CN107693789B - Compound containing cell-targeted antibody and Poly (I: C) and preparation method and application thereof - Google Patents

Compound containing cell-targeted antibody and Poly (I: C) and preparation method and application thereof Download PDF

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CN107693789B
CN107693789B CN201710639942.8A CN201710639942A CN107693789B CN 107693789 B CN107693789 B CN 107693789B CN 201710639942 A CN201710639942 A CN 201710639942A CN 107693789 B CN107693789 B CN 107693789B
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poly
pei
cap
sio
antibody
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CN107693789A (en
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吴珺
邹诗
黄顺梅
杜艳芹
马赛厄斯·埃普尔
维多利亚·索科洛娃
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Union Hospital Tongji Medical College Huazhong University of Science and Technology
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Abstract

The invention provides a compound containing a cell-targeting antibody and Poly (I: C), wherein calcium phosphate nanoparticles are used as an inner core of the compound, PEI is grafted on the surface of the inner core, the Poly (I: C) is adsorbed and loaded on the surface of the inner core, a silicon dioxide shell is arranged outside the inner core, and the silicon dioxide shell is connected with four different cell-targeting antibodies through cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester. Furthermore, the invention also provides a preparation method and medical application of the compound. The invention can effectively realize that poly (I: C) reaches the cells in the liver in a targeted way, thereby combining the poly (I: C) with TLR3, enhancing the production of interferon and proinflammatory factors by activating TLR 3-mediated signal channels of different immune cells and playing a role in resisting HBV viruses.

Description

Compound containing cell-targeted antibody and Poly (I: C) and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a compound CaP/PEI/Poly (I: C)/SiO containing a cell-targeted antibody and Poly (I: C)2-S-Ab and a preparation method and application thereof.
Background
Among various nanoparticles, calcium phosphate nanoparticles have the characteristics of high biocompatibility, high biodegradability, small volume, high affinity, covalent functionalization with some nucleic acids and the like, and have considerable application prospects. The nanoparticles have been used in various fields such as transfection, gene silencing, drug delivery, photodynamic therapy, and the like. Some macromolecular substances often cannot penetrate cell membranes, and therefore, an effective carrier such as a nanoparticle is necessary.
Calcium phosphate nanoparticles, approximately 100 nm in size, are readily taken up by cells and subsequently dissolved in lysosomes. Multiple studies have demonstrated that multi-shell encapsulated calcium phosphate nanoparticles can be used extensively in immunology, for example for prophylactic and therapeutic immunization and specific B cell activation.
As reported by the world health organization, about 20 million people worldwide have been infected with Hepatitis B Virus (HBV), of which about 2 hundred and 4 million people are chronic HBV infected, and about 100 million people die each year from liver failure, cirrhosis and hepatocellular carcinoma due to HBV infection. Chronic hepatitis B is one of the most prominent public health problems in China at the present stage. At present, an effective treatment means is not available for chronic hepatitis B, interferon treatment can only inhibit 20-40% of virus replication of chronic hepatitis B patients, and the drug effect of nucleoside analogues is greatly reduced due to virus tolerance mutation. Recently, more and more researches show that agonists of the natural immune recognition receptor Toll-like receptor (TLR) are expected to become novel medicaments for treating virus infection, inflammatory diseases and tumors as immunomodulators or immunoadjuvants.
The primary target molecule for which innate immunity is directed is called the Pathogen Associated Molecular Pattern (PAMP), and the corresponding recognition receptor is called the Pattern Recognition Receptor (PRR). PAMPs refer primarily to molecules or nucleic acid components of the pathogen cell surface that have been conserved through evolution and are essential for the survival of the pathogen. Nucleic acids from pathogens, including single-and double-stranded RNA, are one of the broad classes of PAMPs. TLRs are a large family of PRRs, and TLR3 is located intracellularly and is capable of recognizing viral double-stranded RNA. The artificially synthesized double-stranded RNA analog polyinosinic acid (poly (I: C)), which is a ligand of TLR3, can be recognized by TLR3 and activate TLR3, and can be recognized by melanoma differentiation related gene 5 (MDA-5).
TLR3 can recruit downstream adaptor proteins MyD88 and TRIF after intracellular poly (I: C) recognition and activation, TLR3 can induce the expression of inflammatory cytokines such as IL-1, TNF-alpha, IL-6 and IL-12 through MyD88 dependent pathway, participate in nonspecific antiviral response, and simultaneously induce the expression of costimulatory molecules CD80 and CD86 and IFN-beta, IP-10 and other antiviral cytokines through MyD88 independent pathway, participate in inducing the differentiation maturation of DC and antiviral immune response. Cytoplasmic MDA-5 can be recruited in the mitochondrial outer coat by the mitochondrial anti-viral signaling protein (MAVS). MDA-5, although using a different adaptor protein than TLR3, has the same downstream signaling pathway and is able to activate a range of transcription factors, including IRF3, IRF7 and NF-. kappa.B, to induce the expression of anti-or pro-inflammatory factors (IL-6, IL-10), type I interferons (IFN-. alpha./beta.) and co-stimulatory molecules.
Relevant experiments demonstrated that poly (I: C) stimulates in vitro hepatocyte (hepatocyte) and non-hepatocyte cells (kupffer cells and antral endothelial cells) to produce IFN- β and inhibit HBV replication. In addition, when HBV infected mice are treated by poly (I: C) high pressure water injection, the result shows that HBV virus can be eliminated by poly (I: C) treatment, the HBV eliminating effect depends on the generation of interferon, and the antiviral effect of poly (I: C) is necessary to activate TLR3 in liver by means of high pressure water injection. In a mouse model, TLR3 in the liver can be activated by high-pressure water injection, but the method cannot be used for human beings, and the poly (I: C) cannot be effectively introduced into the liver of a patient at present. There is therefore a great need for an effective vector that enables poly (I: C) to be targeted by intravenous injection to activate TLR3 in the liver.
Disclosure of Invention
In view of the above, the present invention provides a novel compound, i.e., CaP/PEI/Poly (I: C)/SiO, comprising a cell-targeting antibody and Poly (I: C) using nanoparticles as a carrier2-S-Ab, and a preparation method and medical application thereof.
The invention provides a compound CaP/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2-S-Ab, characterized in that the steps comprise:
s1, preparing CaP/PEI: preparing PEI-calcium phosphate nanoparticles;
s2, preparation of CaP/PEI/poly (I: C): mixing the PEI-calcium phosphate nanoparticles prepared in the step S1 with water-soluble poly (I: C) or poly (I: C) containing fluorescein, and stirring at 15-35 ℃ to obtain PEI-calcium phosphate nanoparticles loaded with poly (I: C);
s3, preparation of CaP/PEI/poly (I: C)/SiO2: fully mixing the CaP/PEI/poly (I: C), tetraethoxysilane, ammonia water and solvent obtained in the step S2, stirring the mixed solution for 14-18 hours at 15-35 ℃, and centrifugally separating to obtain CaP/PEI/poly (I: C)/SiO2Granulating, adding CaP/PEI/poly (I: C)/SiO2Dissolving the particles in ultrapure water;
s4, adding CaP/PEI/poly (I: C)/SiO2Covalent functionalization of particles to make the CaP/PEI/poly (I: C)/SiO2Covalently bonding thiol or amino groups to the surface of the particles;
s5, preparation of CaP/PEI/poly (I: C)/SiO2-S-Ab: covalently functionalized CaP/PEI/poly (I: C)/SiO2Adding the particles into an activated antibody, wherein the antibody comprises one of CD146, F4/80, IgG1 and IgG2, reacting for 20-28 hours at 3-5 ℃, and performing centrifugal separation to obtain CaP/PEI/poly (I: C)/SiO2-S-Ab nanoparticles, CaP/PEI/poly (I: C)/SiO2-S-Ab nanoparticles are dissolved in ultrapure water.
In the research of the invention, the artificially synthesized double-stranded RNA analog polyinosinic acid (poly (I: C)) can be combined with TLR3, the generation of interferon and proinflammatory factors is enhanced by activating a TLR 3-mediated signal channel, and the nanoparticles with cell targeting antibodies can be used as an effective carrier to enable the poly (I: C) to reach the cells in the liver in a targeted mode. The nanometer level medicine carrier is one submicron level medicine carrier conveying system. The drug is encapsulated in submicron particles, so that the release speed can be adjusted, the permeability of a biological membrane is increased, the distribution in a body is changed, the bioavailability is improved, and the like. Therefore, in the invention, the nano-particles are used as carriers to be combined with poly (I: C), and further combined with one of corresponding antibodies CD146, F4/80, IgG1 or IgG 2. CD146 is a surface molecule of Liver Sinus Endothelial Cells (LSEC), F4/80 is a surface molecule of Kuppfer Cells (KC), the two cells account for the majority of liver nonparenchymal cells, and the two cells play an important role in antiviral immune response as antigen-presenting cells in the liver. IgG1 and IgG2 are isotype control antibodies to CD146 and F4/80, respectively. We use the antibodies of the two cells to make the nanoparticles reach specific cells in a targeted way, so that the poly (I: C) carried by the nanoparticles plays an antiviral effect in the liver. And the nanoparticles with isotype control IgG2 antibody were found to have the strongest antiviral effect. Therefore, the compound is expected to be developed into a medicine or health-care product for preventing or treating diseases which can respond to the activation of a TLR3 signal channel in vivo, and the invention also provides the medical application of the compound.
Preferably, the step of preparing PEI-calcium phosphate nanoparticles according to step S1 includes: injecting water-soluble calcium lactate, (NH) into ultrapure water according to the volume ratio of 5:5:74)2HPO4And PEI, and stirring. The concentration of the water-soluble PEI was 2g/L, the concentration of the water-soluble calcium lactate (18mmol/L, pH 10), (NH)4)2HPO4(10.8mmol/L, pH 10); the amount of the ultrapure water is four times of the volume of the water-soluble calcium lactate.
Preferably, the reaction of step S4 is performed by reacting CaP/PEI/poly (I: C)/SiO2The step of covalently bonding thiol groups to the surface of the particles comprises: preparation of CaP/PEI/poly (I: C)/SiO2-SH: dissolving trimethoxy silane in solvent, adding into the solvent the solution of Cap/PEI/poly (I: C)/SiO in ultrapure water2Particles are stirred for 8 to 10 hours at the temperature of between 15 and 35 ℃, and the precipitate is collected by centrifugation to obtainTo Cap/PEI/poly (I: C)/SiO2-SH particles, and adding Cap/PEI/poly (I: C)/SiO2the-SH particles are dissolved in ultrapure water.
Preferably, the centrifugation is performed for 30min at 66000g centrifugal force at an over-speed. Separating out target nano particles by an ultracentrifugation method, and dissolving the target nano particles in ultrapure water by a sound wave crushing method. By the above method, unreacted parent compounds and by-products can be effectively removed.
Preferably, the activated antibody of step S5 is obtained by mixing and activating cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester at 15-35 deg.C for 3-4 hours.
In a second aspect, the invention provides a compound of CaP/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2The compound takes calcium phosphate nanoparticles as an inner core, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed and carried on the surface of the inner core, a silicon dioxide outer shell is arranged outside the inner core, the silicon dioxide outer shell is connected with cell targeting antibodies through cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester, and the cell targeting antibodies comprise CD146 antibody targeting hepatic sinus endothelial cells, F4/80 antibody targeting Kupffer cells, IgG1 isotype control antibody targeting Fc receptor cells expressing IgG1 antibody, and IgG2 isotype control antibody targeting Fc receptor cells expressing IgG2 antibody
Preferably, the compound Cap/PEI/poly (I: C)/SiO2The (E) -S-Ab is prepared by the preparation method.
In a third aspect, the invention provides a compound of Cap/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2-S-Ab, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, for use in the preparation of an agonist of TLR3 in vivo.
In a fourth aspect, the invention provides a compound of Cap/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2-S-Ab, or a pharmaceutically acceptable salt, prodrug or hydrate thereof, for use in the manufacture of a medicament or health product for the prevention or treatment of a disease responsive to a TLR3 agonist in vivo.
In a fifth aspect, the invention provides a method of targeted activation of TLR3 in vivo, comprising the steps of: will be provided withCell expressing TLR3, cell targeting antibody and compound of Poly (I: C), CaP/PEI/Poly (I: C)/SiO2-S-Ab contact.
The invention provides a pharmaceutical composition in a sixth aspect, which comprises the components of a compound CaP/PEI/Poly (I: C)/SiO containing a cell-targeted antibody and Poly (I: C)2-S-Ab or a pharmaceutically acceptable salt, prodrug or hydrate thereof; the pharmaceutical composition is used for treating virus infectious diseases, inflammatory infectious diseases, tumors and the like.
The invention has the beneficial effects that: the invention combines the nano-particles as a carrier with poly (I: C), effectively introduces the poly (I: C) into the body of a patient, further combines with one of corresponding antibodies CD146, F4/80, IgG1 or IgG2, leads the nano-particles to reach specific cells in a targeting way, leads the poly (I: C) carried by the nano-particles to play an anti-virus effect in the liver, combines poly (I: C) with TLR3, and enhances the production of interferon and proinflammatory factors by activating a TLR 3-mediated signal channel.
Drawings
FIG. 1 is a graph showing the results of an antiviral effect test of CaP/PEI-Cy5/Poly (I: C)/SiO2/CD 146;
FIG. 2 is a graph showing the results of an antiviral effect test of CaP/PEI-Cy5/Poly (I: C)/SiO2/IgG 2;
FIG. 3 is a graph showing the results of the experiment of CaP/PEI-Cy5/Poly (I: C)/SiO2/Ab for removing HBV DNA from mouse serum;
FIG. 4 shows CaP/PEI/poly (I: C)/SiO2Graph of experimental results of Ab distribution in mouse liver;
FIG. 5 shows CaP/PEI/poly (I: C)/SiO2-graph of experimental results of Ab distribution in mouse spleen;
FIG. 6 shows CaP/PEI/poly (I: C)/SiO2-graph of experimental results of Ab distribution in mouse lung;
FIG. 7 shows CaP/PEI/poly (I: C)/SiO2Graph of experimental results of Ab distribution in mouse kidney;
FIG. 8 shows CaP/PEI/poly (I: C)/SiO2Graph of experimental results of Ab distribution in mouse lymph nodes;
FIG. 9 shows CaP/PEI/poly (I: C)/SiO2CD146 in miceA graph of statistical test results distributed in different tissues;
FIG. 10 shows CaP/PEI/poly (I: C)/SiO2-graph of statistical test results for distribution of IgG1 in different tissues of mice;
FIG. 11 shows CaP/PEI/poly (I: C)/SiO2-a graph of statistical test results for the distribution of F480 in different tissues of mice;
FIG. 12 shows CaP/PEI/poly (I: C)/SiO2-graph of statistical test results for distribution of IgG2 in different tissues of mice;
FIG. 13 is a flow chart of the preparation process of CaP/PEI-Cy5/Poly (I: C)/SiO 2/Ab.
Detailed Description
The following will describe a compound of CaP/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C) according to the present invention with reference to the specific examples2-S-Ab, methods of preparation and uses thereof are further described. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.
As used in the examples of this invention, the following terms are generally intended to have the following meanings unless the extent is reached such that the context in which they are used indicates otherwise. The following terms have the indicated meanings everywhere:
the term "therapeutically effective amount" or "effective amount" refers to an amount of a compound of the invention that, when administered to a human or non-human patient, is effective for providing a therapeutic benefit such as amelioration of symptoms, slowing of disease progression, or prevention of disease, e.g., a therapeutically effective amount can be an amount sufficient to alleviate symptoms of a disease responsive to a TLR3 agonist. In some embodiments, a therapeutically effective amount is an amount sufficient to reduce the symptoms of viral hepatitis. In some embodiments, a therapeutically effective amount is an amount sufficient to reduce detectable HBsAg, HBeAg, HBV DNA in an organism. In some embodiments, a therapeutically effective amount of a compound is an amount sufficient to prevent a significant increase in or to significantly reduce the detectable levels of hepatitis virus in the blood, serum or tissues of a patient. In the method of the present invention for treating viral hepatitis, a therapeutically effective amount may also be an amount sufficient to detectably slow the progression of the disease when administered to a patient, or to prevent the presence of cirrhosis, liver cancer in the administered patient. In the methods of the invention for treating viral hepatitis, a therapeutically effective amount may also be an amount sufficient to produce a detectable decrease in the amount of marker protein or cell type in the patient's blood or serum. For example, in some embodiments, a therapeutically effective amount is an amount of a compound of the invention sufficient to significantly reduce expression of HBsAg and HBeAg.
The term "activation" indicates a significant increase in the baseline activity of a biological activity or process. By "activation of TLR3," it is meant an increase in TLR3 activity when directly or indirectly responsive to the presence of a compound of the invention relative to TLR3 activity in the absence of a compound of the invention. The increased activity may be attributed to the direct interaction of the compounds of the invention with TLR 3. For example, the presence of a compound of the invention may enhance the activity of TLR3 by binding directly to TLR 3.
The term "viral hepatitis" refers to an infectious disease mainly including liver diseases caused by various hepatitis viruses. Clinically, the symptoms of anorexia, nausea, epigastric discomfort, liver pain and hypodynamia are mainly manifested. Some patients may have jaundice fever and hepatomegaly with liver function impairment. Some patients can become chronic, even develop cirrhosis of the liver, and a few can develop liver cancer.
The term "chronic viral hepatitis B" refers to the hepatitis B virus which is detected as positive, the disease course is more than half a year or the disease date is unclear and chronic hepatitis is clinically manifested. Clinically, it is manifested as lassitude, aversion to food, nausea, abdominal distention, and pain in the liver. Liver is large, medium hardness, and slight tenderness. The serious disease may be accompanied by facial appearance of chronic liver disease, spider nevus, hepatomegaly, splenomegaly, and abnormal or persistent liver function.
The term "diseases responsive to TLR3 agonists" refers to those diseases that can obtain therapeutic benefit such as amelioration of symptoms, delay of disease progression, prevention or delay of disease onset, or activation of aberrant activity of specific cell types (LSEC, KC) by activating the activity of TLR 3.
The term "treating" refers to administering a compound of the invention to a subject to treat, alleviate, slow, alter, cure, affect, ameliorate, or ameliorate a disease, a symptom of a disease, or a precursor of a disease.
The term "patient" refers to an animal, such as a mammal, who has been or will be the subject of treatment, observation or experiment. The methods of the invention are useful in both human therapy and veterinary applications. In some embodiments, the patient is a mammal; in some embodiments, the patient is a human; and in some embodiments, the patient is a mouse.
Example one
This example provides a compound of Cap/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2S-Ab, wherein Ab is a targeting antibody CD146 or IgG1, the compound comprises a calcium phosphate nanoparticle as an inner core, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed on the surface of the inner core, and a silica outer shell is arranged outside the inner core and is connected with the antibody CD146 or IgG1 through cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester.
The compound containing the hepatocyte targeted antibody and Poly (I: C) is CaP/PEI/Poly (I: C)/SiO2S-Ab, i.e. CaP/PEI/poly (I: C)/SiO2-S-CD146 and CaP/PEI/poly (I: C)/SiO2The preparation method of the-S-IgG 1 comprises the following steps:
the nanoparticles were prepared using the following chemicals: branched Polyethylene (PEI), calcium lactate, diammonium phosphate, Tetraethoxysilane (TEOS), trimethoxysilane (MPS), triethoxysilane (APTES), cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester (sulfo-SMCC); ammonia, cross-linked dextran.
The first step is as follows: synthesis of stabilized PEI-calcium phosphate nanoparticles (CaP/PEI)
A glass bottle containing 20mL of ultrasonic water was filled with water-soluble calcium lactate (18mmol/L, pH 10), (NH) at a ratio of 5mL:7mL4)2HPO4(10.8mmol/L,pH＝10) And PEI (2 g/L). Stirred for 20 minutes.
The second step is that: synthesis of poly (I: C) -loaded Cap/PEI (Cap/PEI/poly (I: C))
1mL of CaP/PEI was mixed with 100. mu.L of water-soluble poly (I: C), and stirred at room temperature for 30 minutes.
The third step: silica packaging of a Cap of Cap/PEI/poly (I: C) (Cap/PEI/poly (I: C)/SiO)2)
1mL of CaP/PEI/poly (I: C) was mixed well with 4mL of ethanol, 5. mu.L of TEOS and 2.6. mu.L of aqueous ammonia (30-33%). The reaction solution was stirred at room temperature for 16 hours. The nanoparticles were isolated by ultracentrifugation at 66,000g for 30min and redissolved in 1mL of ultrapure water by sonication. By the above method, unreacted parent compound as well as by-products can be removed (including undissolved poly (I: C)).
The fourth step: covalently functionalizing nanoparticles (Cap/PEI/poly (I: C)/SiO)2-SH)
The nanoparticles are made to carry thiol groups for their covalent functionalization. mu.L of trimethoxysilyl (MPS) was dissolved in 4mL of ethanol. 1mL of nanoparticles was added thereto and stirred at room temperature for 8-10 hours. Collecting functionalized nanoparticles by ultracentrifugation at 66,000g for 30min, discarding the supernatant, dissolving the precipitate in 1mL of ultrapure water to obtain a solution of Cap/PEI/poly (I: C)/SiO2-SH nanoparticles.
The fifth step: nanoparticles covalently bound to different antibodies (Cap/PEI/poly (I: C)/SiO)2-S-Ab)
mu.L of antibody (anti-CD146 or anti-IgG1) was mixed with 40. mu.L of cyclohexane-1-carboxylic acid-3-sulfosuccinimidyl ester (sulfo-SMCC) at room temperature for 3-4 hours to activate the antibody. 1mL of CaP/PEI/poly (I: C)/SiO2the-SH nanoparticles were added to the activated antibody and reacted at 4 ℃ for another 24 hours. After the binding reaction, nanoparticles were collected by ultracentrifugation at 66,000g for 30min, and the precipitate was dissolved in 1mL of ultrapure water to obtain Cap/PEI/poly (I: C)/SiO2-S-CD146 and CaP/PEI/poly (I: C)/SiO2-S-IgG1 nanoparticles.
Example two
This example provides a compound of Cap/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2S-Ab, wherein Ab is a targeting antibody F4/80 or IgG2, the compound comprises calcium phosphate nanoparticles as an inner core, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed on the surface of the inner core, and the outer part of the inner core is provided with a silica outer shell which is connected with the antibody F4/80 or IgG2 through cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester.
The compound containing the cell-targeting antibody and the Poly (I: C) is CaP/PEI/Poly (I: C)/SiO2S-Ab, i.e. CaP/PEI/poly (I: C)/SiO2-S-F4/80 and CaP/PEI/poly (I: C)/SiO2The preparation method of the-S-IgG 2 is basically the same as that of the first embodiment, except that:
the fifth step: nanoparticles covalently bound to different antibodies (Cap/PEI/poly (I: C)/SiO)2-S-Ab)
mu.L of the antibody (anti-mouse F4/80 or anti-mouse IgG2) was mixed with 40. mu.L of cyclohexane-1-carboxylic acid-3-sulfosuccinimidyl ester (sulfo-SMCC) at room temperature for 3 to 4 hours to activate the antibody. 1mL of CaP/PEI/poly (I: C)/SiO2the-SH nanoparticles were added to the activated antibody and reacted at 4 ℃ for another 24 hours. After the binding reaction, nanoparticles were collected by ultracentrifugation at 66,000g for 30min, and the precipitate was dissolved in 1mL of ultrapure water to obtain Cap/PEI/poly (I: C)/SiO2-S-F480 and CaP/PEI/poly (I: C)/SiO2-S-IgG2 nanoparticles.
EXAMPLE III
This example provides a compound of Cap/PEI/Poly (I: C)/SiO comprising a cell-targeting antibody and Poly (I: C)2S-Ab, wherein Ab is a targeting antibody CD146, F4/80, IgG1 or IgG2, the compound comprises an inner core which is calcium phosphate nano-particles, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed on the surface of the inner core, and a silica outer shell is arranged outside the inner core and is connected with the antibody CD146, F4/80, IgG1 or IgG2 through cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester.
The compound containing the cell-targeting antibody and the Poly (I: C) is CaP/PEI/Poly (I: C)/SiO2S-Ab, i.e. CaP/PEI/poly (I: C)/SiO2-S-CD146，CaP/PEI/poly(I:C)/SiO2-S-F4/80，CaP/PEI/poly(I:C)/SiO2-S-IgG1 and CaP/PEI/poly (I: C)/SiO2The preparation method of the-S-IgG 2 is basically the same as that of the first embodiment, except that:
the second step is that: synthesis of poly (I: C) -loaded Cap/PEI (Cap/PEI/poly (I: C))
1mL of CaP/PEI was mixed with 100. mu.L of water-soluble fluorescein-containing poly (I: C) -Cy5 and stirred at room temperature for 30 minutes.
The poly (I: C) -Cy5 used in this example can also be replaced by poly (I: C) -TRITC.
Example four
In this example, the antiviral effects of CaP/PEI-Cy5/Poly (I: C)/SiO2-S-Ab were tested, using CaP/PEI-Cy5/Poly (I: C)/SiO2-S-CD146 and CaP/PEI-Cy5/Poly (I: C)/SiO2-S-IgG1 as the first example, and using CaP/PEI-Cy5/Poly (I: C)/SiO2-S-F480 and CaP/PEI-Cy5/Poly (I: C)/SiO2-S-IgG2 as the second example, the test specifically includes:
1.1 Experimental materials:
male C57bl/6 mice (6-8w) were purchased from Silikecloda laboratory animals, Inc., China.
HBsAg and HBeAg ELISA kits were purchased from Shanghai Kowa bioengineering, Inc. of China.
The hepatitis B virus nucleic acid quantitative detection kit is purchased from Hunan Shengxiang biological technology Co.
1.2 Experimental methods
1.2.1 establishment of Chronic hepatitis B mouse model
A male C57bl/6 (6-8w) mouse with SPF grade is selected, PAAV-HBV1.2 plasmid is given by a tail vein high-pressure water injection mode, and a chronic HBV replication mouse model is established. Mice were injected with normal saline (negative control), nanoparticles with different antibodies, and tail vein high pressure water injected with Poly (I: C) (positive control) 14 days later, respectively.
1.2.2 sample Collection
Serum from each group of mice was collected and expression of HBsAg and HBeAg in mouse serum was detected by HBsAg and HBeAg ELISA kits. The influence of CaP/PEI-Cy5/Poly (I: C)/SiO2-S-Ab on HBV DNA replication in mice is detected by an RT-PCR method through a hepatitis B virus nucleic acid quantitative detection kit.
1.2.3 statistical treatment
The experimental data are expressed by mean values +/-standard error, the statistical software Graphpad Prism6 is used for analyzing, the variance analysis is carried out in an One-Way ANOVA mode, the Dunett method is used for pairwise comparison, and the P <0.05 is the standard with statistical significance difference.
1.3 results of the experiment
The experimental results are shown in FIGS. 1-3, wherein the experiment results are shown in FIG. 1, and the experiment results show that the CaP/PEI-Cy5/Poly (I: C)/SiO2-S-CD146 can target and activate TLR3 with CD146 immune cells and inhibit the expression of serum HBsAg and HBeAg. PAAV-HBV1.2 tail vein high pressure water injection is used for injecting into a mouse body, corresponding nano particles (Poly (I: C) group high pressure water injection) are injected into the tail vein at constant speed after 14 days, and ELISA is used for detecting the expression of HBsAg and HBeAg in serum. FIG. 2 shows that CaP/PEI-Cy5/Poly (I: C)/SiO2-S-IgG2 targeting to the cell surface FcR receptor can target and activate TLR3 with IgG2 immune cells and inhibit the expression of serum HBsAg and HBeAg. PAAV-HBV1.2 tail vein high pressure water injection is used for injecting into a mouse body, corresponding nano particles (Poly (I: C) group high pressure water injection) are injected into the tail vein at constant speed after 14 days, and ELISA is used for detecting the expression of HBsAg and HBeAg in serum. FIG. 3, CaP/PEI-Cy5/Poly (I: C)/SiO2-S-Ab inhibits serum HBV DNA replication. PAAV-HBV1.2 tail vein high pressure water injection is used for injecting the water into a mouse body, corresponding nano particles (Poly (I: C) group high pressure water injection) are injected into the tail vein at constant speed after 14 days, and HBV DNA expression in serum is detected by RT-PCR.
The results show that CaP/PEI-Cy5/Poly (I: C)/SiO2-S-CD146, CaP/PEI-Cy5/Poly (I: C)/SiO2-S-IgG2 injected into tail vein of mice infected with HBV at constant speed can inhibit the expression of HBsAg and HBeAg in the mice and the replication of HBV DNA. And the CaP/PEI-Cy5/Poly (I: C)/SiO2-S-IgG2 nanoparticles have the strongest antiviral effect.
EXAMPLE five
This example is for the preparation of CaP/PEI/poly (I: C)/SiO2Ab distribution in different tissues and organs of mice was tested, including:
1.1 Experimental materials:
male C57bl/6 mice (6-8w) were purchased from Silikecloda laboratory animals, Inc., China.
1.2 Experimental methods
1.2.1 mouse caudal intravenous nanoparticles
SPF grade male C57bl/6 mice (6-8w) were selected and mice were given a constant rate of intravenous saline injection (negative control) along with nanoparticles with different antibodies.
Mice were sacrificed and liver, spleen, lung and lymph node tissues were collected.
SPF grade male C57bl/6 mice (6-8w) were selected and sacrificed 1 and 3 hours after injection of saline or nanoparticles to collect liver, spleen, and lung lymph node tissues.
1.2.3 preparation of frozen sections of each tissue.
Freezing the collected liver, spleen, lung and kidney and lymph node tissues of the mice in liquid nitrogen, wrapping with the frozen stock solution, and making into frozen sections.
1.2.4 fluorescent microscope observations
The cut frozen sections were examined for the distribution of the nanoparticles under a confocal microscope.
1.2.5 statistical treatment
The experimental data are expressed by mean values +/-standard error, the statistical software Graphpad Prism6 is used for analyzing, the variance analysis is carried out in an One-Way ANOVA mode, the Dunett method is used for pairwise comparison, and the P <0.05 is the standard with statistical significance difference.
1.3 results of the experiment
The experimental results are shown in FIGS. 4-10, the red bright spots are nanoparticles with Cy-5 fluorescence taken up in the tissues, the CaP/PEI-Cy5/Poly (I: C)/SiO2-Ab is mainly distributed in the liver tissues, and the nanoparticles are less distributed in other tissues. CaP/PEI-Cy5/Poly (I: C)/SiO2-Ab rapidly reached the liver within 1 hour, and after 3 hours, the nanoparticles in the liver tissue decreased while the nanoparticles in the lung tissue increased.
The results show that after the constant-speed injection of the CaP/PEI-Cy5/Poly (I: C)/SiO2-Ab nanoparticles into tail veins of mice, the nanoparticles have the greatest tendency to reach liver tissues, and the rest tissues are distributed less.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (3)

1. Compound CaP/PEI/Poly (I: C)/SiO containing cell targeting antibody and Poly (I: C)2-S-Ab, characterized in that: the compound takes calcium phosphate nano-particles as an inner core, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed and carried on the surface of the inner core, a silicon dioxide shell is arranged outside the inner core, the silicon dioxide shell is connected with a cell targeting antibody through cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester, and the cell targeting antibody is one of a CD146 antibody targeting hepatic sinus endothelial cells and an F4/80 antibody targeting Kupffer cells.
2. The compound of claim 1 comprising a cell-targeting antibody and Poly (I: C), CaP/PEI/Poly (I: C)/SiO2-S-Ab, comprising the steps of:
s1, preparing CaP/PEI: preparing PEI-calcium phosphate nanoparticles;
s2, preparation of CaP/PEI/poly (I: C): mixing the PEI-calcium phosphate nanoparticles prepared in the step S1 with water-soluble poly (I: C), and stirring at 15-35 ℃ to obtain PEI-calcium phosphate nanoparticles loaded with poly (I: C);
s3, preparation of CaP/PEI/poly (I: C)/SiO2: fully mixing the CaP/PEI/poly (I: C), tetraethoxysilane, ammonia water and ethanol obtained in the step S2, stirring the mixed solution for 14-18 hours at 15-35 ℃, and performing centrifugal separation to obtain CaP/PEI/poly (I: C)/SiO2Granulating, adding CaP/PEI/poly (I: C)/SiO2Dissolving the particles in ultrapure water;
s4, adding CaP/PEI/poly (I: C)/SiO2Covalent functionalization of particles to make the CaP/PEI/poly (I: C)/SiO2Covalently bonding thiol groups to the surface of the particles;
s5, preparation of CaP/PEI/poly (I: C)/SiO2-S-Ab: covalently functionalized CaP/PEI/poly (I: C)/SiO2Granule addingAdding the activated antibody into one of a CD146 antibody targeting hepatic sinus endothelial cells and an F4/80 antibody targeting Kupffer cells, reacting for 20-28 hours at 3-5 ℃, and centrifuging to obtain CaP/PEI/poly (I: C)/SiO2-S-Ab nanoparticles, CaP/PEI/poly (I: C)/SiO2-S-Ab nanoparticles dissolved in ultrapure water;
bringing the CaP/PEI/poly (I: C)/SiO to the solution as described in step S42The step of covalently bonding thiol groups to the surface of the particles comprises: dissolving trimethoxy silane in ethanol, adding into the solution of Cap/PEI/poly (I: C)/SiO dissolved in ultrapure water2The particles are stirred for 8 to 10 hours at the temperature of between 15 and 35 ℃, and precipitate is centrifugally collected to obtain CaP/PEI/poly (I: C)/SiO2-SH particles, and adding Cap/PEI/poly (I: C)/SiO2-SH particles are dissolved in ultrapure water;
the activated antibody in step S5 is obtained by mixing and activating an antibody and cyclohexane-1-carboxylic acid-3-sulfosuccinimide ester at 15-35 ℃ for 3-4 hours.
3. The method of claim 2, wherein: the step of preparing PEI-calcium phosphate nanoparticles according to step S1 includes: injecting water-soluble calcium lactate, (NH) into ultrapure water according to the volume ratio of 5:5:74)2HPO4And PEI, stirring; the concentration of the water-soluble PEI is 2g/L, and the concentration of the water-soluble calcium lactate is 18mmol/L, (NH)4)2HPO4The concentration is 10.8 mmol/L; the amount of the ultrapure water is four times of the volume of the water-soluble calcium lactate.
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