CN107550885B - Nanoparticle carrier containing TLR3 ligand in pattern recognition receptor and preparation method and application thereof - Google Patents
Nanoparticle carrier containing TLR3 ligand in pattern recognition receptor and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a nanoparticle carrier CaP/PEI/poly (I: C)/SiO containing a TLR3 ligand in a pattern recognition receptor2-SH, the compound comprises calcium phosphate nano-particles 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, a silica shell is arranged outside the inner core, the silica shell is functionalized by carrying thiol groups under the action of trimethoxy silane, and different fluorescent dyes such as FITC, Cy5 and the like can be carried by the silica shell. Furthermore, the invention also provides a preparation method and medical application of the compound. The nanoparticle disclosed by the invention is stable in property and low in cytotoxicity, can carry different fluorescent dyes, can be used as a carrier, effectively realizes the combination of poly (I: C) and TLR3, and enhances the generation of interferon and proinflammatory factors by activating a TLR 3-mediated signal channel, so that the nanoparticle is expected to be used as a carrier for in vivo imaging of drugs and vaccines.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a nanoparticle carrier containing a TLR3 ligand in a pattern recognition receptor 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.
There is currently a lack of effective prophylactic or therapeutic vaccines against many viral infections (e.g., hiv, hepatitis b and hepatitis c infections). Vaccines for the prevention or treatment of viral diseases are usually live attenuated vaccines or inactivated pathogens. An ideal vaccine should provide antigens and adjuvants and protect these immune-activating biomolecules from degradation before reaching their target cells. This means that the vaccine delivery system should mimic the components and immune processes of natural pathogens.
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.
Disclosure of Invention
In view of the above, the present invention provides a nanoparticle vector containing a TLR3 ligand in a pattern recognition receptor, and a preparation method and a medical application thereof.
The invention provides a preparation method of a nanoparticle carrier containing a TLR3 ligand in a pattern recognition receptor, which is characterized by comprising the following steps:
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)/SiO2The surface of the particles is covalently bound to thiol groups or amino groups.
We designed calcium phosphate nanoparticles comprising a core carrier of calcium phosphate, Cy5-dye as an imaging tool in vitro and in vivo, and finally poly (I: C) as an adjuvant for stimulation and immunization. We examined the size of the nanoparticles, spectral characteristics, and internalization rates into cells and organs in vitro and in vivo (mice). Furthermore, we also demonstrated the ability of nanoparticles to stimulate mainly relevant cells, namely hepatocytes and Liver Sinus Endothelial Cells (LSECs).
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 nanoparticle can be used as an effective carrier to lead the poly (I: C) to be targeted to activate the TLR3 channel in vitro and in vivo. 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 nanoparticles are used as carriers and combined with poly (I: C), so that poly (I: C) carried by the nanoparticles activates a TLR3 pathway to trigger related natural immune responses, and the nanoparticles can also be combined in specific cells or tissues and can be used as imaging tools in vitro and in vivo. Therefore, the nanoparticle carrier is expected to be developed into a medicine or health-care product for preventing or treating diseases which can respond to activation of a TLR3 signal channel, and can be used as an imaging tool in vitro and in vivo.
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 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 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.
The invention provides a nanoparticle carrier containing a TLR3 ligand in a pattern recognition receptor, which takes calcium phosphate nanoparticles as an inner core, PEI is grafted on the surface of the inner core, 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 thiol groups or amino groups are covalently bonded on the silicon dioxide of the shell.
Preferably, the nanoparticle carrier containing the TLR3 ligand in the pattern recognition receptor is prepared by the preparation method.
In a third aspect, the invention provides the use of a nanoparticle vector comprising a TLR3 ligand in a pattern recognition receptor in the preparation of a TLR3 agonist.
The fourth aspect of the invention provides the use of a nanoparticle vector comprising a TLR3 ligand in a pattern recognition receptor in the manufacture of a medicament or health product for the prevention or treatment of a disease responsive to a TLR3 agonist.
A fifth aspect of the invention provides a method of activating TLR3, the steps comprising: cells expressing TLR3 were contacted with nanoparticle vectors containing the relevant cellular ligand Poly (I: C) and were used as imaging tools in vitro and in vivo.
In a sixth aspect, the invention provides a pharmaceutical composition comprising a nanoparticulate carrier comprising a ligand for TLR3 in a pattern recognition receptor, for use in a viral infectious disease.
The invention has the beneficial effects that: the present invention provides an effective vector for poly (I: C) to activate the TLR3 pathway in vivo and in vitro, and can be used as an imaging tool in vitro and in vivo. The invention combines nanoparticles as a carrier with poly (I: C), effectively activates the TLR3 pathway in vivo and in vitro to trigger related natural immune response, combines poly (I: C) with TLR3, and enhances the production of interferon and proinflammatory factors by activating a TLR 3-mediated signal pathway.
Drawings
FIG. 1 is a scanning electron micrograph of calcium phosphate nanoparticles;
FIG. 2 shows CaP/PEI/poly (I: C)/SiO2-graph of MTT cytotoxicity assay results for SH;
FIG. 3 shows the cell pairs Cap/PEI/poly (I: C)/SiO2-graph of SH uptake capacity assay results;
FIG. 4 shows CaP/PEI/poly (I: C)/SiO2-graph of results of flow cytometry experiments of SH distribution in different tissues and organs of mice;
FIG. 5 shows CaP/PEI/poly (I: C)/SiO2-graph of test results of SH distribution in mouse lungs;
FIG. 6 shows CaP/PEI/poly (I: C)/SiO2-graph of test results of SH distribution in mouse liver;
FIG. 7 shows CaP/PEI/poly (I: C)/SiO2-graph of test results of SH distribution in mouse spleen;
FIG. 8 shows CaP/PEI/poly (I: C)/SiO2-graph of test results of SH distribution in mouse lymph nodes;
FIG. 9 shows CaP/PEI/poly (I: C)/SiO2-a graph of statistical test results of SH distribution in different tissues of mice;
FIG. 10 shows CaP/PEI/poly (I: C)/SiO2-SH cytokine production by hepatocytes;
FIG. 11 shows CaP/PEI/poly (I: C)/SiO2-graphs of results of assays for SH promoting cytokine production in non-parenchymal intrahepatic cells (NPCs);
FIG. 12 shows CaP/PEI/poly (I: C)/SiO2-graphs of results of assays for SH-promoting cytokine production by sinusoidal endothelial cells (LSEC);
FIG. 13 shows CaP/PEI/poly (I: C)/SiO2A process scheme for the preparation of-SH.
Detailed Description
The nanoparticle carrier containing the TLR3 ligand in a pattern recognition receptor provided by the invention, and the preparation method and the application thereof are further described in the following with reference to specific embodiments. 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 "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 "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
The embodiment provides a nanoparticle carrier containing a TLR3 ligand in a pattern recognition receptor, the nanoparticle carrier takes calcium phosphate nanoparticles as an inner core, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed and loaded on the surface of the inner core, the outer part of the inner core is a silica outer shell, and the silica of the outer shell is covalently bonded with thiol groups or amino groups.
The preparation method of the nanoparticle carrier containing the TLR3 ligand in the pattern recognition receptor 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 (18mm, pH 10), (NH) at a ratio of 5mL:7mL4)2HPO4(10.8mm, 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. Will 1mL of nanoparticles were added to this 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 scanning electron micrograph of the obtained CaP/PEI/poly (I: C)/SiO2-SH nanoparticles is shown in the attached figure 1, and the characteristics of the nanoparticle carrier are shown in the table 1.
TABLE 1 characterization of functional calcium phosphate nanoparticles
Example two
The embodiment provides a nanoparticle carrier containing a TLR3 ligand in a pattern recognition receptor, the nanoparticle carrier takes calcium phosphate nanoparticles as an inner core, PEI is grafted on the surface of the inner core, Poly (I: C) is adsorbed and loaded on the surface of the inner core, and the outer part of the inner core is a silica shell, wherein the silica of the shell is covalently bonded with thiol groups.
The preparation method of the nanoparticle carrier containing the TLR3 ligand in the pattern recognition receptor 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 III
This example is for CaP/PEI-Cy5/Poly (I: C)/SiO2-SH cytotoxicity was tested by the specific steps comprising:
1.1 Experimental materials:
6 well plates were purchased from Thermo NUNC, Denmark
RPMI1640 (high sugar), fetal bovine serum, penicillin and streptomycin were purchased from Gibco, USA.
MTT cell proliferation and cytotoxicity kits were purchased from bi yun tian, china.
1.2 Experimental methods:
1.2.1 cell culture
THP-1 cells were mixed with varying concentrations of CaP/PEI-FITC/Poly (I: C)/SiO in RPMI1640 medium containing 10% calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin2Culturing the-SH nano-particles for 1h/24 h.
1.2.2MTT cytotoxicity assay
MTT solution (5mg/ml in PBS, pH 7.4)10ul. was added to each well and incubation continued for 4h, the incubation was terminated and the culture supernatant from the wells was carefully aspirated. Add 100ul DMSO into each well, shake for 10min, make the crystal fully melt. Selecting 490nm wavelength, measuring the light absorption value of each well on an enzyme linked immunosorbent assay, recording the result, and drawing a cell growth curve by taking time as an abscissa and the light absorption value as an ordinate.
1.3 results of the experiment
The results of the experiments are shown in FIG. 2, where THP-1 cells were incubated with nanoparticles with different concentrations of Poly (I: C) or without Poly (I: C) for 1h, 24 h. The cell viability of THP-1 cells at different time points was tested using the MTT cytotoxicity assay. Detection of CaP/PEI-FITC/Poly (I: C)/SiO by MTT cytotoxicity test method2The toxicity of-SH to cells is time and dose dependent, and the larger the dose of nanoparticles, the longer the time of co-incubation with cells, the more toxic to cells, but less toxic to cells within a certain time and dose. Thus, proper dosage is required for in vivo administration.
EXAMPLE III
In this example, the uptake capacity of CaP/PEI-Cy5/Poly (I: C)/SiO2/-SH by cells was tested, and specifically included:
1.1 Experimental materials:
6 well plates were purchased from Thermo NUNC, Denmark
RPMI1640 (high sugar), fetal bovine serum, penicillin and streptomycin were purchased from Gibco, USA.
DAPI was purchased from AAT Bioquest, usa.
1.2 Experimental methods
1.2.1 cell staining
THP-1 cells were incubated with DAPI at a certain concentration overnight in RPMI1640 complex containing 10% calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin, washed 6 times with PBS buffer to wash out unbound DAPI, digested with enzyme and centrifuged to collect cells, and RPMI1640 medium was added to prepare a cell suspension for use.
1.2.2 cell culture
The THP-1 cells with the DAPI dye were mixed with different concentrations of CaP/PEI-FITC/Poly (I: C)/SiO2Culturing the-SH nano-particles for 1h/24 h.
1.2.3 fluorescent microscope observations
The cultured cells were observed under a fluorescent microscope.
1.3 results of the experiment
The results of the experiment are shown in FIG. 3, and nanoparticles with different concentrations of Poly (I: C) were incubated with THP-1 cells for 1h and 24 h. The ability of THP-1 cells to take up nanoparticles at different time points was observed with a fluorescence microscope. Cell pairs of Cap/PEI-FITC/Poly (I: C)/SiO by fluorescent microscope2The uptake capacity of SH is time and dose dependent, the larger the dose of nanoparticles, the longer the time of co-incubation with cells, the more nanoparticles the cells take up.
Example four:
this example is for the preparation of CaP/PEI/poly (I: C)/SiO2-SH distribution in different tissues and organs of mice is tested, and the method specifically comprises the following steps:
1.1 Experimental materials:
male C57bl/6 mice (6-8w) were purchased from Silikecloda laboratory animals, Inc., China.
Anti-mouse CD146 flow antibody was purchased from BD Pharmingen, USA
Collagenase was purchased from Roche, switzerland.
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 cell suspensions from each tissue.
The liver and lung tissues of the mice collected in the above were digested with collagenase at 37 ℃ for 60 minutes and 45 minutes, respectively, and then filtered through a 70 μm filter to obtain cell suspensions. Spleen and lymph node tissues are ground and filtered by a 70-micron filter screen, and a cell suspension is obtained after red blood cell lysate is added and PBS is added to wash cells.
1.2.4 flow cytometry analysis.
And (3) after the obtained cell suspension of each tissue organ is stained by using a corresponding flow antibody, analyzing the distribution of the nanoparticles in each tissue organ by using a flow cytometry.
1.2.5 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.
1.2.6 fluorescent microscope observations
The cut frozen sections were examined for the distribution of the nanoparticles under a confocal microscope. And randomly selecting ten visual fields, counting the number of the nano particles in each visual field, and calculating an average value.
1.3 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.
1.4 results of the experiment
The experimental results are shown in FIGS. 4-9, the red bright spots are nanoparticles with Cy-5 fluorescence, which are taken up in the tissues, and the CaP/PEI-Cy5/Poly (I: C)/SiO2-SH are mainly distributed in the liver and lung tissues, and are distributed most in the lung. After 1 hour of injection, the nanoparticles in the lung were maximal, followed by liver tissue, which decreased after 3 hours while the nanoparticles in the lung continued to increase.
The results show that after the constant-speed injection of the CaP/PEI-Cy5/Poly (I: C)/SiO2-SH nanoparticles into the tail vein of the mouse, the nanoparticles tend to reach lung tissues most and liver tissues second, and the rest tissues are distributed less.
EXAMPLE five
In this example, the experiment of CaP/PEI-Cy5/Poly (I: C)/SiO2-SH on promoting the production of cytokines by different cells in the liver was carried out, which specifically includes:
1.1 Experimental materials:
male C57bl/6 mice (6-8w) were purchased from Silikecloda laboratory animals, Inc., China.
IFN- β, IFN- γ, TNF- α, IL-6, and IP-10 primers were purchased from Invivogen, USA.
The perfusion enzyme Liberase TM was purchased from Roche, Switzerland.
One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) Kit was purchased from TaKaRa, Japan.
CD146 MicroBeads, mouse magnetic beads were purchased from Miltenyi, Germany.
DMEM, fetal bovine serum, penicillin and streptomycin were purchased from Gibco, USA.
RNAioso Plus (Total RNA extraction reagent) was purchased from TaKaRa, Japan.
1.2 Experimental methods:
1.2.1 isolation of hepatocytes, non-parenchymal hepatocytes (NPC) and sinusoidal endothelial cells (LSEC)
Selecting SPF male C57bl/6 (6-8w) mice, separating the liver cells, liver cells and non-parenchymal liver cells (NPC) of the mice by a digestive enzyme Liberase TM perfusion method, and separating Liver Sinus Endothelial Cells (LSEC) by a magnetic bead sorting method.
1.2.2 cell culture
Hepatocytes, NPC and LSEC cells were incubated for 6h with nanoparticles of CaP/PEI-Cy5/Poly (I: C)/SiO2-SH (1ug/ml) in a DMEM solution containing 10% calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin.
1.2.3 cellular RNA extraction
RNA of the cultured cells is extracted with RNAioso Plus (Total RNA extraction reagent), chloroform, isopropanol, or the like.
1.2.4 RT-PCR
The extracted cell RNA is applied to a RT-PCR detection method to detect IFN-beta, IFN-gamma, TNF-alpha, IL-6 and IP-10 cell factors expressed by the cells.
1.3 results of the experiment
As shown in FIGS. 10 to 12, hepatocytes, non-parenchymal intrahepatic cells (NPC) and sinusoidal endothelial cells (LSEC) were isolated from the liver of wild-type C57 male mice (6-8w), and the three cells were stimulated with CaP/PEI-FITC/Poly (I: C)/SiO2-SH for 6 hours, respectively. And extracting RNA of the stimulated cells, and detecting the expression of cytokines IFN-beta, IFN-gamma, TNF-alpha, IL-6 and IP-10 in the three cells by using an RT-PCR method. As a result, it was found that CaP/PEI-Cy5/Poly (I: C)/SiO2-SH promoted the production of cytokines (FN-. beta., IFN-. gamma., TNF-. alpha., IL-6 and IP-10) by various cells in the liver.
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. A nanoparticle vector comprising a TLR3 ligand in a pattern recognition receptor, wherein: the nano particle carrier 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, and thiol groups are covalently bonded on silicon dioxide of the shell.
2. A method of preparing the nanoparticle vector of claim 1 comprising a TLR3 ligand in a pattern recognition receptor, the steps comprising:
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;
the step of covalently bonding thiol groups to the surfaces of the particles of Cap/PEI/poly (I: C)/SiO2 of step S4 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)/SiO2the-SH particles are dissolved in ultrapure water.
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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| CN102258793A (en) * | 2011-07-14 | 2011-11-30 | 长春理工大学 | Application of core-shell nano particle |
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| Cell targeting by antibody-functionalized calcium phosphate nanoparticles;D Kozlova 等;《Journal of Materials Chemistry》;20121231;第22卷(第2期);摘要；第396-400页 * |
| Poly(I:C) treatment leads to interferon-dependent clearance of hepatitis B virus in a hydrodynamic injection mouse model;Wu J 等;《J Virol》;20140611;第88卷(第18期);摘要 * |
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