CN117883562A - Aluminum hydroxide nano-particles taking bacterial outer membrane vesicles as templates and preparation method and application thereof - Google Patents
Aluminum hydroxide nano-particles taking bacterial outer membrane vesicles as templates and preparation method and application thereof Download PDFInfo
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- CN117883562A CN117883562A CN202311780036.1A CN202311780036A CN117883562A CN 117883562 A CN117883562 A CN 117883562A CN 202311780036 A CN202311780036 A CN 202311780036A CN 117883562 A CN117883562 A CN 117883562A
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- outer membrane
- bacterial outer
- aluminum hydroxide
- aluminum
- membrane vesicles
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01F—COMPOUNDS OF THE METALS BERYLLIUM, MAGNESIUM, ALUMINIUM, CALCIUM, STRONTIUM, BARIUM, RADIUM, THORIUM, OR OF THE RARE-EARTH METALS
- C01F7/00—Compounds of aluminium
- C01F7/02—Aluminium oxide; Aluminium hydroxide; Aluminates
- C01F7/30—Preparation of aluminium oxide or hydroxide by thermal decomposition or by hydrolysis or oxidation of aluminium compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/03—Particle morphology depicted by an image obtained by SEM
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/61—Micrometer sized, i.e. from 1-100 micrometer
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Thermal Sciences (AREA)
- Geology (AREA)
- Inorganic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the field of biological medicine, and in particular relates to aluminum hydroxide nano particles taking bacterial outer membrane vesicles as templates, and a preparation method and application thereof. The method successfully prepares the aluminum hydroxide nano-particles by a protein biomineralization method, synthesizes the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicles as templates, simply and efficiently applies the bacterial outer membrane vesicles to a nanoadjuvant system, and expands biomedical application of the bacterial outer membrane vesicles. The aluminum hydroxide nano particles prepared by the method have the advantages of high surface activity, more surface active centers, strong adsorption capacity and the like due to small particle size and rapid increase of specific surface area, and compared with the traditional aluminum hydroxide adjuvant, the aluminum hydroxide nano particles can remarkably reduce the aluminum consumption and effectively reduce local adverse reactions. The aluminum hydroxide nano-particles prepared by the invention have the immunity stimulating capability without additional antigens, can induce strong immune response, enrich the performance of aluminum hydroxide adjuvant, and are novel composite adjuvant.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to aluminum hydroxide nano particles taking bacterial outer membrane vesicles as templates, and a preparation method and application thereof.
Background
Aluminum hydroxide adjuvants are the most long-used vaccine adjuvants, which have been used for almost centuries to greatly promote humoral (antibody) responses and Th2 cell immune responses, and are widely used in various human and veterinary vaccines. As a vaccine adjuvant approved for use in humans by the U.S. food and drug administration (FoodandDrugAdministration, FDA), its effectiveness and safety has been fully validated for clinical use for more than 30 years. However, conventional aluminum adjuvants have certain limitations in that they induce a strong Th2 humoral immune response, but lack a Th1 cellular immune response. This has limited the use of conventional aluminum adjuvants and has not been satisfactory for use in vaccines for HIV, tuberculosis, malaria, tumors, etc. And the traditional aluminum salt adjuvant has high aluminum content and large size, is not easy to metabolize at the injection site, and can cause some local adverse reactions, such as erythema, subcutaneous nodules, contact hypersensitivity, granuloma and the like. Therefore, a new aluminum adjuvant capable of inducing both strong humoral immunity and cellular immunity and reducing toxic and side effects is urgently needed to be studied.
In order to enrich the functionality of aluminium hydroxide adjuvants, a number of aluminium hydroxide based complex adjuvants have been developed and applied in vaccine preparation in recent years. AS04 adjuvants, such AS those prepared by adsorption of monophosphoryl liposome a (MPL) onto Aluminum hydroxide adjuvants, are effective in inducing T cell immune responses and increasing the frequency of memory B cell production and releasing high levels of antibodies (DIDIERLAURENTAM, MOREL S, LOCKMAN L, et al as04, an Aluminum Salt-and TLR4 agnist-Based Adjuvant System, induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity1[ J ]. The Journal ofImmunology,2009,183 (10): 6186-97.); unmethylated cytosine-phosphate-guanine oligonucleotides (CpG-ODNs) were mixed with aluminum hydroxide adjuvant to elicit multiple immune responses (Zhang Y, zheng X, shaping W, et al, alum/CpG Adjuvanted Inactivated COVID-19Vaccine with Protective Efficacy against SARS-CoV-2and Variants (Basel). 2022;10 (8): 1208.Published 2022Jul 29.). With the development of nano technology, the application of nano materials in the biomedical field has been greatly advanced since the 80 th century of 20 th century. Compared with the traditional aluminum hydroxide adjuvant, the nanometer aluminum hydroxide adjuvant has the advantages of small particle size, rapid increase of specific surface area, high surface activity, more surface active centers, strong adsorption capacity and the like, so that more antigens (Li X, aldayel AM, cui Z.aluminum hydroxide nanoparticles show a stronger vaccine adjuvant activity than traditional aluminum hydroxide micro parts.J Control release.2014; 173:148-157) can be adsorbed under the same aluminum content. In addition, the nano aluminum hydroxide particles have smaller size, are easier to internalize by antigen presenting cells (such as dendritic cells), are beneficial to the intake, processing and presentation of extracellular antigens by the antigen presenting cells, can remarkably promote the secretion of important cytokines such as IL-12, TNF-alpha, IFN-gamma and the like, and induce strong antibody responses and Th1 cellular immune responses (Orr MT, khandharAP, seydoux E, et al, reprograming the adjuvant properties ofaluminum oxyhydroxide with nanoparticle technology, NPJVAccines.2019;4:1.Published 2019Jan 3.). Early nanometer aluminum hydroxide adjuvants were prepared directly mainly by the following two strategies: (1) gradually reducing the size of aluminum hydroxide particles or hydrogel to prepare nano aluminum hydroxide; (2) is formed by coprecipitation of aluminum salt and stabilizer and gradually grows to the required size. In recent years, protein biomineralization techniques using proteins as templates have been used to guide the synthesis of inorganic nanoparticles. As the protein is rich in functional groups such as rich amino, carboxyl, sulfhydryl and the like, the protein has a plurality of metal ion binding sites, and can regulate and control the growth of nanocrystals through coordination with metal ions (Wang W, liu X, zheng X, jin HJ, li X. Biomeralation: an Opportunity and Challenge of Nanoparticle Drug Delivery Systems for Cancer therapeutic. Adv health Mater.2020;9 (22): e 2001117.). Therefore, the protein biomineralization technology using protein as a template can be adopted to construct the nano aluminum hydroxide adjuvant.
Disclosure of Invention
At present, aluminum hydroxide-based compound adjuvants are complex in composition and high in cost, are limited in that the larger size of the compound adjuvants is difficult to internalize by antigen presenting cells, are insufficient for inducing strong immune responses, and still are difficult to effectively activate anti-tumor immunity. The existing technology for directly preparing the nano aluminum hydroxide has the defects of complicated preparation process, complex prescription components and higher requirements on process equipment, often needs to add excipients or stabilizers, possibly needs to remove residual organic matters in the subsequent purification process, adds additional cost and restricts the application of the nano aluminum hydroxide adjuvant in vaccines to a certain extent. However, the existing indirect preparation method based on biomineralization technology often introduces nonfunctional protein templates, and the adjuvant has single function.
Aiming at the defects or improvement demands of the prior art, the invention aims to provide a preparation method and application of aluminum hydroxide nano-particles taking bacterial outer membrane vesicles as templates. Bacterial outer membrane vesicles (outer membrane vesicles, OMVs) are vesicles secreted predominantly by gram-negative bacteria, contain various endogenous immunostimulatory components from their parent cells, such as lipopolysaccharide, outer membrane proteins and lipoproteins, and do not have replication capacity at 20-250 nm. Can directly activate the innate immune pattern recognition receptor of most hosts and induce immune response, and is an immune adjuvant with great application prospect.
According to the invention, the aluminum hydroxide nano particles are constructed by using the protein biomineralization technology with the bacterial outer membrane vesicle as a template for the first time, so that the advantages of the bacterial outer membrane vesicle and the aluminum hydroxide adjuvant are integrated, and the composite adjuvant function is provided. In addition, the synthesis method of the invention has simple operation, green and pollution-free reaction process, and the prepared aluminum hydroxide with the bacterial outer membrane vesicle as a template has regular morphology, can play an immune activation role as a whole, can be used for immunotherapy, and has good clinical application prospect.
In order to solve the technical problems, the application provides the following technical scheme:
the invention provides a preparation method of aluminum hydroxide nano particles with bacterial outer membrane vesicles as templates, which comprises the following steps:
(1) Respectively preparing an aluminum salt aqueous solution, an acidity regulator and a 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution;
(2) Dispersing the bacterial outer membrane vesicles in the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to obtain a bacterial outer membrane vesicle solution;
(3) And adding an aluminum salt water solution into the bacterial outer membrane vesicle solution, incubating, and ultra-filtering and washing to obtain the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicle as a template.
Preferably, the bacterial outer membrane vesicles are selected from bacterial outer membrane vesicles of escherichia coli origin or bacterial outer membrane vesicles of attenuated salmonella origin.
Preparation of bacterial outer membrane vesicles reference previous studies (Qing S, lyu C, zhu L, et al, biomineralized Bacterial Outer Membrane Vesicles Potentiate Safe and Efficient Tumor Microenvironment Reprogramming forAnticancer treatment.AdvMater.2020; 32 (47): e 2002085.)
Coli was purchased from the industrial microorganism strain engineering technical research center of Henan, north Nanology, model Escherichia coli Nissle1917.
Attenuated salmonella, specifically Salmonella typhimurium YS1646, was purchased from Baozi & American type culture Collection with strain deposit information of VNP20009.
Preferably, the particle size of the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicle as a template is 50-250nm.
Preferably, in the aluminum salt aqueous solution, the aluminum salt is selected from aluminum sulfate, aluminum chloride or aluminum nitrate, and the mass ratio of the aluminum salt to water is 5-10:1.
preferably, the acidity regulator is sodium hydroxide aqueous solution; the concentration of the sodium hydroxide aqueous solution is 1-2mmol/L.
Preferably, in the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid is 10-20mmol/L, and the pH value is 7.0-7.5.
Preferably, the pH of the 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer is adjusted to 7.0-7.5 with aqueous sodium hydroxide.
Preferably, in the step (2), the dispersing method is to stir by magnetic force of 500-600rpm for 2-4 hours.
Preferably, in the bacterial outer membrane vesicle solution, the protein concentration of the bacterial outer membrane vesicle is 30-50 mug/mL.
Preferably, in the step (3), the aqueous aluminum salt solution is added to the bacterial outer membrane vesicle solution, and the volume of the aqueous aluminum salt solution is 10-200. Mu.L.
Preferably, in the step (3), the incubation is performed by stirring for 2-4 hours, heating and stirring for 2-4 hours at 35-40 ℃ and stirring for 12-24 hours.
Preferably, the stirring and the heating stirring are both magnetic stirring.
Preferably, the rotational speed of the stirring and the heating stirring is 500-600rpm.
Preferably, in the step (3), the ultrafiltration washing method comprises the following steps: the sample was concentrated by centrifugation at 4000-5000rpm for 10-20min, the filtrate was discarded, 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer was added to the concentrate, and the original volume of the sample was recovered and repeated three times.
Preferably, a ultrafiltration tube with a molecular weight cut-off of 100K is used for the centrifugal concentration.
The invention also provides aluminum hydroxide nano-particles with the bacterial outer membrane vesicles prepared by the preparation method as templates.
The invention also provides an immunoadjuvant, which adopts the aluminum hydroxide nano-particles with the bacterial outer membrane vesicle as a template.
Compared with the prior art, the technical scheme of the invention has the following advantages:
compared with the prior art, the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicles as the templates are prepared by taking the bacterial outer membrane vesicles as the templates for the first time, and the obtained nano-aluminum hydroxide whole not only has the unique immunostimulating capability of the bacterial outer membrane vesicles, but also has the antigen adsorption capability of an aluminum hydroxide adjuvant, thereby being a unique novel composite adjuvant and being applicable to immunotherapy.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention successfully prepares the aluminum hydroxide nano-particles by a protein biomineralization method.
2. In the invention, the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicles as templates are synthesized, so that the bacterial outer membrane vesicles are simply and efficiently applied to a nano-adjuvant system, and the biomedical application of the nano-adjuvant system is expanded. In addition, compared with unmethylated cytosine-phosphate-guanine oligonucleotides (CpG-ODNs), polyinosinic acid-polycytidylic acid (poly (I: C)) and other adjuvants, the bacterial outer membrane vesicles are easy to prepare and low in cost.
3. The aluminum hydroxide nano particles prepared by the method have the advantages of high surface activity, more surface active centers, strong adsorption capacity and the like due to small particle size and rapid increase of specific surface area, and compared with the traditional aluminum hydroxide adjuvant, the aluminum hydroxide nano particles can remarkably reduce the aluminum consumption and effectively reduce local adverse reactions.
4. The aluminum hydroxide nano-particles prepared by the invention have the immunity stimulating capability without additional antigens, can induce strong immune response, enrich the performance of aluminum hydroxide adjuvant, and are novel composite adjuvant.
Drawings
FIG. 1 is a scanning electron microscope picture of the Al@OMV prepared in example 1.
FIG. 2 is a transmission electron microscope picture of the Al@OMV prepared in example 1.
FIG. 3 is an infrared spectrum of the Al@OMV prepared in example 1.
FIG. 4 is an X-ray diffraction pattern of the Al@OMV prepared in example 1.
FIG. 5 is a graph showing the hydrated particle size distribution of the Al@OMV prepared in example 1 and its control groups 1 and 2.
FIG. 6 is a transmission electron microscope picture of the Al@OMV prepared in example 2.
FIG. 7 is a transmission electron microscope picture of the Al@OMV prepared in example 3.
FIG. 8 is a transmission electron microscope picture of the Al@OMV prepared in example 4.
FIG. 9 is a transmission electron microscope picture of the Al@OMV prepared in example 5.
FIG. 10 is a transmission electron microscope picture of the Al@OMV prepared in example 6.
FIG. 11 is a transmission electron microscope picture of the Al@OMV prepared in example 7.
FIGS. 12 and 13 are both graphs showing the adsorption capacity of Al@OMV antigen and the activation of BMDCs prepared in example 9.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
In the invention, aluminum hydroxide nano-particles taking bacterial outer membrane vesicles as templates are abbreviated as follows: al@OMV.
In the following examples, the preparation protocol for each bacterial outer membrane vesicle is as follows:
coli nisdle 1917 and attenuated salmonella typhimurium VNP20009 were grown overnight in solid LB medium at 37 ℃ in a 250rpm shaker, respectively. One single colony was then inoculated into LB medium. After inoculation, the bacteria were continued to be cultured at 37℃and 250rpm for 6 hours, and then diluted 1:100 with LB medium. Shake flask culture until the OD600 of the bacterial solution reached about 1.0.
The medium was centrifuged at 8000g for 20min to remove bacteria, and then filtered with a 0.45 μm vacuum filter. The filtrate was then concentrated by centrifugation using a ultrafiltration tube with a molecular weight cut-off of 100K. Then ultracentrifuged at 110,000g for 3 hours at 4 ℃. The supernatant was discarded, the bacterial outer membrane vesicles were resuspended in 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, 10 mM) buffer and filtered with a 0.45 μm pore size filter to avoid bacterial or cell debris contamination. The BCA protein concentration assay kit was used to determine total protein concentration. The samples were stored in a-80℃refrigerator for further use.
Example 1
The preparation method of the aluminum hydroxide nano-particles taking the outer membrane vesicles of the escherichia coli Nissle1917 as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum sulfate, adding 10mL of deionized water, and stirring for 10min until the aluminum sulfate is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) The outer membrane vesicles of the escherichia coli Nissle 1917-derived bacteria with the protein quantity of 60 mu g are dispersed in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, and the magnetic stirring is carried out for 2 hours, wherein the magnetic stirring rotating speed is 600rpm;
(5) Respectively adding 20 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, and magnetically stirring for 2 hours at the magnetic stirring rotating speed of 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
Aluminum hydroxide (abbreviated as AlOH) was not added to the bacterial outer membrane vesicle as control group 1 in the same experimental procedure. Simple bacterial outer membrane vesicles (abbreviated as OMVs) were used as control group 2.
Table 1Al@OMV and hydrated particle size distribution and Zeta potential of control groups 1 and 2 thereof
The scanning electron microscope picture of the Al@OMV prepared in the embodiment is shown in a figure 1, the transmission electron microscope picture is shown in a figure 2, and the particle size of the Al@OMV is 50-250 nm; the infrared spectrum is shown in figure 3, which shows that the Al@OMV has an Al-O-H infrared characteristic peak; the X-ray diffraction pattern is shown in FIG. 4, which shows that substances with crystal structures are attached to the surfaces of outer membrane vesicles of bacteria; the hydrated particle size distribution and Zeta potential of Al@OMV and control groups 1 and 2 thereof are shown in Table 1 and FIG. 5, which show that aluminum hydroxide nanoparticles can be successfully prepared by taking bacterial outer membrane vesicles as templates, and the surface charge of the bacterial outer membrane vesicles can be reversed from negative to positive. In addition, the aluminum content in the nano particles is 24.96 mug/mL by adopting an inductively coupled plasma emission spectrometry (ICP-OES); protein concentration in the nanoparticle was determined to be 41.19 μg/mL using the BCA protein concentration determination kit.
Example 2
The preparation method of the aluminum hydroxide nano-particles taking the outer membrane vesicles of the escherichia coli Nissle1917 as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum chloride, adding 10mL of deionized water, and stirring for 10min until the aluminum chloride is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) The outer membrane vesicles of the escherichia coli Nissle 1917-derived bacteria with the protein quantity of 60 mu g are dispersed in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, and the magnetic stirring is carried out for 2 hours, wherein the magnetic stirring rotating speed is 600rpm;
(5) Adding 50 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, magnetically stirring for 2 hours, wherein the magnetic stirring speed is 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
The transmission electron microscope picture of the Al@OMV prepared in the example is shown in FIG. 6, and the particle size of the Al@OMV is 50-250nm.
Example 3
The preparation method of the aluminum hydroxide nano-particles taking the outer membrane vesicles of the escherichia coli Nissle1917 as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum nitrate, adding 10mL of deionized water, and stirring for 10min until the aluminum nitrate is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) The outer membrane vesicles of the escherichia coli Nissle 1917-derived bacteria with the protein quantity of 60 mu g are dispersed in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, and the magnetic stirring is carried out for 2 hours, wherein the magnetic stirring rotating speed is 600rpm;
(5) Adding 50 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, magnetically stirring for 2 hours, wherein the magnetic stirring speed is 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
The transmission electron microscope picture of the Al@OMV prepared in the example is shown in FIG. 7, and the particle size of the Al@OMV is 50-250nm.
Example 4
The preparation method of the aluminum hydroxide nano-particles taking the attenuated salmonella typhimurium VNP20009 outer membrane vesicles as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum sulfate, adding 10mL of deionized water, and stirring for 10min until the aluminum sulfate is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) Dispersing bacterial outer membrane vesicles of attenuated salmonella typhimurium VNP20009 source with a protein quantity of 60 mu g in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, magnetically stirring for 2h, wherein the magnetic stirring rotating speed is 600rpm;
(5) Adding 20 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, and magnetically stirring for 2 hours at the magnetic stirring rotating speed of 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
The transmission electron microscope picture of the Al@OMV prepared in the example is shown in FIG. 8, and the particle size of the Al@OMV is 50-250nm.
Example 5
The preparation method of the aluminum hydroxide nano-particles taking the attenuated salmonella typhimurium VNP20009 outer membrane vesicles as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum chloride, adding 10m of L deionized water, and stirring for 10min until the aluminum chloride is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) Dispersing bacterial outer membrane vesicles of attenuated salmonella typhimurium VNP20009 source with a protein quantity of 60 mu g in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, magnetically stirring for 2h, wherein the magnetic stirring rotating speed is 600rpm;
(5) Adding 50 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, magnetically stirring for 2 hours, wherein the magnetic stirring speed is 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
The transmission electron microscope picture of the Al@OMV prepared in the example is shown in FIG. 9, and the particle size of the Al@OMV is 50-250nm.
Example 6
The preparation method of the aluminum hydroxide nano-particles taking the attenuated salmonella typhimurium VNP20009 outer membrane vesicles as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum nitrate, adding 10mL of deionized water, and stirring for 10min until the aluminum nitrate is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) Dispersing bacterial outer membrane vesicles of attenuated salmonella typhimurium VNP20009 source with a protein quantity of 60 mu g in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, magnetically stirring for 2h, wherein the magnetic stirring rotating speed is 600rpm;
(5) Adding 50 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, magnetically stirring for 2 hours, wherein the magnetic stirring speed is 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
The transmission electron microscope pictures of the Al@OMVs prepared in this example are shown in FIG. 10, which shows that the particle sizes of the Al@OMVs are 50-250nm.
Example 7
The preparation method of the aluminum hydroxide nano-particles taking the attenuated salmonella typhimurium VNP20009 outer membrane vesicles as a template comprises the following specific steps:
(1) Weighing 100mg of aluminum nitrate, adding 10mL of deionized water, and stirring for 10min until the aluminum nitrate is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) Dispersing bacterial outer membrane vesicles of attenuated salmonella typhimurium VNP20009 source with a protein quantity of 60 mu g in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, magnetically stirring for 2h, wherein the magnetic stirring rotating speed is 600rpm;
(5) Adding 100 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, magnetically stirring for 2 hours, wherein the magnetic stirring speed is 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
The transmission electron microscope picture of the Al@OMV prepared in the embodiment is shown in FIG. 11, and the particle size of the Al@OMV is 50-250 nm; in comparison with example 6, the aluminum content in the prepared al@omv can be significantly increased by increasing the quality of the aluminum salt under other conditions.
Example 8Al@OMV antigen adsorption Capacity and activation of BMDCs
(1) Weighing 100mg of aluminum sulfate, adding 10mL of deionized water, and stirring for 10min until the aluminum sulfate is completely dissolved;
(2) 400mg of sodium hydroxide is weighed, 10mL of deionized water is added, and stirring is carried out for 10min until complete dissolution;
(3) Weighing 24.7mg of 4-hydroxyethyl piperazine ethane sulfonic acid, adding 10mL of deionized water, stirring for 10min to completely dissolve, and adjusting the pH value of the 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution to 7.0 by using sodium hydroxide aqueous solution;
(4) The outer membrane vesicles of the escherichia coli Nissle 1917-derived bacteria with the protein quantity of 60 mu g are dispersed in 2mL of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, and the magnetic stirring is carried out for 2 hours, wherein the magnetic stirring rotating speed is 600rpm;
(5) Respectively adding 50 mu L of aluminum salt aqueous solution into the bacterial outer membrane vesicle solution, and magnetically stirring for 2 hours at the magnetic stirring rotating speed of 600rpm; then heating in water bath and magnetically stirring for 2h, wherein the water bath temperature is 37 ℃ and the magnetic stirring rotating speed is 600rpm; finally magnetically stirring at room temperature for 12h, wherein the magnetic stirring speed is 600rpm. Concentrating the sample by centrifugation at 4000rpm for 20min using a ultrafiltration tube having a molecular weight cut-off of 100K, discarding the filtrate, adding 4-hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer to the concentrate, recovering the original volume of the sample, and repeating the process three times.
Bone marrow cells were flushed from the bone marrow cavity of the femur and tibia of 6-8 week old female C57BL/6J mice with 1mL PMI 1640 medium. Cell suspension was filtered through nylon mesh, centrifuged to obtain cell pellet, red blood cells were lysed with red blood cell lysate, washed 1 time with PBS at 1500rpm for 3min, centrifuged, and the obtained cells were cultured in RPMI 1640 medium containing 10ng/mL GM-CSF for 3 days, half-exchanged with RPMI 1640 medium containing 10ng/mLGM-CSF on day 3, and cultured continuously until day 7 to obtain immature BMDCs. PBS, ovalbumin (OVA), universal Rehydragel@LV aluminum adjuvant (Rehydragel@LV) +OVA, al@OMV+OVA, LPS were added to the corresponding wells, respectively, and treated for 24h. After cell culture was terminated, each group of cells was collected, incubated with anti-CD 11c, anti-CD 80 and anti-CD 86 antibodies for 1h, and finally BMDCs activation was detected by flow cytometry. BMDCs with Al@OMV+OVA at 5. Mu.g/mL (protein amount) were added as control group 1, BMDCs with PBS treatment as control group 2, BMDCs with OVA at 5. Mu.g/mL (protein amount) were added as control group 2, BMDCs with Rehydro@LV+OVA at the same aluminum content (16.6. Mu.g/mL) as control group 3, BMDCs with LPS treatment at 5. Mu.g/mL were added as positive control group 4.
Experimental results the commercial aluminum adjuvants, rehydagel@lv+ova and al@omv+ova, significantly increased CD80 relative to the control OVA group + CD86 + The ratio of BMDCs (as shown in the figures) demonstrates that the aluminum adjuvant can adsorb soluble antigen OVA to enhance its pair BStimulation of MDCs; the Al@OMV+OVA group compares to the Rehydragel@LV+OVA group, CD80 + CD86 + The proportion of BMDCs (as shown in the figure) increased significantly, indicating that al@omvs had a greater ability to stimulate maturation of BMDCs. The results demonstrate that Al@OMVs can adsorb exogenous soluble antigens, promote the maturation of BMDCs and play a role of an immune agonist. And the Al@OMV antigen adsorption capacity and the activation effect on BMDCs are superior to those of a commercial aluminum adjuvant Rehydragel@LV.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. The preparation method of the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicles as templates is characterized by comprising the following steps of:
(1) Respectively preparing an aluminum salt aqueous solution, an acidity regulator and a 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution;
(2) Dispersing the bacterial outer membrane vesicles in the 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution to obtain a bacterial outer membrane vesicle solution;
(3) And adding an aluminum salt water solution into the bacterial outer membrane vesicle solution, incubating, and ultra-filtering and washing to obtain the aluminum hydroxide nano-particles taking the bacterial outer membrane vesicle as a template.
2. The method of claim 1, wherein the bacterial outer membrane vesicles are selected from the group consisting of escherichia coli-derived bacterial outer membrane vesicles and attenuated salmonella-derived bacterial outer membrane vesicles.
3. The method of claim 1, wherein the aluminum hydroxide nanoparticles templated by bacterial outer membrane vesicles have a particle size of 50-250nm.
4. The preparation method according to claim 1, wherein in the aqueous aluminum salt solution, aluminum salt is selected from aluminum sulfate, aluminum chloride or aluminum nitrate, and the mass ratio of aluminum salt to water is 5-10:1.
5. the method of claim 1, wherein the acidity regulator is an aqueous sodium hydroxide solution; the concentration of the sodium hydroxide aqueous solution is 1-2mmol/L.
6. The preparation method according to claim 1, wherein the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid in the 4-hydroxyethyl piperazine ethanesulfonic acid buffer is 10-20mmol/L, and the pH value is 7.0-7.5.
7. The method of claim 1, wherein the concentration of the protein in the bacterial outer membrane vesicle solution is 30-50 μg/mL.
8. The method of claim 1, wherein in the step (3), the incubation is performed by stirring for 2-4 hours, heating and stirring for 2-4 hours at 35-40 ℃ and stirring for 12-24 hours.
9. An aluminum hydroxide nanoparticle as a template of the bacterial outer membrane vesicle prepared by the preparation method of any one of claims 1 to 8.
10. An immunoadjuvant, characterized by using the aluminum hydroxide nanoparticle of the bacterial outer membrane vesicle of claim 9 as a template.
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