers), so that the local temperature of tumor tissues is raised, tumor cells are killed, and an anti-tumor immune effect is induced. However, the effect of PTT is limited by limited tissue penetration of light, poor photothermal conversion efficiency and light stability of photothermal materials, insufficient tumor accumulation capacity of photothermal materials, and the like.
In recent years, with the continuous development of nanotechnology, nanomaterials with unique optical properties have been applied to tumor PTT, such as gold nanomaterials, carbon nanotubes, semiconductor nanoparticles, and organic polymer nanomaterials. Researches show that the CuS nanoparticles have the advantages of low cost, simple preparation, stable photo-thermal performance and the like, are separated from numerous materials, become research hotspots in photo-thermal treatment, and are widely applied to anti-tumor treatment.
The common synthesis method of the CuS nanoparticles is a ligand method, and common ligand agents comprise sodium citrate, povidone, gelatin and the like. However, the preparation of CuS nanoparticles by using the above ligand usually requires high temperature and other conditions, and the obtained CuS is not easy to be post-modified to enhance tumor targeting property, biocompatibility and the like. The biological safety of the photo-thermal material is the key of clinical PTT application, and the ligand agent has great influence on the biocompatibility of the photo-thermal material CuS nanoparticles. In recent years, protein biomineralization techniques using proteins as ligands have been used to guide the synthesis of inorganic nanoparticles. Because the protein is rich in amino, carboxyl, sulfydryl and other functional groups, the growth of the nano crystal can be regulated and controlled by the coordination of various metal ion binding sites and metal ions, and another method is provided for constructing functional metal sulfides so as to meet the requirements of biomedical application. In the prior art, a biological membrane (such as a tumor cell membrane, a red cell membrane and the like) is usually modified on the surface of the CuS nanoparticle by adopting an extrusion or ultrasonic mode to endow the CuS nanoparticle with a biological bionic function, but the existing modes are complicated and can damage membrane protein to influence the realization of the function. Other situations that the nanoparticles are modified by chemical substances often require further physical or chemical methods, that is, corresponding chemical substances are synthesized first, and then the chemical substances are modified on the surfaces of the nanoparticles by physical or chemical reactions, so that the realization that the process is complicated and the functions of the materials are possibly damaged also exists.
Bacterial Outer Membrane Vesicles (OMVs) are vesicles of 30-200nm that are secreted mainly by gram-negative bacteria and are not replication competent. OMVs contain antigens from bacteria and various pathogen-associated molecular patterns such as lipopolysaccharides, outer membrane proteins, lipoproteins, etc., and are promising immunological adjuvants. In addition, because OMVs have easy preparation, good biocompatibility, good stability, unique tumor targeting ability, etc., they have been widely used in the research of antitumor drug delivery.
Therefore, the research and development of the nano copper sulfide taking OMVs as the ligand has the advantages that the preparation method is simple and easy to realize, the performance of the nano copper sulfide can be enriched, and the important clinical application value is realized.
Disclosure of Invention
In view of the above defects or improvement needs of the prior art, an object of the present invention is to provide a method for preparing nano copper sulfide with bacterial outer membrane vesicles as ligands and an application thereof, wherein the nano copper sulfide with bacterial outer membrane vesicles as ligands is obtained by using nano copper sulfide as a matrix and using bacterial outer membrane vesicles as ligands for the first time, such that the nano copper sulfide with bacterial outer membrane vesicles as ligands is directly located on the nano copper sulfide, and the nano copper sulfide has immune activation effect and strong photothermal conversion effect under irradiation of near infrared light, thereby achieving immune/photothermal synergistic therapy of tumors. In addition, the synthetic method is simple and convenient to operate, the reaction process is green and pollution-free, and the prepared nano copper sulfide with the bacterial outer membrane vesicle serving as the ligand has regular shape, a near infrared absorption function, excellent photo-thermal conversion performance and good biocompatibility; meanwhile, the nano copper sulfide with the bacterial outer membrane vesicle as the ligand can play a role in immune activation on the whole and has tumor targeting capability. The nano copper sulfide with the prepared bacterial outer membrane vesicle as the ligand is used for photo-thermal and immunotherapy of tumors, and has good clinical application prospect.
In order to achieve the above object, according to one aspect of the present invention, there is provided a nano copper sulfide with bacterial outer membrane vesicles as ligands, comprising a nano copper sulfide matrix, and ligand bacterial outer membrane vesicles directly on the matrix.
In a further preferred embodiment of the present invention, the bacterial outer membrane vesicle is preferably a bacterial outer membrane vesicle derived from escherichia coli or a bacterial outer membrane vesicle derived from attenuated salmonella.
In a further preferred embodiment of the present invention, the nano copper sulfide with the bacterial outer membrane vesicle as a ligand has an overall size of 30 to 200 nm.
According to another aspect of the present invention, the present invention provides a method for preparing nano copper sulfide with the bacterial outer membrane vesicle as a ligand, comprising the following steps:
(S1) preparing a copper salt aqueous solution and a sodium sulfide aqueous solution respectively by using a copper salt as a copper source material and sodium sulfide as a sulfur source material;
(S2) dispersing the bacterial outer membrane vesicles in a phosphate buffer to obtain a bacterial outer membrane vesicle solution;
(S3) adding the aqueous copper salt solution to the bacterial outer membrane vesicle solution for incubation; and then, dropwise adding the sodium sulfide aqueous solution into the incubated solution system, stirring for reaction, centrifuging, and washing to obtain the nano copper sulfide with the bacterial outer membrane vesicle as the ligand.
In a further preferred embodiment of the present invention, in the step (S3), the stirring reaction is performed at a temperature of 20 to 25 ℃, and the reaction time is 24 to 36 hours.
As a further preferable mode of the present invention, in the step (S1), the copper salt is specifically copper acetate, the copper salt aqueous solution is specifically obtained by dissolving copper acetate in deionized water, and a ratio of a substance amount of the copper acetate to a volume of the deionized water is 0.01:1mmol/mL to 0.02:1 mmol/mL;
or the copper salt is specifically copper chloride, the copper salt aqueous solution is specifically obtained by dissolving copper chloride in deionized water, and the ratio of the amount of the copper chloride to the volume of the deionized water is 0.01:1 mmol/mL-0.02: 1 mmol/mL;
or the copper salt is copper sulfate, the copper salt aqueous solution is obtained by dissolving copper sulfate in deionized water, and the ratio of the amount of the copper sulfate substance to the volume of the deionized water is 0.01:1 mmol/mL-0.02: 1 mmol/mL;
in the step (S1), the sodium sulfide aqueous solution is obtained by dissolving sodium sulfide nonahydrate in deionized water, and the ratio of the amount of the sodium sulfide nonahydrate to the volume of the deionized water is 0.1:1mmol/mL to 0.2:1 mmol/mL;
in the step (S3), the amount of the aqueous copper salt solution and the amount of the aqueous sodium sulfide solution satisfy: the ratio of the amount of the copper element in the copper salt aqueous solution to the amount of the sulfur element in the sodium sulfide aqueous solution is 1:1 to 1: 4.
In a more preferred aspect of the present invention, in the step (S2), the concentration of the phosphate buffer is 5mmol/L to 9 mmol/L;
in the step (S3), the incubation time of the incubation is 30-60 min;
in the step (S3), the centrifugation speed is 5000-6000 rpm/min, and the centrifugation time is 15-30 min.
According to another aspect of the invention, the invention provides an application of the nano copper sulfide with the bacterial outer membrane vesicle as the ligand in preparing an anti-tumor medicine.
As a further preferred aspect of the present invention, the anti-tumor drug is specifically a photothermal anti-tumor drug, and the light action waveband of the photothermal anti-tumor drug is preferably a near-infrared light waveband; preferably, the tumor is breast cancer, oral squamous carcinoma or skin cancer.
As a further preferred aspect of the present invention, the anti-tumor drug is specifically an anti-tumor immune activation drug; preferably, the anti-tumor drug is an anti-tumor drug capable of realizing photothermal/immune combination therapy.
Through the technical scheme, compared with the prior art, the nano copper sulfide with the bacterial outer membrane vesicles as the ligand is designed and synthesized, the bacterial outer membrane vesicles are used as the ligand for the first time, the nano copper sulfide is matched with the nano copper sulfide as a matrix, the ligand bacterial outer membrane vesicles are directly positioned on the nano copper sulfide, and the correspondingly obtained nano copper sulfide with the bacterial outer membrane vesicles as the ligand is a whole, so that the nano copper sulfide can be used as a photo-thermal material for anti-tumor treatment (for example, under the irradiation of near infrared light), can also be used as an immune drug for anti-tumor treatment, and particularly can realize photo-thermal/immune combined treatment of tumors.
The invention relates to synthesis of nano copper sulfide with bacterial outer membrane vesicles as ligands, which is different from the prior art that the bacterial outer membrane vesicles are modified on the surfaces of copper sulfide nano particles prepared by taking povidone, gelatin, sodium citrate and the like as ligands (the copper sulfide nano particles are prepared by proper ligands are needed because the ligands can stabilize Cu ions and S ions to obtain CuS nanocrystals, otherwise, only CuS precipitates can be obtained). And (3) the obtained nano copper sulfide with the bacterial outer membrane vesicles as ligands, wherein the components of the bacterial outer membrane vesicles are directly positioned on the nano copper sulfide, and no other ligands exist except the bacterial outer membrane vesicles. The OMVs contain rich protein structures, and the metal ion binding sites can regulate and control the growth of the nanocrystals through the coordination with metal ions, so that after the co-incubation of the OMVs and a copper salt aqueous solution is finished, a sulfur source aqueous solution is added, and the nano copper sulfide integral material taking the bacterial outer membrane vesicles as ligands can be effectively generated. However, if extrusion or ultrasonic method is adopted, in addition to the complicated process, the topological structure of the OMVs protein is also likely to be destroyed in the synthesis process, which affects the function of OMVs. In addition, in the development process of the present invention, an attempt was made to incubate the bacterial outer membrane vesicles in an aqueous sulfur source solution, but since the aqueous sulfur source solution affects the morphology of the bacterial outer membrane vesicles and causes negative effects, a synthesis process in which the bacterial outer membrane vesicles are incubated in an aqueous copper source solution, and then the aqueous sulfur source solution is added dropwise thereto was finally confirmed.
Specifically, the present invention can achieve the following advantageous effects:
(1) the nano copper sulfide with the bacterial outer membrane vesicle serving as the ligand has tumor targeting capacity, good biocompatibility and stable photo-thermal conversion and immunologic adjuvant performance;
(2) the nano copper sulfide with the bacterial outer membrane vesicle serving as the ligand can particularly realize the tumor photothermal/immune synergistic treatment effect, and has no obvious toxic or side effect on organisms.
(3) The nano copper sulfide with the bacterial outer membrane vesicle as the ligand can be obtained by a simple synthesis method, and the synthesis method can be particularly carried out at room temperature; in addition, compared with gold which is a noble metal, the bacterial outer membrane vesicle is nano copper sulfide of the ligand, the matrix of the bacterial outer membrane vesicle is nano copper sulfide, and the bacterial outer membrane vesicle is composed of Cu and S with low cost, so that the cost is greatly reduced.
In application, for example, the nano copper sulfide with the bacterial outer membrane vesicle as a ligand obtained in the present invention may be dispersed in any physiological saline (e.g. 0.9% sodium chloride solution) or buffer (e.g. phosphate buffer with pH 7-9) suitable for clinical application, and administered into an organism in the form of an injection (the injection may be intravenous injection, intratumoral injection, etc.); the nano copper sulfide with the bacterial outer membrane vesicles as ligands has an immune activation effect, and can convert light energy into heat energy under the irradiation of near infrared light to realize photothermal therapy of tumors, and particularly can inhibit tumor growth by utilizing photothermal/immune synergistic therapy.
In conclusion, the nano copper sulfide with the bacterial outer membrane vesicle as the ligand is prepared by a simple, convenient and green pollution-free method, has the characteristics of regular shape, near infrared absorption function, photo-thermal conversion performance and good biocompatibility, can be effectively enriched at a tumor part, and realizes photo-thermal and immunotherapy of targeted tumors.
Drawings
FIG. 1 is a transmission electron micrograph of CuS-OMVs prepared in example 1.
FIG. 2 is a UV-VIS-NIR absorption spectrum of the CuS-OMVs prepared in example 1.
FIG. 3 is a transmission electron micrograph of the CuS-OMVs prepared in example 2.
FIG. 4 is a UV-VIS-NIR absorption spectrum of the CuS-OMVs prepared in example 2.
FIG. 5 is a transmission electron micrograph of the CuS-OMVs prepared in example 3.
FIG. 6 shows the CuS-OMVs prepared in example 3 at a power of 1W/cm 2 Temperature rise curve chart under 1064nm near infrared light irradiation.
FIG. 7 is a transmission electron micrograph of the CuS-OMVs prepared in example 4.
FIG. 8 is a graph of temperature rise for different concentrations of CuS-OMVs in example 5.
FIG. 9 is a graph showing the effect of different concentrations of CuS-OMVs on the proliferation potency of breast cancer cells under near infrared light irradiation in example 6.
FIG. 10 is a graph showing the activation of BMDCs by CuS-OMVs in example 7; wherein A, B, C in FIG. 10 are the expression changes of BMDCs activation-associated costimulatory molecules CD80, CD86 and MHC II, respectively (the horizontal axis in the figure is sequentially a control group 1, a control group 2, an experimental group and a control group 3 from left to right).
FIG. 11 is a graph of the effect of CuS-OMVs on reverse polarization of M2-type macrophages in example 8; wherein A, B in FIG. 11 is the expression change of M1-related surface characteristic molecule CD80 and CD86 after the M2 macrophage cell is reversely polarized by CuS-OMVs, and C in FIG. 11 is the expression change of M2-related surface characteristic molecule CD206 after the M2 macrophage cell is reversely polarized by CuS-OMVs (the horizontal axis in the figure is a control group 1, a control group 2 and an experimental group from left to right in sequence).
FIG. 12 is the results of the distribution of CuS-OMVs in tumor-bearing mice in example 9; wherein, A in figure 12 is the result of in vivo imaging, and B in figure 12 is the result of distribution of CuS-OMVs in different tissues of tumor-bearing mice.
FIG. 13 is a graph of the photothermal/immunotherapy effect of CuS-OMVs in vivo in example 10; wherein, a in fig. 13 is a temperature rise graph of the in situ tumor of the mouse in the laser irradiation treatment group, B in fig. 13 is a growth curve of the in situ tumor of the mouse in different treatment groups, and C in fig. 13 is a graph of the effect result of different treatment groups on the survival time of the mouse.
FIG. 14 is a toxicity study of CuS-OMVs in example 11; wherein A in FIG. 14 corresponds to the body weight of the mouse, B in FIG. 14 corresponds to the glutamic-pyruvic transaminase content in the serum of the mouse, and C in FIG. 14 corresponds to the creatinine content in the serum of the mouse.
FIG. 15 is the results of the inhibition of tumor growth by CuS-OMVs in example 12 in a SCC-7 mouse oral squamous cell carcinoma model.
FIG. 16 is a graph of the results of the inhibition of tumor growth by CuS-OMVs in the B16-F10 melanoma model in example 13.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The preparation method of the nano copper sulfide (hereinafter abbreviated as CuS-OMVs) with the bacterial outer membrane vesicle as the ligand can comprise the following steps:
step 1: dissolving copper acetate in deionized water, and stirring until the copper acetate is completely dissolved to obtain a copper acetate solution;
step 2: dissolving sodium sulfide nonahydrate in deionized water, and stirring until the sodium sulfide nonahydrate is completely dissolved to obtain a sodium sulfide aqueous solution;
and step 3: dispersing the bacterial outer membrane vesicles in a phosphate buffer;
and 4, step 4: adding a copper acetate solution into the bacterial outer membrane vesicles, and incubating;
and 5: and (4) dropwise adding the sodium sulfide solution into the solution in the step (4), stirring at room temperature for reaction, centrifuging, and washing to obtain CuS-OMVs.
The following examples employ the following main reagents and material sources:
copper acetate, sodium sulfide nonahydrate, and sodium chloride (beijing limited, drug controlled stock); yeast extract, tryptone (OXOID, UK); enhanced BCA protein detection kit, BeyoGoldTM vacuum filter (picuonsy biotechnology limited); dialysis bags (source leaves); CCK8 (homalomene, japan); pancreatin, fetal bovine serum (Gibco Life Technologies, usa); 1640 medium, DMEM (1X) medium, penicillin-streptomycin double antibody (HyClone, usa); 96-well plates and 100mm petri dishes were purchased from Corning; microplate readers (Shanghai three-family instruments, Inc.); flow cytometry (Cytoflex, usa); mouse breast cancer 4T1 cell line, SCC-7 mouse oral squamous carcinoma cell line, B16-F10 melanoma cell line were purchased from American Type Culture Collection (Manassas, Va., USA); BALB/C mice, C57/6J mice were purchased from Experimental animals technology, Inc. of Wei Tony, Beijing.
Example 1: a preparation method of nano copper sulfide with outer membrane vesicles of Escherichia coli Nissle 1917 as ligands comprises the following specific steps:
(1) weighing 1.2mg of copper acetate, adding 0.3mL of deionized water, and stirring for 10min until the copper acetate is completely dissolved;
(2) weighing 5.76mg of sodium sulfide nonahydrate, adding 0.12mL of deionized water, and stirring for 10min until the sodium sulfide nonahydrate is completely dissolved;
(3) dispersing outer membrane vesicles of bacteria derived from Escherichia coli Nissle 1917 in 9mmol/L phosphate buffer solution; (4) adding a copper acetate solution into the bacterial outer membrane vesicles, and incubating for 1 h;
(5) and (4) dropwise adding the sodium sulfide solution into the solution in the step (4), stirring and reacting for 12 hours at room temperature, centrifuging, and washing to obtain CuS-OMVs.
The transmission electron microscope picture of the CuS-OMVs prepared in the embodiment is shown in figure 1, and the figure shows that the particle size of the CuS-OMVs is 30-200 nm; the UV-Vis-NIR absorption spectra of CuS-OMVs are shown in FIG. 2, which shows that the CuS-OMVs of the present invention have strong absorption in the NIR region.
Example 2: a preparation method of nano copper sulfide with outer membrane vesicles of Escherichia coli Nissle 1917 as ligands comprises the following specific steps:
(1) weighing 1.2mg of copper acetate, adding 0.3mL of deionized water, and stirring for 10min until the copper acetate is completely dissolved;
(2) weighing 2.88mg of sodium sulfide nonahydrate, adding 0.06mL of deionized water, and stirring for 10min until the sodium sulfide nonahydrate is completely dissolved;
(3) dispersing the outer bacterial membrane vesicles derived from escherichia coli Nissle 1917 in 5mmol/L phosphate buffer solution;
(4) adding a copper acetate solution into the bacterial outer membrane vesicles, and incubating for 0.5 h;
(5) and (4) dropwise adding the sodium sulfide solution into the solution in the step (4), stirring and reacting at room temperature for 12 hours, centrifuging, and washing to obtain CuS-OMVs.
The transmission electron microscope picture of the CuS-OMVs prepared in the example is shown in FIG. 3, which shows that the particle size of the CuS-OMVs is 30-200 nm; the UV-Vis-NIR absorption spectra of CuS-OMVs are shown in FIG. 4. it can be seen that the CuS-OMVs of the present invention have a strong absorption in the NIR region.
Example 3: a preparation method of nano copper sulfide with attenuated salmonella (guanosine tetraphosphate synthesis defect) outer membrane vesicles as ligands comprises the following specific steps:
(1) weighing 1.2mg of copper acetate, adding 0.3mL of deionized water, and stirring for 10min until the copper acetate is completely dissolved;
(2) weighing 5.76mg of sodium sulfide nonahydrate, adding 0.12mL of deionized water, and stirring for 10min until the sodium sulfide nonahydrate is completely dissolved;
(3) dispersing the attenuated salmonella-derived bacterial outer membrane vesicles in 9mmol/L phosphate buffer solution;
(4) adding a copper acetate solution into the bacterial outer membrane vesicles, and incubating for 1 h;
(5) and (4) dropwise adding the sodium sulfide solution into the solution in the step (4), stirring and reacting for 12 hours at room temperature, centrifuging, and washing to obtain CuS-OMVs.
The transmission electron microscope picture of the CuS-OMVs prepared in the embodiment is shown in FIG. 5, and the picture shows that the particle size of the CuS-OMVs is 30-200 nm; the photothermal temperature rise curve of CuS-OMVs is shown in FIG. 6, and it can be seen that the temperature of the CuS-OMVs dispersion gradually rises with the laser irradiation time, showing a time dependence, which indicates that the CuS-OMVs has good photothermal conversion performance.
Example 4: a preparation method of nano copper sulfide with attenuated salmonella (guanosine tetraphosphate synthesis defect) outer membrane vesicles as ligands comprises the following specific steps:
(1) weighing 1.2mg of copper acetate, adding 0.3mL of deionized water, and stirring for 10min until the copper acetate is completely dissolved;
(2) weighing 2.88mg of sodium sulfide nonahydrate, adding 0.06mL of deionized water, and stirring for 10min until the sodium sulfide nonahydrate is completely dissolved;
(3) dispersing the attenuated salmonella-derived bacterial outer membrane vesicles in 5mmol/L phosphate buffer solution;
(4) adding a copper acetate solution into the bacterial outer membrane vesicles, and incubating for 0.5 h;
(5) and (4) dropwise adding the sodium sulfide solution into the solution in the step (4), stirring and reacting at room temperature for 12 hours, centrifuging, and washing to obtain CuS-OMVs.
A transmission electron micrograph of the CuS-OMVs prepared in this example is shown in FIG. 7, which shows that the particle size of the CuS-OMVs is 30 to 200 nm.
Example 5: photo-thermal heating effect of CuS-OMVs with different concentrations
The CuS-OMVs prepared in example 1 were used as a 50. mu.g/mL dispersion stock solution, which was diluted to 10. mu.g/mL and 30. mu.g/mL dispersions, respectively, and the CuS-OMVs dispersions of different concentrations were applied to a 1064nm laser at a wavelength of 0.76W/cm 2 Irradiating with power for 5min, and recording temperature change with thermal imager, with the result shown in FIG. 8;
according to experimental results, under the same laser irradiation condition, CuS-OMVs solutions with different concentrations all show a certain temperature rise phenomenon, and the temperature rise is more remarkable along with the increase of the content of CuS-OMVs, and concentration dependence is shown, so that the CuS-OMVs has better photo-thermal conversion performance under 1064nm laser irradiation.
Example 6: effect of CuS-OMVs prepared in example 1 on proliferation potency of Breast cancer cells under irradiation with near Infrared light
Collecting 4T1 cells in logarithmic growth phase, digesting with 0.05% pancreatin for 2min, stopping digestion with complete culture medium, centrifuging at 1000r/min for 3min, discarding supernatant, adding 9mL complete culture medium, gently blowing to obtain single cell suspension, and mixingCell suspension was subjected to cell counting, and cell concentration was adjusted at 2X 10 4 The cells were plated in 96-well plates at cell densities of 0, 2, 5, 10, 20, 30. mu.g/mL for the different concentrations of the groups of CuS-OMVs. After 12h, the culture medium was discarded, DMEM without serum was replaced, CuS-OMVs were added at different concentrations, 20. mu.L per well, and the mixture was cultured in an incubator for 12 h. After 12h, the medium was aspirated and washed 3 times with PBS, replaced with fresh serum-free DMEM, and irradiated at 1064nm with a laser (0.76W/cm) 2 5min), and then culturing is continued for 24 h. After the culture is stopped, 10 mu L of CCK8 solution is added into each hole, after incubation for 2-4h in the dark, the absorbance value of each group is detected at the wavelength of 450nm by using an enzyme-labeling instrument, and the survival rate of the cells is calculated.
The experimental result shows that CuS-OMVs with different concentrations have no obvious influence on the proliferation capacity of breast cancer cells under the condition of no laser irradiation, which indicates that the CuS-OMVs have no obvious toxicity on the breast cancer cells; under the laser irradiation condition, the low-concentration CuS-OMVs can inhibit the proliferation capacity of the breast cancer cells to a certain degree, and the inhibition capacity of the CuS-OMVs on the proliferation of the breast cancer cells is obviously enhanced along with the increase of the concentration of the CuS-OMVs, so that the CuS-OMVs has certain concentration dependence, and the CuS-OMVs has a better photo-thermal killing effect on the breast cancer cells under the laser irradiation of 1064 nm. The results of the experiment are shown in FIG. 9.
Example 7: activation of BMDCs by CuS-OMVs prepared in example 1
Bone marrow cells were washed from the bone marrow cavity of femur and tibia of 5-6 week old female BALB/c mice with 1mL RPMI 1640 medium. The cell suspension was filtered with a nylon mesh, centrifuged to obtain a cell pellet, red blood cells were lysed with a red blood cell lysate, washed 1 time with PBS, centrifuged, and the obtained cells were cultured in RPMI 1640 medium containing 10ng/mL GM-CSF and 10ng/mL IL-4 for 6d to obtain immature BMDCs. PBS, OMVs, CuS-OMVs and LPS are added into the corresponding wells respectively for 24 h. After cell culture was terminated, each group of cells was collected, incubated for 30min with anti-CD 11c, anti-CD 80, and anti-CD 86 antibodies, and finally activation of BMDCs was detected by flow cytometry. BMDCs from CuS-OMVs were added at 7.5. mu.g/mL (protein amount) as experimental group, PBS-treated BMDCs as control group 1, BMDCs from bacterial outer membrane vesicles at 7.5. mu.g/mL (protein amount) as control group 2, and 200ng/mL LPS-treated BMDCs as control group 3.
Experimental results, OMVs and CuS-OMVs significantly increased CD80 + BMDCs、CD86 + BMDCs (shown as A in FIG. 10 and B in FIG. 10) and MHC II + The proportion of BMDCs (as shown by C in fig. 10) was slightly stronger than that of the negative control PBS group and that of the positive control LPS group. The above results demonstrate that CuS-OMVs effectively activate DCs to act as an immune agonist.
Example 8: effect of CuS-OMVs prepared in example 1 to reverse-polarize M2-type macrophages
Bone marrow cells were washed from the bone marrow cavity of femur and tibia of 5-6 week old female BALB/c mice with 1mL RPMI 1640 medium. The cell suspension was filtered with a nylon mesh, centrifuged to obtain a cell pellet, red blood cells were lysed with a red blood cell lysate, washed 1 time with PBS, centrifuged, and the resulting cells were cultured in RPMI 1640 medium containing 20ng/mL M-CSF for 5d to obtain immature BMDMs. The immature BMDMs are treated by 20ng/mL IL-4 for 24h to obtain M2 type BMDMs. PBS, OMVs and CuS-OMVs were added to the corresponding wells for 24 h. After cell culture was terminated, each group of cells was collected, incubated for 30min with anti-CD 206, anti-CD 80 and anti-CD 86 antibodies, and finally the reverse polarization effect of M2 BMDMs was examined by flow cytometry. BMDMs from CuS-OMVs were added at 7.5. mu.g/mL (protein amount) as experimental group, PBS-treated BMDMs as control group 1, and BMDMs from bacterial outer membrane vesicles at 7.5. mu.g/mL (protein amount) as control group 2.
Experimental results, OMVs and CuS-OMVs significantly increased CD80 + BMDMs and CD86 + The proportion of BMDMs significantly reduced CD206 relative to the negative control PBS group (shown as A in FIG. 11 and B in FIG. 11) + Ratio of BMDMs (as shown by C in FIG. 11). The results show that CuS-OMVs reversibly polarize M2 type BMDMs into M1 type BMDMs, and improve immunosuppressive microenvironment.
Example 9: detection of accumulation of CuS-OMVs prepared in example 1 in tumor tissues by 4T1 mouse Breast carcinoma in situ tumor animal model
(1) Mice: SPF-grade BALB/C female mice, 5-6 weeks old, 18-22 g;
(2) constructing an in-situ breast cancer model: taking 4T1 breast cancer cells in logarithmic growth phase,digesting with 0.05% pancreatin for 1min, centrifuging at 1000rpm for 5min, removing supernatant, counting, washing with physiological saline for 3 times, and adjusting the concentration to 1 × 10 by resuspending with physiological saline 7 one/mL, 50. mu.L of cell suspension was injected at the right mammary fat pad of the mouse;
(3) the volume of the tumor to be treated is as long as 100- 3 Tumor-bearing mice are randomly divided into 2 groups, namely, mice treated by IR 780-labeled OMVs are used as a control group 1, mice treated by IR 780-labeled CuS-OMVs are used as an experimental group, and 3 mice are used in each group;
(4) the administration mode is tail vein injection;
(5) after 24 hours of administration, the two groups of mice were placed in a small animal imager, respectively; and detecting the distribution of IR780 fluorescence in tumor-bearing mice.
(6) After the live imaging was completed, the mice were sacrificed and hearts, livers, spleens, lungs, kidneys, and tumors were imaged and weighed, respectively.
The experimental results are shown in FIG. 12, wherein the A result in FIG. 12 shows that CuS-OMVs can target and accumulate in tumor tissues as well as OMVs, which indicates that CuS-OMVs prepared by using OMVs as a ligand still has the ability of targeting tumor tissues by OMVs; the results B in fig. 12 further demonstrate that CuS-OMVs efficiently accumulate at the tumor site compared to normal tissues (heart, liver, spleen, lung, kidney).
Example 10: in vivo photothermal/immunotherapy effects of CuS-OMVs prepared in example 1 were examined by 4T1 mouse breast cancer orthotopic tumor animal model
(1) Mice: SPF-grade BALB/C female mice, 5-6 weeks old, 18-22 g;
(2) constructing an in-situ breast cancer model: collecting 4T1 breast cancer cells in logarithmic growth phase, digesting with 0.05% pancreatin for 1min, centrifuging at 1000rpm for 5min, removing supernatant, counting, washing with normal saline for 3 times, and adjusting concentration to 1 × 10 by using normal saline to resuspend 7 one/mL, 50. mu.L of cell suspension was injected at the right mammary fat pad of the mouse;
(3) when the tumor volume is 50-100mm 3 Tumor-bearing mice were randomly divided into 6 groups, i.e., (i) PBS-treated mice as control group 1, (ii) OMVs-treated mice as control group 2, and (iii) CuS-OMVs-treated miceMice as a control group 3, PBS + near-infrared laser irradiation treated mice as a control group 4, OMVs + near-infrared laser irradiation treated mice as a control group 5, and sixth CuS-OMVs + near-infrared laser irradiation treated mice as experimental groups, each group having 6 mice;
(4) the administration mode is tail vein injection;
(5) after 24 hours after the administration, the tumor parts of the mice are respectively placed at 1W/cm 2 Irradiating for 10min under 1064nm near-infrared light; and detecting the change relation of the temperature in the tumor with time after the tumor-bearing mouse is irradiated by laser.
(6) After each group was treated, the group was fed in the normal feeding mode, and the tumor volume was recorded every other day for 15 days.
The experimental result is shown in figure 13, the tumor temperature of the tumor-bearing mice increases along with the increase of the time after the tumor-bearing mice are irradiated by the laser, the result is shown in figure 13A, and the tumor temperature of the mice of the group IV and V does not increase along with the increase of the time after the tumor-bearing mice are irradiated by the laser. The tumor growth curves of the mice in each treatment group are obviously different after treatment, the tumor volume of the mice in the CuS-OMVs + near infrared laser irradiation group is obviously smaller than that in the third group, the tumor volume of the mice in the CuS-OMVs + near infrared laser irradiation group is obviously smaller than that in the fifth group, the growth trends of the tumors in the other five groups are not obviously different, the fifth group has a certain inhibition effect on the tumor volume (shown as B in figure 13), and the survival time of the mice in the CuS-OMVs + near infrared laser irradiation group can be prolonged (shown as C in figure 13). The results show that the CuS-OMVs prepared by the method have good photo-thermal/immunotherapy effect.
Example 11: EXAMPLE 1 toxicity Studies of the CuS-OMVs prepared
(1) Mice: SPF-grade BALB/C female mice, 5-6 weeks old, 18-22 g;
(2) constructing an in-situ breast cancer model: collecting 4T1 breast cancer cells in logarithmic growth phase, digesting with 0.05% pancreatin for 1min, centrifuging at 1000rpm for 5min, removing supernatant, counting, washing with normal saline for 3 times, and adjusting concentration to 1 × 10 by using normal saline to resuspend 7 one/mL, 50. mu.L of cell suspension was injected at the right mammary fat pad of the mouse;
(3) volume of tumor to be treatedThe length of the glass is 50-100mm 3 Dividing the tumor-bearing mice into 6 groups at random, namely, taking the mice treated by PBS as a control group 1, taking the mice treated by OMVs as a control group 2, taking the mice treated by CuS-OMVs as a control group 3, taking the mice treated by PBS + near-infrared laser irradiation as a control group 4, taking the mice treated by OMVs + near-infrared laser irradiation as a control group 5, and taking the mice treated by CuS-OMVs + near-infrared laser irradiation as experimental groups, wherein each group comprises 6 mice;
(4) the administration mode is tail vein injection;
(5) after 24 hours after the administration, the tumor parts of the mice are respectively placed at 1W/cm 2 Irradiating for 10min under 1064nm near infrared light;
(6) after each group was treated, the mice were fed in a normal feeding manner, and the body weights of the mice were recorded every other day for 15 days.
The experimental results show that the body weight of the mice in each treatment group is not obviously different after treatment (shown as A in figure 14), and the glutamic-pyruvic transaminase (shown as B in figure 14) and the creatinine (shown as C in figure 14) of the mice in each treatment group are not obviously changed, which indicates that the near-infrared laser and the CuS-OMVs do not generate obvious side effects on the mice.
Example 12: in vivo photothermal/immunotherapy effects of CuS-OMVs prepared in example 1 were examined by SCC-7 oral squamous cell carcinoma mouse transplanted tumor animal model
(1) Mice: an SPF-grade BALB/C male mouse is aged for 5-6 weeks and 18-22 g;
(2) constructing an oral squamous carcinoma mouse transplantation tumor model: collecting SCC-7 cells in logarithmic growth phase, digesting with 0.05% pancreatin for 1min, centrifuging at 1000rpm for 5min, removing supernatant, counting, washing with physiological saline for 3 times, and resuspending with physiological saline to adjust concentration to 5 × 10 7 One per mL. Each mouse was injected subcutaneously with 0.2mL of cell suspension;
(3) the tumor volume is 50-100mm 3 Tumor-bearing mice were randomly divided into 6 groups, i.e., (i) PBS-treated mice as control group 1, (ii) OMVs-treated mice as control group 2, (iii) CuS-OMVs-treated mice as control group 3, (iv) PBS + near-infrared laser irradiation-treated mice as control group 4, (v) OMVs + near-infrared laser irradiation-treated miceThe mice used as a control group 5, and mice treated by the CuS-OMVs + near-infrared laser irradiation used as experimental groups, wherein each group comprises 6 mice;
(4) the administration mode is tail vein injection;
(5) after 24 hours after the administration, the tumor parts of the mice are respectively placed at 1W/cm 2 Irradiating for 10min under 1064nm near-infrared light; and detecting the change relation of the temperature in the tumor with time after the tumor-bearing mouse is irradiated by laser.
(6) After each group was treated, the group was fed in the normal feeding mode, and the tumor volume was recorded every other day for 15 days.
The experimental result is shown in figure 15, the tumor growth curves of the mice in each treatment group are obviously different after treatment, the tumor volume of the mice in the CuS-OMVs + near infrared laser irradiation group is obviously smaller than that of the mice in the third group, the fifth group is not obviously different from that of the mice in the other five groups, and the fifth group has a certain inhibition effect on the tumor volume. The results show that the CuS-OMVs prepared by the method have good photo-thermal/immunotherapy effect.
Example 13: in vivo photothermal/immunotherapy efficacy of CuS-OMVs prepared in example 1 was examined by B16-F10 melanoma animal models
(1) Mice: SPF grade C57/6J female mouse, 5-6 weeks old, 18-22 g;
(2) construction of mouse melanoma model: collecting B16-F10 melanoma cells in logarithmic growth phase, digesting with 0.05% pancreatin for 1min, centrifuging at 1000rpm for 5min, removing supernatant, counting, washing with normal saline for 3 times, and adjusting concentration to 1 × 10 by using normal saline to resuspend 7 one/mL, 50 μ L of cell suspension per mouse was injected subcutaneously;
(3) the tumor volume is 50-100mm 3 Dividing the tumor-bearing mice into 6 groups at random, namely, taking the mice treated by PBS as a control group 1, taking the mice treated by OMVs as a control group 2, taking the mice treated by CuS-OMVs as a control group 3, taking the mice treated by PBS + near-infrared laser irradiation as a control group 4, taking the mice treated by OMVs + near-infrared laser irradiation as a control group 5, and taking the mice treated by CuS-OMVs + near-infrared laser irradiation as experimental groups, wherein each group comprises 6 mice;
(4) the administration mode is tail vein injection;
(5) after 24 hours after the administration, the tumor parts of the mice are respectively placed at 1W/cm 2 Irradiating for 10min under 1064nm near-infrared light; and detecting the change relation of the temperature in the tumor with time after the tumor-bearing mouse is irradiated by laser.
(6) After each group was treated, the group was fed in the normal feeding mode, and the tumor volume was recorded every other day for 15 days.
The experimental result is shown in figure 16, the tumor growth curves of the mice in each treatment group are obviously different after treatment, the tumor volume of the mice in the CuS-OMVs + near infrared laser irradiation group is obviously smaller than that of the mice in the third group, the fifth group is not obviously different from that of the mice in the other five groups, and the fifth group has a certain inhibition effect on the tumor volume. The results show that the CuS-OMVs prepared by the method have good photo-thermal/immunotherapy effect.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. The nano copper sulfide with bacterial outer membrane vesicle as ligand is characterized by comprising a nano copper sulfide matrix and ligand bacterial outer membrane vesicle directly positioned on the matrix.
2. The nano-copper sulfide with a ligand of the bacterial outer membrane vesicle of claim 1, wherein the bacterial outer membrane vesicle is preferably a bacterial outer membrane vesicle derived from escherichia coli or an attenuated bacterial outer membrane vesicle derived from salmonella.
3. The nano-copper sulfide with the bacterial outer membrane vesicle as the ligand according to claim 1, wherein the overall size of the nano-copper sulfide with the bacterial outer membrane vesicle as the ligand is 30-200 nm.
4. The method for preparing nano copper sulfide with the bacterial outer membrane vesicle as the ligand according to any one of claims 1-3, which comprises the following steps:
(S1) preparing a copper salt aqueous solution and a sodium sulfide aqueous solution respectively by using a copper salt as a copper source material and sodium sulfide as a sulfur source material;
(S2) dispersing the bacterial outer membrane vesicles in a phosphate buffer to obtain a bacterial outer membrane vesicle solution;
(S3) adding the aqueous copper salt solution to the bacterial outer membrane vesicle solution for incubation; and then, dropwise adding the sodium sulfide aqueous solution into the incubated solution system, stirring for reaction, centrifuging, and washing to obtain the nano copper sulfide with the bacterial outer membrane vesicle as the ligand.
5. The method according to claim 4, wherein in the step (S3), the stirring reaction is carried out at a temperature of 20 ℃ to 25 ℃ for 24 hours to 36 hours.
6. The preparation method according to claim 4, wherein in the step (S1), the copper salt is copper acetate, the aqueous copper salt solution is obtained by dissolving copper acetate in deionized water, and the ratio of the amount of the copper acetate to the volume of the deionized water is 0.01:1mmol/mL to 0.02:1 mmol/mL;
or the cupric salt is specifically cupric chloride, the cupric salt aqueous solution is specifically obtained by dissolving cupric chloride in deionized water, and the ratio of the amount of cupric chloride to the volume of the deionized water is 0.01:1 mmol/mL-0.02: 1 mmol/mL;
or the copper salt is copper sulfate, the copper salt aqueous solution is obtained by dissolving copper sulfate in deionized water, and the ratio of the amount of the copper sulfate substance to the volume of the deionized water is 0.01:1 mmol/mL-0.02: 1 mmol/mL;
in the step (S1), the aqueous sodium sulfide solution is obtained by dissolving sodium sulfide nonahydrate in deionized water, and the ratio of the amount of the sodium sulfide nonahydrate to the volume of the deionized water is 0.1:1mmol/mL to 0.2:1 mmol/mL;
in the step (S3), the amount of the aqueous copper salt solution and the amount of the aqueous sodium sulfide solution satisfy: the ratio of the amount of the copper element in the copper salt aqueous solution to the amount of the sulfur element in the sodium sulfide aqueous solution is 1:1 to 1: 4.
7. The method according to claim 4, wherein in the step (S2), the concentration of the phosphate buffer solution is 5mmol/L to 9 mmol/L;
in the step (S3), the incubation time of the incubation is 30-60 min;
in the step (S3), the centrifugation speed is 5000-6000 rpm/min, and the centrifugation time is 15-30 min.
8. The use of the nano copper sulfide with the bacterial outer membrane vesicle as a ligand according to any one of claims 1-3 in the preparation of an anti-tumor medicament.
9. The use according to claim 8, wherein the antineoplastic drug is in particular a photothermal antineoplastic drug, the photothermal antineoplastic drug having a light-affected waveband, preferably a near-infrared light waveband; preferably, the tumor is breast cancer, oral squamous carcinoma or skin cancer.
10. The use according to claim 8, wherein the antineoplastic drug is in particular an antineoplastic immune-activating drug; preferably, the anti-tumor drug is an anti-tumor drug capable of realizing photothermal/immune combination therapy.
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