CN114949246A - Toll-like receptor agonist nanoparticles and preparation method and application thereof - Google Patents
Toll-like receptor agonist nanoparticles and preparation method and application thereof Download PDFInfo
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- CN114949246A CN114949246A CN202210421734.1A CN202210421734A CN114949246A CN 114949246 A CN114949246 A CN 114949246A CN 202210421734 A CN202210421734 A CN 202210421734A CN 114949246 A CN114949246 A CN 114949246A
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Abstract
The invention relates to the technical field of biological medicines, in particular to Toll-like receptor agonist nanoparticles and a preparation method and application thereof. The nano particle is prepared by encapsulating an imidazoquinoline agonist by a zeolite imidazole framework 8, the preparation method is simple, and the problems of poor water solubility and low bioavailability of the agonist are solved. Compared with free agonist, the nano particles are more effectively enriched in liver organs, break immune tolerance, activate antigen presenting cells, promote the secretion of Hepatitis B Virus (HBV) surface antibodies, effectively eliminate HBV surface antigens in serum of mice infected by hepatitis B virus, and show excellent hepatitis B virus eliminating effect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method and application of Toll-like receptor agonist nanoparticles.
Background
Chronic infection with Hepatitis B Virus (HBV) is a serious public health problem worldwide and is one of the most important inducing factors of primary liver cancer. Currently, only less than 1% of patients are cured each year in spite of various HBV therapeutic drugs including IFNs, nucleoside analogs, and the like. And CD8 in patients with chronic hepatitis B + Depletion of the function of various immune effector cells such as T cells and NK cells is a major cause of persistent HBV infection. Toll-like receptors (TLRs) are an important class of pattern recognition receptors involved in innate immunity, which bridges innate and adaptive immunity. In recent years, multiple studies show that Toll-like receptor (TLR) mediated immune response in chronic hepatitis B patients is inhibited, and a TLR agonist can improve the innate immune response and adaptive immunity of hosts to HBV infection, thereby showing good application prospects. However, TLR agonists can diffuse rapidly into blood vessels after entering the body, leading to side effects such as gastrointestinal inflammation, cytokine storm, etc. in most treated patients. The nonspecific immune reaction causes insufficient immune response of the liver, and cannot effectively play an immune regulation role. How to target TLR agonist specificity to liver, stimulate liver tissue innate immune response and improveCD8 caused by HBV chronic infection + The exhaustion state of immune effector cells such as T cells and NK cells is an important bottleneck of TLR agonist in treating HBV chronic infection.
Disclosure of Invention
Aiming at the defects of the existing treatment scheme, the invention provides a Toll-like receptor agonist nanoparticle and a preparation method and application thereof, the ZIF-8 is used for packaging the imidazoquinoline Toll-like receptor agonist (IMDQ @ ZIF-8), so that the problems of poor water solubility and low bioavailability of the imidazoquinoline Toll-like receptor agonist are solved, the efficiency of the medicament entering the liver is improved, the toxic and side effects are reduced, and the hepatitis B virus is effectively eliminated.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the invention, the Toll-like receptor agonist nanoparticles are composed of an imidazoquinoline agonist and a zeolite imidazole skeleton 8, wherein the imidazoquinoline agonist is encapsulated in pores of the zeolite imidazole skeleton 8.
In a second aspect of the present invention, there is provided a method for preparing the Toll-like receptor agonist nanoparticles of the first aspect, comprising: dissolving an imidazoquinoline agonist in an organic reagent, uniformly mixing the imidazoquinoline agonist with polyethylene glycol and zinc nitrate aqueous solution at room temperature, adding 2-methylimidazole aqueous solution, stirring for reaction, and centrifuging.
In a third aspect of the invention, there is provided a pharmaceutical composition comprising the Toll-like receptor agonist nanoparticles of the first aspect.
In a fourth aspect of the invention, there is provided a pharmaceutical formulation comprising a Toll-like receptor agonist nanoparticle of the first aspect or a pharmaceutical composition of the third aspect.
In a fifth aspect of the invention, there is provided a drug delivery system comprising a Toll-like receptor agonist nanoparticle of the first aspect.
In a sixth aspect of the present invention, an application of the Toll-like receptor agonist nanoparticles, the pharmaceutical composition, the pharmaceutical preparation or the drug delivery system in the preparation of a drug for preventing and/or treating hepatitis b virus is provided.
One or more of the above technical solutions have the following beneficial effects:
1) the Toll-like receptor agonist nanoparticles prepared by the invention are nanoparticles (IMDQ @ ZIF-8NPs) formed by packaging imidazoquinoline Toll-like receptor agonists (IMDQ) in ZIF-8 pores, are prepared by a one-pot method, are simple in method and mild in reaction conditions, and solve the problems of poor water solubility and low bioavailability of IMDQ.
2) The IMDQ @ ZIF-8NPs prepared by the method has high drug loading, uniform particle size and high stability, can be efficiently enriched in the liver, and solves the problems that small-molecule drugs are easy to diffuse in vivo and induce systemic inflammatory reaction.
3) Compared with other control groups, the IMDQ @ ZIF-8NPs show better immune system activation and better eliminate hepatitis B virus.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a transmission electron microscope image of IMDQ @ ZIF-8 nanoparticles prepared in example 1, with a scale of 200 nm;
FIG. 2 is a scanning electron microscope image of IMDQ @ ZIF-8 nanoparticles prepared in example 1, with a scale of 200 nm;
FIG. 3 is an ultraviolet spectrum of IMDQ @ ZIF-8 nanoparticles before and after degradation of IMDQ and ethylenediaminetetraacetic acid (EDTA) in example 1;
FIG. 4 is a graph showing the results of the IMDQ @ ZIF-8 nanoparticles stimulating BMDC cell maturation in example 3;
FIG. 5 is a graph showing the results of the IMDQ @ ZIF-8 nanoparticles in example 4 regulating HBsAg in mice infected with hepatitis B virus;
FIG. 6 is the result of IMDQ @ ZIF-8 nanoparticles in example 4 regulating HBsAb in mice infected with hepatitis B virus.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions or according to conditions recommended by the manufacturers.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Toll-like receptor agonists generally have the defects of poor water solubility, easy removal of molecular states, lack of targeting property and the like, and are easy to cause systemic inflammatory response. In order to solve the problems, the invention provides Toll-like receptor agonist nanoparticles and a preparation method and application thereof.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the invention, the Toll-like receptor agonist nanoparticles are composed of an imidazoquinoline agonist and a zeolite imidazole skeleton 8, wherein the imidazoquinoline agonist is encapsulated in pores of the zeolite imidazole skeleton 8.
After the nanoparticles enter the body, the nanoparticles are mainly enriched in the liver due to the liver antrum structure and the mononuclear phagocyte system, and myeloid cells such as kupffer cells are mainly located in the liver antrum. Therefore, the TLR agonist delivered by the nano-carrier technology can improve liver targeting, reduce toxic and side effects, promote the combination of Antigen Presenting Cells (APCs) and the TLR agonist and improve the curative effect of the medicament. Zeolite imidazole framework 8(ZIF-8) is a novel porous material, is easy to decompose under acidic conditions (pH 5.0-6.0), and is beneficial to controlling the pH value of the drug to be transferred and released.
The agonist is packaged in ZIF-8, so that the problems of poor water solubility and low bioavailability of the imidazoquinoline agonist are solved, the problems that the small-molecular Toll-like receptor agonist is easy to diffuse in vivo and induces systemic inflammatory reaction are solved, and a better hepatitis B virus clearing effect is shown.
In one or more embodiments of the invention, the imidazoquinoline-based agonists are a class of Toll-like receptor 7/8 agonists, including any one or more of imiquimod (R837) IMDQ, reisimmod (R848), Gardiquimod (CAS 1020412-43-4); more preferably, the imidazoquinoline agonist is imiquimod (R837).
In a second aspect of the present invention, there is provided a method for preparing Toll-like receptor agonist nanoparticles, comprising:
dissolving an imidazoquinoline agonist in an organic solvent to form an imidazoquinoline agonist solution, uniformly mixing the imidazoquinoline agonist solution with polyethylene glycol and a zinc nitrate aqueous solution at room temperature, adding a 2-methylimidazole aqueous solution to form a mixed solution, stirring for reaction, and separating.
In one or more embodiments of the present invention, the concentration of the imidazoquinoline-based agonist solution is 1-6mg/mL, preferably 4 mg/mL.
In one or more embodiments of the present invention, the organic solvent is any one or more of dimethylsulfoxide, N-dimethylformamide, dichloromethane, acetonitrile, ethyl acetate.
Preferably, the volume ratio of the organic solvent to the aqueous solution in the reaction system is 1:7 to 1:2, more preferably 1: 3.
In one or more embodiments of the invention, the polyethylene glycol is a mineralizer, the molecular weight of the polyethylene glycol is 2-100kDa, and the addition amount of the polyethylene glycol in the mixed solution is 1-5 mg/mL; the polyethylene glycol is straight chain, four-arm or eight-arm polyethylene glycol.
Preferably, the polyethylene glycol is eight-arm polyethylene glycol hydroxyl, the molecular weight is 20-80kDa, and the addition amount of the polyethylene glycol is 2-3 mg/mL.
In one or more embodiments of the invention, the concentration of the 2-methylimidazole solution is 40 to 640mmol/L, and preferably, the concentration of the 2-methylimidazole solution is 320 mmol/L.
In one or more embodiments of the invention, the concentration of the zinc nitrate solution is 20 to 80mmol/L, and preferably, the concentration of the zinc nitrate solution is 40 mmol/L.
In one or more embodiments of the invention, the molar ratio of 2-methylimidazole to zinc nitrate is from 8:1 to 2:1, and preferably the molar ratio of 2-methylimidazole to zinc nitrate is 4: 1.
In one or more embodiments of the present invention, the reaction temperature is 4 to 60 ℃ and the reaction time is 0.25 to 2 hours, preferably, the reaction temperature is 25 to 30 ℃ and the reaction time is 0.5 to 1 hour; further preferably, the reaction temperature is 25 ℃ and the reaction time is 0.5 to 1 hour.
In one or more embodiments of the invention, centrifugal separation is adopted for the separation, the centrifugal force used for the centrifugal separation is 5000-; preferably, the centrifugal force used for centrifugal separation is 6000-8000g, and the centrifugal time is 3 min.
In one or more embodiments of the present invention, the obtained nanoparticles have a particle size of 150-500nm, preferably, the particle size of the nanoparticles is 200-400 nm.
In order to further improve the purity of the nano particles, the method also comprises the operations of water washing and sterilization after centrifugation.
In a third aspect of the invention, there is provided a pharmaceutical composition or formulation comprising Toll-like receptor agonist nanoparticles.
In one or more embodiments of the invention, the medicament is an intravenous injection.
In a fourth aspect of the invention, there is provided a drug delivery system comprising Toll-like receptor agonist nanoparticles.
In a fifth aspect of the invention, the invention provides an application of Toll-like receptor agonist nanoparticles, a pharmaceutical composition, a pharmaceutical preparation and a drug delivery system in preparation of drugs for preventing and/or treating hepatitis B virus.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1: preparation of nanoparticles
The preparation method of the Toll-like receptor agonist nanoparticles (IMDQ @ ZIF-8NPs) comprises the following steps:
(1) IMDQ was precisely weighed by an analytical balance and dispersed in dimethyl sulfoxide (DMSO) to prepare a 4mg/mL IMDQ solution. Uniformly mixing 1mL of IMDQ solution, eight-arm polyethylene glycol (10mg) with the molecular weight of 40kDa and 2mL of zinc nitrate aqueous solution (40mM), adding 1mL of 2-methylimidazole aqueous solution (320mM), and stirring and reacting for 0.5h at normal temperature;
(2) after the reaction is finished, the mixed solution is centrifuged (8000g, 3min), the operation is repeated for 4 times, 4mL of ultrapure water is added after each centrifugation to remove unreacted substances, and the nanoparticles are prepared after sterilization.
Example 2: characterization of the physical and chemical Properties of the nanoparticles
The ultraviolet spectrum was measured by an ultraviolet-visible spectrophotometer (Shimadzu, UV-2600). The morphology of the nanoparticles was characterized by transmission electron microscopy (TEM, JEOL JEM-1400) and scanning electron microscopy (SEM, Carl Zeiss G300). The results are shown in FIGS. 1-3, and the prepared nanoparticles have regular morphology and uniform size, and the particle size is about 200 nm. Comparing the changes of the ultraviolet spectrum of the IMDQ, IMDQ @ ZIF-8 and IMDQ @ ZIF-8+ EDTA (EDTA can compete for coordination, so that the nano particles are disassembled and the internally packaged molecules are released), and confirming that the IMDQ is successfully packaged in the nano particles. The quantitative results showed that 39.5% of the IMDQ was successfully encapsulated in the nanoparticles.
Example 3: nanoparticle-activated immune cell maturation assay
Extracting and stimulating to obtain mouse bone marrow-derived dendritic cell BMDC, adding into 24-well plate (1 × 10) 5 Cells/well). Control (blank medium), free IMDQ (15. mu.g/mL), IMDQ @ ZIF-8(IMDQ dose 15. mu.g/mL) were added to the cell supernatant and incubation continued in the incubator for 24 h. The surface maturation factor expression was detected by flow cytometry and the results are shown in FIG. 4. Compared with free IMDQ, IMDQ @ ZIF-8 effectively promotes the mature differentiation of BMDC cells, which is caused by the fact that nanoparticles are more easily taken up by antigen presenting cells.
Example 4: in vivo hepatitis B Virus eradication Studies
1. Establishment of animal model
A male C57BL/6 mouse with age of 6-8 weeks is injected with pAAV-HBV1.2 adeno-associated virus vector HBV expression plasmid (professor of Ding-Shinn Chen) under high pressure and water power to establish an HBV infection model.
2. Hepatitis B Virus removal assay in animals
4 days after the mouse tail vein high pressure injection, the mice were randomly divided into 4 groups: blank plasmid control + physiological saline group, HBV + IMDQ @ ZIF-8NPs group. Serum HBV surface antigen (HBsAg), HBV surface antibody (HBsAb) levels were measured by tail vein injection of 2mg/kg (in terms of IMDQ) with physiological saline and two forms of IMDQ, once every three days. As shown in figures 5 and 6, compared with free IMDQ, the IMDQ encapsulated by the ZIF-8 nano-particles can obviously reduce the content of HBsAg in serum of mice, and can obviously improve the secretion level of HBsAb in infected mice, which indicates that the IMDQ has good effect of removing hepatitis B virus.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A Toll-like receptor agonist nanoparticle is characterized by consisting of an imidazoquinoline agonist and a zeolite imidazole framework 8, wherein the imidazoquinoline agonist is encapsulated in pores of the zeolite imidazole framework 8.
2. The Toll-like receptor agonist nanoparticle of claim 1, wherein the imidazoquinoline agonist is a Toll-like receptor 7/8 agonist comprising any one or more of imiquimod, ranisimmod, Gardiquimod; preferably, the imidazoquinoline agonist is imiquimod.
3. The method for preparing Toll-like receptor agonist nanoparticles as claimed in claim 1 or 2, comprising: dissolving an imidazoquinoline agonist in an organic solvent to form an imidazoquinoline agonist solution, uniformly mixing the imidazoquinoline agonist solution with polyethylene glycol and a zinc nitrate aqueous solution at room temperature, adding a 2-methylimidazole aqueous solution to form a mixed solution, stirring for reaction, and separating.
4. The process according to claim 3, wherein the concentration of the imidazoquinoline agonist solution is 1 to 6mg/mL, preferably 4 mg/mL.
5. The preparation method according to claim 3, wherein the organic solvent is any one or more of dimethyl sulfoxide, N-dimethylformamide, dichloromethane, acetonitrile and ethyl acetate;
or, in the reaction system, the volume ratio of the organic solvent to the aqueous solution is 1:7-1:2, and more preferably 1: 3;
the polyethylene glycol is a mineralizer, and the polyethylene glycol with the molecular weight of 2-100kDa is linear chain, four-arm or eight-arm polyethylene glycol; the concentration of the polyethylene glycol in the mixed solution is 1-5 mg/mL;
more preferably, the polyethylene glycol is eight-arm polyethylene glycol, the molecular weight is 20-80kDa, and the concentration of the polyethylene glycol in the mixed solution is 2-3 mg/mL.
6. The method according to claim 3, wherein the concentration of the 2-methylimidazole solution is 40 to 640mmol/L, and preferably, the concentration of the 2-methylimidazole solution is 320 mmol/L;
or the concentration of the zinc nitrate solution is 20-80mmol/L, preferably, the concentration of the zinc nitrate solution is 40 mmol/L;
or, the molar ratio of 2-methylimidazole to zinc nitrate is 8:1 to 2:1, preferably, the molar ratio of 2-methylimidazole to zinc nitrate is 4: 1;
or the reaction temperature is 4-60 ℃, the reaction time is 0.25-2 hours, preferably, the reaction temperature is 25-30 ℃, and the reaction time is 0.5-1 hour; more preferably, the reaction temperature is 25 ℃ and the reaction time is 0.5 to 1 hour;
or, the separation adopts centrifugal separation, the centrifugal force is 5000-10000g, and the centrifugal force used for the solution centrifugal separation is preferably 6000-8000 g. Preferably, the centrifugal force used for centrifugal separation is 6000-8000g, and the centrifugal time is 3 min.
7. A pharmaceutical composition comprising a Toll-like receptor agonist nanoparticle according to any one of claims 1 and 2.
8. A pharmaceutical formulation comprising a Toll-like receptor agonist nanoparticle according to claim 1 or 2 or a pharmaceutical composition according to claim 7.
9. A drug delivery system comprising the Toll-like receptor agonist nanoparticle according to claim 1 or 2.
10. Use of the Toll-like receptor agonist nanoparticles of claim 1 or 2, the pharmaceutical composition of claim 7, the pharmaceutical formulation of claim 8 or the drug delivery system of claim 9 for the manufacture of a medicament for the prophylaxis and/or treatment of hepatitis b virus.
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| CN111704722A (en) * | 2020-05-27 | 2020-09-25 | 山东大学 | Preparation method and application of zeolite imidazole type metal organic framework material with polyethylene glycol as mineralizer |
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