CN113350514B

CN113350514B - Hybrid material of bacteria and MOF-based carrier, and preparation method and application thereof

Info

Publication number: CN113350514B
Application number: CN202110562765.4A
Authority: CN
Inventors: 陈巍海; 梁军龙; 王佳玮; 雒国凤; 张先正
Original assignee: SUZHOU Institute OF WUHAN UNIVERSITY; Wuhan University WHU
Current assignee: SUZHOU RESEARCH INSTITUTE OF WUHAN UNIVERSITY; Wuhan University WHU
Priority date: 2021-05-24
Filing date: 2021-05-24
Publication date: 2022-06-07
Anticipated expiration: 2041-05-24
Also published as: CN113350514A

Abstract

The invention discloses a hybrid material of bacteria and MOF-based carrier, a preparation method and application thereof, wherein the method comprises the following steps: loading the MOF nanoparticles with a therapeutic agent to obtain a MOF carrier loaded with a chemotherapeutic drug; wrapping hyaluronic acid on the chemotherapeutic drug loaded MOF carrier to obtain a surface electronegative MOF carrier; modifying polyacrylamide hydrochloride on the surface of Shewanella to obtain a Shewanella electropositive solution on the surface; and (3) combining the surface electronegative MOF carrier with the surface electropositive Shewanella solution through electrostatic interaction to obtain the hybrid material of the bacteria and the MOF-based carrier. The hybrid material of the bacteria and the MOF-based carrier has high accuracy in anti-tumor treatment and can be used for multi-mode combined treatment, not only combines the advantages of each monotherapy, but also can make up the respective defects, thereby generating the anti-tumor effect with high efficiency and low toxicity.

Description

Hybrid material of bacteria and MOF-based carrier, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a hybrid material of bacteria and MOF-based carriers, and a preparation method and application thereof.

Background

Cancer, one of the most fatal diseases in the world, has been a serious threat to human life and health. Currently, chemotherapy, radiation therapy, surgical therapy and high-intensity focused ultrasound therapy are widely used in the clinic and have had great success in inhibiting tumor proliferation and prolonging patient survival. However, current clinical treatments have met with limited success due to the complexity, diversity and heterogeneity of tumors. For example, to meet the need for malignant proliferation of tumor cells, tumor cells often exhibit aberrant energy metabolism characteristics, primarily manifested by greatly increased glucose uptake and enhanced glycolysis, ultimately resulting in the massive accumulation of the metabolite, lactate, i.e., the Warburg effect. These accumulated lactic acids are not only glycolytic metabolites, but also are involved in regulating many biological functions of tumors. Meanwhile, lactic acid accumulation is apparently present in metastatic tumor patients, and exogenous lactic acid accumulated in large amounts can promote tumor cell invasion and metastasis by inducing the expression of matrix metalloproteases and integrins. Meanwhile, lactic acid also has the effects of promoting tumor immune escape, increasing tumor angiogenesis, inducing tumor drug resistance, resisting radiotherapy and the like. Therefore, the search for new approaches and strategies for tumor therapy, which are different from the conventional therapeutic strategies, is urgent. Metal-organic framework (MOF) materials have been studied in recent years due to their structural specificity. At present, common transition metal nodes (such as Fe, Cu, Mn, Co, Zr, Zn and the like) are combined with organic ligands such as carboxyl, amino, sulfonic acid and the like in different modes to obtain MOF materials with diversified functional characteristics, such as ZIF-8, MIL-101, UiO-66, HKUST-1 and the like. Due to the advantages of designability, diversity, easy functionalization and the like of the structure, the MOF material has great application potential in the fields of catalysis, adsorption, separation, anti-tumor nano-carriers and the like. For example, zinc-imidazole ZIF-8 nano MOF materials have been used as good controlled release carriers to load chemotherapeutic drugs, enzymes, photosensitizers, etc. to tumor sites due to their good biocompatibility and degradability under acidic conditions, thereby specifically killing tumor cells. These MOF-based delivery systems not only have diverse structures, but also can effectively deliver therapeutic agents to tumor sites, greatly reducing the toxic side effects of drug molecules.

However, the conventional nano therapeutic agent has the defects of poor precision and single treatment mode, so how to develop a high-precision multi-anti-tumor nano material with multiple treatment modes becomes a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a hybrid material of bacteria and MOF-based carriers, a preparation method and application thereof, which have high precision and can carry out multi-mode combined treatment, not only combines the advantages of each monotherapy, but also can make up the respective defects, thereby generating high-efficiency and low-toxicity anti-tumor effect.

In a first aspect of the invention, there is provided a method for preparing a hybrid material of a bacterium and a MOF-based carrier, the method comprising:

loading therapeutic agent on MOF nano particles to obtain a MOF carrier loaded with chemotherapeutic drug;

wrapping hyaluronic acid on the chemotherapeutic drug loaded MOF carrier to obtain a surface electronegative MOF carrier;

modifying polyacrylamide hydrochloride on the surface of Shewanella to obtain a Shewanella electropositive solution on the surface;

and (3) combining the surface electronegative MOF carrier with the surface electropositive Shewanella solution through electrostatic interaction to obtain the hybrid material of the bacteria and the MOF-based carrier.

Further, the MOF nanoparticles are synthesized by a rate-controlled titration method, which specifically comprises:

respectively adding the metal precursor solution and the ligand precursor solution into a mixed solvent of DMF/water at a rate of 10-30 mL/h for reaction, centrifuging and washing to obtain MIL-101-NH₂(Fe) MOFs, i.e., said MOF nanoparticles;

wherein the metal precursor solution comprises one of a ferric chloride aqueous solution, a zirconium oxychloride N' N-dimethylformamide solution and a manganese chloride aqueous solution; the ligand precursor solution comprises one of 2-amino-terephthalic acid DMF/water solution, 2-carboxyl-terephthalic acid DMF/water solution, 2-nitro-terephthalic acid DMF/water solution, 2-bromo-terephthalic acid DMF/water solution and 2-chloro-terephthalic acid DMF/water solution.

Further, the loading of the MOF nanoparticles with a therapeutic agent to obtain a chemotherapeutic drug loaded MOF carrier comprises:

ultrasonically dispersing the nano particles in an aqueous solution to obtain a nano MOF dispersion liquid;

and adding the water solution of the anticancer drug into the nano MOF dispersion liquid, and uniformly mixing in a dark place to obtain the chemical therapy drug loaded MOF carrier.

Further, the mass-to-volume ratio of the MOF nanoparticles to the aqueous solution is (5-15) mg: 10 mL; the mass-volume ratio of the anti-cancer drug to the nano MOF dispersion liquid is (0.1-1) mg: 10 mL.

Further, wrapping hyaluronic acid on the chemotherapeutic drug loaded MOF carrier to obtain a surface electronegative MOF carrier, comprising:

adding hyaluronic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the chemical therapy medicine-loaded MOF carrier to carry out a light-shielding reaction, and then centrifuging and washing to obtain the surface electronegative MOF carrier.

Further, the mass ratio of the hyaluronic acid to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is (5-50): (7-70): (12-20).

Further, modifying polyacrylamide hydrochloride on the surface of the Shewanella to obtain a Shewanella electropositive solution, which comprises the following steps:

adding aqueous solution containing Shewanella into aqueous solution containing polyacrylamide hydrochloride, mixing uniformly, centrifuging and washing to obtain surface electropositive Shewanella solution;

wherein the number of bacterial colonies in the aqueous solution containing Shewanella is 10⁷～10⁹CFU/mL, wherein the concentration of the aqueous solution containing the polyacrylamide hydrochloride is 10-40 mg/mL, and the volume ratio of the aqueous solution containing the Shewanella to the aqueous solution containing the polyacrylamide hydrochloride is (0.5-5): (1-3).

Further, combining the surface electronegative MOF carrier and the surface electropositive Shewanella solution through electrostatic interaction to obtain the hybrid material of the bacteria and the MOF-based carrier, which comprises the following steps:

ultrasonically dispersing the surface electronegative MOF carrier in a NaCl solution, adding the surface electronegative Shewanella solution, uniformly mixing, centrifuging and washing to obtain a hybrid material of bacteria and MOF-based carrier;

wherein the mass-volume ratio of the surface electronegative MOF carrier to the NaCl solution to the surface electropositive Shewanella solution is as follows: (2-10) mg: 5mL of: (0.5-2) mL, wherein the mass fraction of the NaCl solution is 0.89-0.91%.

In a second aspect of the invention, a hybrid material of the bacteria and the MOF-based carrier prepared by the method is provided.

In a third aspect of the invention, the application of the hybrid material of the bacteria and the MOF-based carrier in serving as an anti-tumor drug is provided.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. according to the hybrid material of the bacteria and the MOF-based carrier, provided by the invention, different MOF-based drug-loaded nano-preparations are carried to hypoxic tumor parts by utilizing the specific tumor colonization performance of Shewanella, so that the targeted tumor treatment effect is improved. Meanwhile, Shewanella consumes the lactic acid accumulated excessively in the tumor tissue by mediating the redox reaction between the lactic acid and the metal ions in the MOF, so as to realize the metabolic treatment of the tumor; the redox reaction mediated by bacteria can consume lactic acid accumulated by tumor tissues in a targeted manner, so that tumor metabolic treatment is realized, and the tumor inhibition efficiency is improved;

2. according to the hybrid material of the bacteria and the MOF-based carrier, in the process of tumor metabolic treatment, the generated electrons are transferred to high-valence metal ions in the MOF by the bacteria, so that the degradation of the MOF-based carrier system is induced, and finally, the sustained release of low-valence metal ions and a therapeutic agent is caused. The positive-driven targeting-metabolic regulation-multi-mode combined treatment strategy of the active life body overcomes the defects of poor accuracy and single treatment mode of the traditional nano therapeutic agent, and provides efficient and accurate targeted treatment for tumors. The multi-mode combination therapy not only combines the advantages of each monotherapy, but also can make up the respective deficiencies, thereby generating high-efficiency and low-toxicity anti-tumor effect and realizing the enhanced tumor therapy curative effect of 1+1> 2;

3. according to the bacteria and MOF-based carrier hybrid material and the preparation method thereof, metal MOF is combined with bacteria, so that the targeting accuracy of a therapeutic agent can be effectively improved under the drive of active bacteria, and off-target toxic and side effects are reduced;

4. according to the hybrid material of the bacteria and the MOF-based carrier and the preparation method thereof, the extracellular electron transfer of the bacteria is combined with the activity of metal ions, so that the organic combination between the bacteria and the biological material is obviously enhanced; thereby remarkably enhancing the tumor inhibition effects of chemotherapy, immunotherapy, photodynamic therapy and the like;

5. the preparation method of the hybrid material of the bacteria and the MOF-based carrier is simple, simple and convenient in process and high in operability.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 shows the construction of the SO @ MDH hybrid therapeutic system and its anti-tumor mechanism;

FIG. 2 is a representation of the MOF @ Shewanella hybrid material obtained in example 1; wherein, FIG. 2A is a SEM image; FIG. 2B is the crystal structure, FIG. 2C is the TEM image, FIG. 2D is the result of the particle size test, FIG. 2E is the result of the particle size test, FIG. 2F is the Shewanella coated PAH and MIL-101-NH₂The results of the potential changes before and after the modification of HA are shown in FIG. 2G, which is the results of the number of colonies of Shewanella bacteria before and after the modification of the iron-based MOF;

FIG. 3 shows the cytotoxicity and lactic acid-consuming capacity of the MOF @ Shewanella hybrid material obtained in example 1; wherein, FIG. 3A is the result of cytotoxicity of different materials on 3T3, and FIG. 3B is the result of cytotoxicity of hybrid active material SO @ MDH on 4T1 tumor;

FIG. 4 shows the tumor targeting ability and biocompatibility of the MOF @ Shewanella hybrid material obtained in example 1 in mice; wherein FIG. 4A is the result of in vivo fluorescence imaging and FIG. 4B is the result of biosafety analysis;

FIG. 5 shows the in vivo anti-tumor effect of the MOF @ Shewanella hybrid material obtained in example 1; wherein, fig. 5A is a tumor decimal picture, and fig. 5B is a weight change result; FIG. 5C shows the results of tumor size changes;

FIG. 6 is a flow chart of a preparation method of the hybrid material of bacteria and MOF-based carrier provided by the embodiment of the invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

The embodiment of the invention provides a hybrid material of bacteria and an MOF-based carrier, which has the following general idea:

according to another exemplary embodiment of the present invention, there is provided a method for preparing a hybrid material of bacteria and a MOF-based carrier, as shown in fig. 6, the method comprising:

s1, loading the MOF nanoparticles with a therapeutic agent to obtain a MOF carrier loaded with chemotherapeutic drugs;

as an alternative embodiment, the MOF nanoparticles are synthesized by a rate-controlled titration method, which specifically comprises:

synthesis of MIL-101-NH by controlled Rate titration₂(Fe) MOF, specifically as follows: respectively adding 10-50 mM metal precursor solution and 10-50 mM ligand precursor solution into a reactor containing 8-16 mL DMF/water mixed solvent through an independent pipeline by an injection pump at a rate of 10-30 mL/h, and reacting under stirring at 50 ℃. After the reaction is finished, taking out the reaction solution, centrifuging (10000-12000 rpm, 10-20 min), and washing to obtain the MIL-101-NH with regular shape and size₂(Fe) MOFs, i.e., said MOF nanoparticles;

wherein, the metal precursor solution can be ferric chloride aqueous solution, zirconium oxychloride N' N-Dimethylformamide (DMF) solution or manganese chloride aqueous solution; the ligand precursor solution may be a 2-amino-terephthalic acid DMF/water (V/V ═ 1/1) solution, an M2-carboxy-terephthalic acid DMF/water (V/V ═ 1/1) solution, a 2-nitro-terephthalic acid DMF/water (V/V ═ 1/1) solution, a 2-bromo-terephthalic acid DMF/water (V/V ═ 1/1) solution, or a 2-chloro-terephthalic acid DMF/water (V/V ═ 1/1) solution.

The reason why the adding rate is controlled to be 10-30 mL/h is as follows: the particle size of the MOF nano particles is controlled, and if the adding speed is less than 10mL/h, the particle size of the synthesized MOF nano particles is increased, so that the MOF nano particles are not beneficial to being modified on the surface of bacteria; if the adding speed is more than 30mL/h, the particle size of the MOF nano particles is very small, and the yield is low;

if the concentrations of the metal precursor liquid and the ligand precursor liquid are less than 10mM, the particle size of the MOF nano-particle may become larger; if it exceeds 50mM, the particle size of the MOF nanoparticles may become smaller;

the volume ratio of DMF to water in the DMF/water mixed solvent is preferably 1: 3: the solubility of the organic ligand can be improved, and in other embodiments, other volume ratios can be selected to dissolve the organic ligand;

the volume ratio of DMF/aqueous solution of DMF to water in the metal precursor solution and the ligand precursor solution is preferably 1:1, so that the solubility of the ligand can be improved, and in other embodiments, other volume ratios can be selected to dissolve the organic ligand;

as an optional implementation manner, the step S1 specifically includes:

The mass-to-volume ratio of the MOF nanoparticles to the aqueous solution is (5-15) mg: 10 mL; the mass-to-volume ratio is too small, and the MOF concentration is too low, so that the yield of the obtained medicine-carrying MOF nano particles is reduced; the MOF with too large mass-to-volume ratio has too high concentration, poor dispersibility and easy aggregation, and is not beneficial to entrapping the medicine;

the anti-cancer drug comprises one of adriamycin, paclitaxel and photosensitizer Ce 6; here the MOF nanoparticles can be loaded with different types of therapeutic agents such as: adriamycin, paclitaxel and photosensitizer Ce6, but not limited to one of the three; the mass-volume ratio of the anti-cancer drug to the nano MOF dispersion liquid is (0.1-1) mg: 10 mL. The mass-to-volume ratio is too small, the drug concentration is too small, and the drug loading rate is low; too large, resulting in low drug encapsulation efficiency;

s2, coating hyaluronic acid on the MOF carrier loaded with the chemotherapeutic drug to obtain a MOF carrier with negative surface charge;

in step S2, the MOF carrier loaded with the chemotherapeutic drug is subjected to surface modification of Hyaluronic Acid (HA) with different amounts to enable the surface of the HA to be electronegative so as to facilitate subsequent combination with bacteria.

The step S2 specifically includes:

adding hyaluronic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the chemical therapy drug loaded MOF carrier to perform a light-shielding reaction, and then centrifuging and washing to obtain a surface electronegative MOF carrier, namely the chemical therapy drug loaded nano-particle (MIL-101-NH)₂(Fe)-DOX-HA)。

The mass ratio of the hyaluronic acid to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is (5-50): (7-70): (12-20). The excessive addition of hyaluronic acid easily causes the solution to be viscous (or form gel), which is not beneficial to the activation of the carboxyl functional group of hyaluronic acid; too little addition results in too little hyaluronic acid modified on the MOF surface, and medicine leakage is easily caused; the cost is increased due to excessive addition of N-hydroxysuccinimide, and the product loss is serious and the yield is reduced due to increased washing times; the addition of the acid is too little to completely activate the carboxyl on the hyaluronic acid, so that the condensation reaction is not facilitated; the cost is increased by adding too much 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride, and the product loss is serious and the yield is reduced due to the increase of the washing times; the carboxyl on the hyaluronic acid can not be completely activated due to too little addition, which is not favorable for the condensation reaction;

the hyaluronic acid, the N-hydroxysuccinimide and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride can be added separately or mixed to prepare a mixed solution, and the volume ratio of the chemical therapy medicine loaded MOF carrier to the mixed solution is 10: (4-6).

S3, modifying polyacrylamide hydrochloride on the surface of the Shewanella to obtain a Shewanella electropositive solution on the surface;

in step S3, the aqueous solution of bacteria and the aqueous solution of allylamine hydrochloride (PAH) are mixed such that the bacterial surface is rendered electropositive for subsequent binding to MOFs.

The step S3 specifically includes:

The number of bacterial colonies in the aqueous solution containing Shewanella is less than 10⁷CFU/mL, Shewanella concentration is too low, resulting in excessive modified PAH, and further affecting bacterial activity; if it is greater than 10⁹CFU/mL, too little PAH modified on the surface of bacteria, weak positive electricity on the surface, and unfavorable for combining with the MOF nano particles with negative electricity through electrostatic interaction;

if the concentration of the aqueous solution containing the polyacrylamide hydrochloride is less than 10mg/mL, the PAH modified on the surface of the bacteria is too little, the electropositivity of the surface is weak, and the combination with the negatively charged nanoparticles through the electrostatic action is not facilitated; if the concentration is more than 40mg/mL, excessive modified PAH is caused, and the activity of bacteria is further influenced;

the volume ratio of the aqueous solution containing the Shewanella to the aqueous solution containing the polyacrylamide hydrochloride is (0.5-5): (1-3) reasons for: within this ratio, PAH-modified bacteria can be obtained without affecting the activity of the bacteria, and too small or too large a volume ratio is not favorable for obtaining the desired PAH-modified bacteria;

as an alternative embodiment, the aqueous solution containing shewanella is prepared by the following steps: shewanella (S. oneidensis MR-1) was cultured aerobically in Luria-Bertani (LB) medium (10mg/mL trypsin, 5mg/mL yeast extract and 0.5mg/mL NaCl) overnight at 30 ℃. And when the bacteria grow to a plateau stage, taking a culture medium containing the bacteria, centrifuging to collect the bacteria, then re-suspending with sterile PBS, centrifuging again (centrifugally collecting the bacteria at the rotating speed of 1000-5000 rpm for 5-20 min), repeating for three times, and dispersing the bacteria in water for later use.

The microbial agent purchased from Shewanella facultative anaerobe (S. oneidensis MR-1) can effectively transfer electrons from certain organic matters to Fe-containing substances³⁺、Mn⁴⁺Thereby realizing the conversion of the metal ions from a high valence state to a low valence state. Also, such bacteria can selectively utilize lactic acid as an energy source to produce non-carcinogenic acetate rather than glucose. Based on the method, the S.oneidensis MR-1 can be used as a biological therapeutic agent for targeting lactic acid metabolism in a tumor microenvironment, and can realize the purpose of accurately regulating and controlling tumor metabolism through unique physiological metabolism of bacteria, thereby providing a new idea for realizing accurate treatment of tumors.

S4, combining the surface electronegative MOF carrier with the surface electropositive Shewanella solution through electrostatic interaction to obtain the hybrid material of the bacteria and the MOF-based carrier.

The step S4 specifically includes:

ultrasonically dispersing the surface electronegative MOF carrier in a NaCl solution, adding the surface electronegative Shewanella solution, uniformly mixing, centrifuging (the rotating speed is 1000-5000 rpm, and the time is 5-20 min), and washing to obtain a hybrid material of bacteria and an MOF-based carrier;

The NaCl solution with the mass fraction of 0.89-0.91% is adopted during dispersion because the sodium chloride aqueous solution with the concentration is equivalent to the osmotic pressure of body fluid of mammals, adverse reaction cannot be caused by tail vein injection, and other solutions can be adopted for substitution, such as HEPES biological buffer solution and the like;

the mass-volume ratio of the surface electronegative MOF carrier to the NaCl solution to the surface electropositive Shewanella solution is as follows: (2-10) mg: 5mL of: (0.5-2) mL: under the condition of the mass-volume ratio, the SO @ MDH biological hybrid active material with good appearance and function can be prepared; too little or too much surface electronegative MOF carriers and too little or too much surface electropositive Shewanella solution are not favorable for preparing ideal SO @ MDH biological hybrid active materials, SO that too much or too little MOF nano particles modified by the surface functions of the bacteria are caused, or the biological activity of the bacteria is influenced;

in summary, the technical scheme of the invention overcomes the technical difficulties by the following important steps:

in step S3, the bacterial colony count of the aqueous solution containing Shewanella is adjusted to 10⁷～ 10⁹CFU/mL, wherein the concentration of the aqueous solution containing the polyacrylamide hydrochloride is 10-40 mg/mL, and the volume ratio of the aqueous solution containing the Shewanella to the aqueous solution containing the polyacrylamide hydrochloride is (0.5-5): (1-3) optimizing and selectively modifying the amount of PAH on the surface of Shewanella at unit concentration so as to obtain bacteria with enough positive charges on the surface and without influencing the activity of Shewanella;

in step S4, in the embodiment of the present invention, the mass-to-volume ratio of the surface electronegative MOF carrier, the NaCl solution, and the surface electropositive shewanella solution is adjusted to: (2-10) mg: 5mL of: (0.5-2) mL to regulate and control the grafting ratio of the PAH-modified Shewanella and the hyaluronic acid functionalized MOF nanoparticles, SO as to obtain an SO @ MDH hybrid material with excellent performance;

further, in the step S1, the morphology and size of the MOF nanoparticles are controlled by optimizing the preparation conditions (selection of different metal ions and organic ligands), the solvent ratio, the reaction temperature, the dropping speed, and the like of the MOF nanoparticles.

According to another exemplary embodiment of the invention, a hybrid material of bacteria and MOF-based carriers is provided, which is prepared by the method.

According to another exemplary embodiment of the invention, the use of the hybrid material of bacteria and MOF-based carrier as an antitumor drug is provided.

The hybrid material of the bacteria and the MOF-based carrier utilizes the specific tumor colonization performance of Shewanella to carry different MOF-based drug-loaded nano-preparations to the hypoxic tumor part, thereby improving the treatment effect of the targeted tumor. Meanwhile, Shewanella consumes the lactic acid accumulated excessively in the tumor tissue by mediating the redox reaction between the lactic acid and the metal ions in the MOF, so that the tumor metabolic treatment is realized. During this process, the bacteria transfer the generated electrons to the higher valent metal ions in the MOF, inducing degradation of the MOF-based drug-carrier system, ultimately resulting in sustained release of the lower valent metal ions and therapeutic agent. As shown in fig. 1, the positive-driven targeting-metabolic regulation-multi-mode combined therapy strategy of the active living body overcomes the defects of poor accuracy and single treatment mode of the traditional nano therapeutic agent, and provides efficient and accurate targeted therapy for tumors.

A detailed description of a hybrid material of bacteria and MOF-based carrier according to the present application will be given below with reference to examples and experimental data.

Example 1 Shewanella binding iron-based MOF hybrid active Material and method for preparing the same

(1) A metal precursor solution (40mM ferric chloride aqueous solution) and a ligand precursor solution (40mM 2-amino-terephthalic acid DMF/water (V/V. 1/1) solution) were prepared, and then the above precursor solutions were added to a reactor containing 10mL DMF/water (1/3) mixed solvent at 20mL/h through separate tubes by syringe pumps, respectively, and reacted for 24h with stirring at 50 ℃. Then centrifuging (11000rpm, 15min), washing to obtain the MIL-101-NH with regular shape and size₂(Fe)MOF。

(2) Subjecting 10mg of MIL-101-NH obtained in the above (1)₂(Fe) MOF was uniformly dispersed in 10mL of an aqueous solution and sonicated for 10min to fully disperse. Then, adding 1mL of aqueous solution of DOX (0.5mg/mL) into the nano MOF dispersion liquid, and stirring at room temperature in the dark for 24h to obtain MIL-101-NH loaded with chemotherapeutic drug DOX₂(Fe)-DOX。

(3) 5mL of a mixture solution containing hyaluronic acid (25mg), N-hydroxysuccinimide (NHS, 35mg), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC, 60mg) was added to the above MIL-101-NH2(Fe) -DOX solution, and reacted at room temperature with exclusion of light for 24 hours. After the reaction is finished, centrifuging (11000rpm, 15min), washing with water, and collecting precipitates to obtain the chemotherapeutic drug loaded nano-particle MIL-101-NH₂(Fe)-DOX-HA。

(4) Shewanella (S.oneidensis MR-1) was cultured aerobically in Luria-Bertani (LB) medium (10mg/mL trypsin, 5mg/mL yeast extract and 0.5mg/mL NaCl) overnight at 30 ℃. When the bacteria grow to the plateau stage, taking the culture medium containing the bacteria, centrifuging (5000rpm, 5min) to collect the bacteria, then re-centrifuging after being resuspended by sterile PBS, and dispersing the bacteria in water for standby after repeating three times.

(5) Subsequently, 1mL of an aqueous solution (10) containing the bacteria was added⁸CFU/mL) was added to 2mL of an aqueous solution (20mg/mL) containing poly (allylamine hydrochloride) (PAH), vortexed gently for one minute, centrifuged (5000rpm, 5min), washed with water three times, and bacteria (shewanella @ PAH) having PAH coated on the surface were collected.

(6) Subjecting 10mg of MIL-101-NH obtained in the above (3)₂(Fe) -DOX-HA was ultrasonically dispersed in 5mL of an aqueous solution, to which was subsequently added 2mL of an aqueous solution of bacteria @ PAH. Vortexed for 3min, centrifuged (5000rpm, 5min), washed three times with ultrapure water, and the hybrid active material (SO @ MDH) obtained by combining the bacteria and the MOF is collected.

Example 2 Shewanella-binding iron-based MOF hybrid active Material and method for preparing the same

(1) A metal precursor solution (10mM ferric chloride aqueous solution) and a ligand precursor solution (10mM 2-amino-terephthalic acid DMF/water (V/V ═ 1/1) solution) were prepared, and then the above precursor solutions were added to a reactor containing 8mL of mixed solvent of mf/water (1/3) at 10mL/h by a syringe pump through independent pipes, and reacted for 24 hours with stirring at 50 ℃. Then centrifuging (11000rpm, 15min), washing to obtain the MIL-101-NH with regular shape and size₂(Fe)MOF。

(2) Mixing 5mg of MIL-101-NH obtained in the above (1)₂(Fe) MOF was homogeneously dispersed in 10mL of aqueous solution, ultraThe mixture was sonicated for 10min to disperse it thoroughly. Then, adding 1mL of aqueous solution of DOX (0.1mg/mL) into the nano MOF dispersion liquid, and stirring at room temperature in the dark for 24h to obtain MIL-101-NH loaded with chemotherapeutic drug DOX₂(Fe)-DOX。

(3) 5mL of a mixture solution containing hyaluronic acid (5mg), N-hydroxysuccinimide (NHS, 7mg), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC, 12mg) was added to the above MIL-101-NH2(Fe) -DOX solution, and reacted at room temperature with exclusion of light for 24 hours. After the reaction is finished, centrifuging (11000rpm, 15min), washing with water, and collecting precipitates to obtain the chemotherapeutic drug loaded nano-particle MIL-101-NH₂(Fe)-DOX-HA。

(4) Shewanella (S.oneidensis MR-1) was aerobically cultured in Luria-Bertani (LB) medium (10mg/mL of trypsin, 5mg/mL of yeast extract and 0.5mg/mL of NaCl) at 30 ℃ overnight. When the bacteria grow to the plateau stage, taking the culture medium containing the bacteria, centrifuging (5000rpm, 5min) to collect the bacteria, then re-suspending the bacteria by sterile PBS, centrifuging again, repeating the steps for three times, and dispersing the bacteria in water for standby.

(5) Subsequently, 0.5mL of an aqueous solution (10) containing the bacteria was added⁷CFU/mL) was added to 2mL of an aqueous solution (10mg/mL) containing poly (allylamine hydrochloride) (PAH), vortexed gently for one minute, centrifuged (5000rpm, 5min), washed with water three times, and bacteria (shewanella @ PAH) having PAH coated on the surface were collected.

(6) 2mg of MIL-101-NH obtained in the above (3)₂(Fe) -DOX-HA was dispersed ultrasonically in 0.5mL of an aqueous solution, to which was subsequently added 2mL of an aqueous solution of bacteria @ PAH. Vortexed for 3min, centrifuged (5000rpm, 5min), washed three times with ultrapure water, and the hybrid active material (SO @ MDH) obtained by combining the bacteria and the MOF is collected.

Example 3 Shewanella binding iron-based MOF hybrid active Material and method for preparing the same

(1) A metal precursor solution (50mM aqueous solution of ferric chloride) and a ligand precursor solution (50mM solution of 2-amino-terephthalic acid DMF/water (V/V) ═ 1/1) were prepared, and then the above precursor solutions were added to a reactor containing 16mL of a DMF/water (1/3) mixed solvent at 30mL/h by a syringe pump through independent pipes, respectively, and reacted for 24 hours with stirring at 50 ℃. Subsequently, the process of the present invention,centrifuging (11000rpm, 15min), washing to obtain the MIL-101-NH with regular shape and size₂(Fe)MOF。

(2) Subjecting 15mg of MIL-101-NH obtained in the above (1)₂(Fe) MOF was uniformly dispersed in 10mL of an aqueous solution and sonicated for 10min to fully disperse. Then, adding 1mL of aqueous solution of DOX (1mg/mL) into the nano MOF dispersion liquid, and stirring at room temperature in the dark for 24h to obtain MIL-101-NH loaded with chemotherapeutic drug DOX₂(Fe)-DOX。

(3) 5mL of a mixture solution containing hyaluronic acid (50mg), N-hydroxysuccinimide (NHS, 70mg), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC, 120mg) was added to the above MIL-101-NH2(Fe) -DOX solution, and reacted at room temperature with exclusion of light for 24 hours. After the reaction is finished, centrifuging (11000rpm, 15min), washing with water, and collecting precipitates to obtain the chemotherapeutic drug loaded nano-particle MIL-101-NH₂(Fe)-DOX-HA。

(4) Shewanella (S.oneidensis MR-1) was cultured aerobically in Luria-Bertani (LB) medium (10mg/mL trypsin, 5mg/mL yeast extract and 0.5mg/mL NaCl) overnight at 30 ℃. When the bacteria grow to the plateau stage, taking the culture medium containing the bacteria, centrifuging (5000rpm, 5min) to collect the bacteria, then re-suspending the bacteria by sterile PBS, centrifuging again, repeating the steps for three times, and dispersing the bacteria in water for standby.

(5) Subsequently, 5mL of an aqueous solution (10) containing the bacteria was added⁹CFU/mL) was added to 2mL of an aqueous solution (40mg/mL) containing poly (allylamine hydrochloride) (PAH), vortexed gently for one minute, centrifuged (5000rpm, 5min), washed with water three times, and bacteria (shewanella @ PAH) having PAH coated on the surface were collected.

(6) Subjecting 10mg of MIL-101-NH obtained in the above (3)₂(Fe) -DOX-HA was dispersed ultrasonically in 5mL of an aqueous solution, to which was subsequently added 2mL of an aqueous solution of bacteria @ PAH. Vortexed for 3min, centrifuged (5000rpm, 5min), washed three times with ultrapure water, and the hybrid active material (SO @ MDH) obtained by combining the bacteria and the MOF is collected.

Experimental example 1 structural characterization and Performance testing of Shewanella-binding iron-based MOF hybrid active materials

Using a Transmission Electron Microscope (TEM), a Scanning Electron Microscope (SEM), anda Field Emission Scanning Electron Microscope (FESEM) represents various MOF nano materials and the shapes and sizes of the MOF nano materials after the MOF nano materials are hybridized with bacteria; as shown in FIG. 2, the MOF nano material and the morphology, size and element mapping change of the MOF nano material after hybridization with bacteria prove that the material is successfully prepared. Measuring the size and potential change of the prepared MOF carrier material and the combined bacteria by using a dynamic scatterometer (DLS) so as to judge whether the surface modification is successful and the dispersibility of the material; as shown in FIG. 2F, Shewanella encapsulates PAH and MIL-101-NH₂The potential change before and after the modification of HA further proves that the hybrid material can be prepared by electrostatic adsorption. Characterizing a crystal structure in the nanomaterial using powder X-ray diffraction (PXRD); as shown in FIG. 2B, MIL-101-NH₂The nano particles and the Shewanella combined iron-based MOF hybrid active material can keep the crystal structure of MIL-101MOF, and further prove that MIL-101-NH₂Successful synthesis and stability of the material during functionalization. Furthermore, as shown in FIG. 2G, the number of colonies of Shewanella bacteria did not change before and after modification of the iron-based MOF, demonstrating that the activity of Shewanella bacteria was not affected during the functionalization process.

Application example 1 in vitro experiment

In-vitro co-culture experiments are carried out by using 3T3 normal cells, 4T1 tumor cells and the prepared MOF @ bacteria hybrid active materials, and in-vitro anti-tumor performance of the active living body hybrid materials is systematically researched.

The method comprises the following steps: normal cell toxicity. Different hybrid materials and normal 3T3 cells are co-cultured for 24h, and the cytotoxicity of the series of hybrid materials is respectively detected by an MTT method. First, 3T3 cells were seeded in 24-well plates (5X 10)⁴Individual cells/well) were incubated in 1640RPMI medium for 24 h. Then, 100. mu.g/mL of the materials of the different groups were added, incubated for 24h, carefully aspirated, washed 2 times with PBS, fresh medium containing CCK8 solution was added, incubated for 2 hours, and absorbance at 450nm was measured with a microplate reader. The different groups of materials were tested for cytotoxicity against 3T 3. As shown in FIG. 3A, the viability of normal cells of 3T3 was greater than 80%, demonstrating that the material was almost non-toxic to 3T3 cells, indicating that the material had good biocompatibility.

Tumor cell toxicity. Firstly, the method4T1 cells were seeded in 24-well plates (5X 10)⁴Individual cells/well) were incubated in 1640RPMI medium for 24 h. To verify that Shewanella can selectively consume lactic acid in the culture environment for metabolism, cells of the hybrid active material SO @ MDH and 4T1 were co-cultured with the cells under culture conditions containing glucose or lactic acid for 24h, followed by careful aspiration, PBS washing for 2 times, addition of fresh medium containing CCK8 solution, incubation for 2 h, and absorbance at 450nm wavelength measured with a microplate reader. The hybrid active material SO @ MDH was assayed for its cytotoxicity against 4T1 tumor cells. As shown in FIG. 3B, when the culture medium contains lactic acid, a large amount of 4T1 tumor cells are killed by the hybrid material SO @ MDH, and when the culture medium only contains glucose, the SO @ MDH has no obvious toxicity to the 4T1 tumor cells, which indicates that the SO @ MDH can selectively consume the lactic acid in the culture environment for metabolism and remarkably enhance the chemotherapy curative effect.

In-vivo tumor targeting capability and biocompatibility of application example 2 SO @ MDH hybrid material

The invention takes BALB/c mouse as model animal to construct 4T1 tumor-bearing mouse model, and systematically studies the in vivo tumor targeting ability and biocompatibility of the hybrid material after tail vein injection; the method comprises the following steps:

(1) tumor targeted enrichment

When the tumor grows to 150mm³On the left and right, Cy 5-labeled SO, SO @ MDH, SO + MDH, MDH (SO: 5X 10)⁷CFU, MDH:0.53mg) hybrid material was injected into mice via tail vein, and the anesthetized mice were placed in Maestro in vivo fluorescence imaging system (CRI, Woburn, MA, USA) for observation at predetermined time points (0, 2, 4, 6, 8, 10 h). Cy5, Ex 640nm, Em 680 nm. As shown in fig. 4A, the synthesized hybrid active material SO @ MDH showed significant fluorescence at the tumor site and the fluorescence intensity gradually increased over time, indicating that the bacteria can perform targeting effect to carry MOF to the tumor site accurately.

(2) Biosafety analysis

The indexes of the mouse organism after the material treatment, including blood biochemical analysis, immunotoxicity, biocompatibility and the like, are comprehensively evaluated, and the biological safety and the clinical application potential of the hybrid material constructed by the invention in vivo are detected. As shown in fig. 4B, after the injection of the hybrid active material, the indexes of the mice are not significantly different from those of the normal control mice, indicating that the MOF-based bacterial hybrid material has good biological safety and clinical application potential in vivo.

In vivo anti-tumor performance research of application example 3 SO @ MDH hybrid material

The invention takes BALB/c mice as model animals to construct a 4T1 tumor-bearing mouse model, and systematically studies the treatment effect of the hybrid material on tumors after tail vein injection. When the tumor grows to 50-100 mm³Meanwhile, tumor-bearing mice were randomly grouped, and the corresponding materials were injected into the mice through tail vein on the first day, the fourth day, the seventh day, and the tenth day, respectively. Changes in mouse body weight (measured with a balance) and tumor size (tumor major axis (L) and minor axis (W) measured with a vernier caliper) were recorded every two days, tumor volume was determined using Still's formula: v — W2 × L/2. After treatment, mice were sacrificed and all tumors were collected, weighed and photographed.

As shown in fig. 5, compared with other control materials (the control materials include PBS, DOX and MDH), after the tumor-bearing mice receive the treatment of the hybrid active material SO @ MDH, the growth of the tumor is obviously inhibited, and the volume and weight of the tumor are both obviously smaller than those of the other materials, which indicates that the SO @ MDH hybrid material can effectively inhibit the growth of the tumor and has good anti-tumor effect. The MDH is a pure MOF drug-loaded nanoparticle, and the treatment effect is poor; meanwhile, the therapeutic effect of the physical mixture of bacteria and MDH is also inferior to that of SO @ MDH; optimal therapeutic effect can only be achieved by targeted delivery of nanoparticles to the tumor area by bacteria and metabolism of lactate resulting in degradation of MDH, releasing DOX.

Finally, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method for preparing a hybrid material of bacteria and a MOF-based carrier, the method comprising:

wrapping hyaluronic acid on the chemotherapeutic drug-loaded MOF carrier to obtain a surface electronegative MOF carrier;

adding aqueous solution containing Shewanella into aqueous solution containing polyacrylamide hydrochloride, mixing uniformly, centrifuging and washing to obtain surface electropositive Shewanella solution; wherein the number of bacterial colonies in the aqueous solution containing Shewanella is 10⁷～10⁹CFU/mL, wherein the concentration of the aqueous solution containing the polyacrylamide hydrochloride is 10-40 mg/mL, and the volume ratio of the aqueous solution containing the Shewanella to the aqueous solution containing the polyacrylamide hydrochloride is (0.5-5): (1-3);

combining the surface electronegative MOF carrier with the surface electropositive Shewanella solution through electrostatic interaction to obtain a hybrid material of bacteria and MOF-based carrier;

wherein the Shewanella is S. oneidedensis MR-1;

the MOF nano-particles are synthesized by adopting a speed-controlled titration method, and specifically comprise the following steps:

respectively adding the metal precursor solution and the ligand precursor solution into a mixed solvent of DMF/water at a rate of 10-30 mL/h for reaction, centrifuging and washing to obtain MIL-101-NH₂(Fe) MOF, i.e. said MOFNanoparticles;

wherein the metal precursor solution is ferric chloride aqueous solution; the ligand precursor solution is 2-amino-terephthalic acid DMF/water solution.

2. The preparation method of the hybrid material of the bacteria and the MOF-based carrier, according to claim 1, is characterized in that the MOF nanoparticles are loaded with the therapeutic agent to obtain the MOF carrier loaded with the chemotherapeutic drug, and the preparation method comprises the following steps:

3. The preparation method of the hybrid material of the bacteria and the MOF-based carrier, according to claim 2, wherein the mass-to-volume ratio of the MOF nanoparticles to the aqueous solution is (5-15) mg: 10 mL; the mass-volume ratio of the anti-cancer drug to the nano MOF dispersion liquid is (0.1-1) mg: 10 mL.

4. The method for preparing the hybrid material of the bacteria and the MOF-based carrier according to claim 1, wherein the method for coating the chemical therapy drug loaded MOF carrier with hyaluronic acid to obtain the MOF carrier with electronegativity on the surface comprises the following steps:

5. A method for preparing hybrid material of bacteria and MOF-based carrier according to claim 4,

the mass ratio of the hyaluronic acid to the N-hydroxysuccinimide to the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride is (5-50): (7-70): (12-20).

6. The preparation method of the hybrid material of the bacteria and the MOF-based carrier, according to claim 1, is characterized in that the surface electronegative MOF carrier and the surface electropositive Shewanella solution are combined through electrostatic interaction to obtain the hybrid material of the bacteria and the MOF-based carrier, and comprises the following steps:

wherein the mass-volume ratio of the surface electronegative MOF carrier to the NaCl solution to the surface electropositive Shewanella solution is as follows: (2-10) mg: 5mL of: (0.5-2) mL, and the mass fraction of the NaCl solution is 0.89-0.91%.

7. A hybrid material of bacteria and MOF-based carrier prepared by the method of any one of claims 1 to 6.

8. Use of the bacteria and MOF-based carrier hybrid material according to claim 7 for the preparation of an anti-tumor drug.