CN108283719B

CN108283719B - Nano-particles and preparation method and application thereof

Info

Publication number: CN108283719B
Application number: CN201710014816.3A
Authority: CN
Inventors: 刘瑞田; 张凌宵; 刘栋群
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2017-01-09
Filing date: 2017-01-09
Publication date: 2021-02-12
Anticipated expiration: 2037-01-09
Also published as: CN108283719A

Abstract

The invention relates to the technical field of medicines, in particular to a nano particle and a preparation method and application thereof. The nanoparticle can co-deliver hydrotalcite nanoparticles of indoleamine 2,3-dioxygenase small interfering RNA and tyrosinase related antigen peptide 2, the particles have the functions of targeting DC cells, breaking immune tolerance, activating an immune system and causing stronger cellular immune response, and compared with a vaccine or IDO-siRNA which is based on a tumor antigen specific epitope Trp2, the nanoparticle has the advantage that the inhibition effect on the growth of melanoma is obviously enhanced.

Description

Nano-particles and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a nanoparticle, a preparation method and application thereof, and specifically relates to preparation and application of a hydrotalcite nanoparticle for co-delivering small interfering RNA and tumor-associated epitope.

Background

Tyrosinase-related antigenic peptide 2(Trp2) is a tumor-specific epitope, and can be prepared into vaccines to effectively inhibit the growth of melanoma. Trp2 is a small molecular polypeptide consisting of 9 amino acids, has short sequence and weak immunogenicity, and is difficult to cause effective toxic T cell response. Vectors are therefore commonly used to boost the immunogenicity of Trp 2. The over-expression of IDO gene in dendritic cell can result in the increase of IL-12 and TGF-beta level of its downstream products, and these cytokines are important factors for the immune tolerance of organism. Indoleamine 2,3-dioxygenase small interfering RNA (IDO siRNA) interaction with mRNA, the transcription product of the IDO gene, silences the IDO gene in the target cell. Through targeted delivery of IDO siRNA to dendritic cells, IDO genes in the cells are silenced, so that the immune tolerance of an organism can be broken to a certain extent, a cellular immune system is activated, and the growth of tumors is inhibited.

The carriers for the above drug targeted delivery include liposomes, cationic nanoparticles, and the like. Zeng, H.Jiang, T.Wang, Z.Zhang, T.Gong, X.Sun, Cationic cell delivery of Trp2 peptide for effective viral priming and enhanced cytotoxic T-lymphocyte responses, Journal of controlled release 200(2015)1-12. Chen et al, J.Koropatnick, Targeted siRNA encapsulation of Indolamine 2,3-Dioxygenase in antibiotic-presenting Cells Using nanoparticles-conjugated Liposomes A Novel Strategy for Strategy of Melanoma, J Immunother 37(2014) 123. sub.134. "Chen et al nanoparticles prepared by embedding IDO-siRNA with Mannose-modified Liposomes can be Targeted for delivery to dendritic Cells. These carriers have the disadvantages of high preparation cost, difficult large-scale production or high biological toxicity, etc. The vaccine vector has the advantages of low price, easy preparation and high biocompatibility, and has better application prospect.

In recent years, studies on the delivery of biopharmaceuticals using layered double hydroxide composite metal oxides (LDH, hydrotalcite) have received much attention. LDH has positive charge, high specific surface area, and has the characteristics of endosome escape capability, good ion exchange capability, interlayer anion exchange performance and the like. Biomolecules carrying negative charges, such as RNA, enter LDH layers in an intercalation mode under certain conditions, but have small influence on the surface charge property of the particles, so that LDH nanoparticles still have the capacity of adsorbing the negative charge molecules on the surfaces of the particles, and a structural basis is provided for constructing IDO-siRNA and Trp2 co-delivery nanoparticles.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a nanoparticle and a preparation method and application thereof, and particularly relates to preparation and application of a hydrotalcite nanoparticle for co-delivering a small interfering RNA and a tumor-associated antigen epitope.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a nanoparticle, which takes a layered double-hydroxyl composite metal oxide as a carrier, wherein the carrier loads small interfering RNA and epitope peptide, the small interfering RNA is inserted into the carrier, and the epitope peptide is adsorbed on the surface of the carrier.

In the invention, the chemical formula of the nanoparticle is epitope peptide/LDH/IDO-siRNA, free epitope peptide or IDO-siRNA is unstable and easily degraded in vivo and is difficult to be effectively phagocytized by cells, and the epitope peptide and the IDO-siRNA can be presented into the cells simultaneously to play a synergistic effect by using the nanoparticle, and the specific mechanism is as follows: the nanoparticles are phagocytized by dendritic cells through a clathrin mediated pathway to form an endosome, and in the process of acidification of the endosome, hydrotalcite is dissolved, and then the concentration of ions in cells is increased, and water molecules flow in to cause rupture of the endosome. Meanwhile, the IDO-siRNA loaded by hydrotalcite and the epitope peptide are released in an endosome, and when the endosome is broken, the medicaments enter cytoplasm at the same time to generate a synergistic effect: on one hand, IDO-siRNA inhibits dendritic cell differentiation to immune tolerance cell caused by IDO gene overexpression by acting on IDO mRNA to silence IDO gene expression in dendritic cell, thereby effectively presenting antigen epitope and promoting T cell proliferation and activation; on the other hand, tumor epitopes enter the cytoplasm to bind to MHC class I molecules and are presented to the cell surface, triggering the development of a Th1 immune response. Briefly, the IDO-siRNA maintains the function of presenting tumor epitopes by dendritic cells, and inhibits the occurrence of immune tolerance, thereby ensuring that tumor epitopes are presented and activating cellular immunity.

According to the invention, the small interfering RNA (IDO-siRNA) is indoleamine 2,3-dioxygenase small interfering RNA.

Preferably, the sense strand of the small interfering RNA is shown as SEQ ID NO.1, and the antisense strand of the small interfering RNA is shown as SEQ ID NO. 2.

The nucleic acid sequence is as follows:

sense strand (SEQ ID NO. 1): 5'-GGGCUUCUUCCUCGUCUCUTT-3', respectively;

antisense strand (SEQ ID NO. 2): 5'-AGAGACGAGGAA GAAGCCCTT-3' are provided.

Preferably, the epitope peptide is any one or the combination of at least two of tyrosinase epitope peptide, Melan-A/MART-1 epitope peptide, gp100 epitope peptide, chicken egg albumin, HER-2/neu tumor-associated antigen or MUC1 antigen peptide.

In the invention, the tyrosinase epitope peptide, the Melan-A/MART-1 epitope peptide, the gp100 epitope peptide and the chicken ovalbumin are mainly directed to melanoma, and the HER-2/neu tumor-associated antigen and the MUC1 antigen peptide are mainly directed to breast cancer.

Preferably, the amino acid sequence of the tyrosinase epitope peptide is shown in SEQ ID NO.3, and the amino acid sequence (SEQ ID NO.3) is SVYDFFVWL.

In a second aspect, the present invention provides a method for preparing nanoparticles as described in the first aspect, comprising the steps of:

(1) preparing a dihydroxy composite metal oxide;

(2) mixing the dihydroxy composite metal oxide carrier prepared in the step (1) with small interfering RNA, and standing after mixing to obtain nanoparticles containing small interfering RNA intercalation;

(3) and (3) mixing the small interfering RNA intercalation-containing nanoparticles prepared in the step (2) with epitope peptide, mixing, standing, and centrifuging to obtain the small interfering RNA and epitope peptide-containing nanoparticles.

According to the present invention, the preparation of the dihydroxy composite metal oxide in step (1) is a conventional technical means in the field, and the skilled person can adjust various conditions of synthesis as required, and the preparation of the dihydroxy composite metal oxide according to the present invention comprises the following specific steps:

mixing magnesium nitrate with the molar concentration of 0.01-0.1M, preferably 0.03M and aluminum nitrate with the molar concentration of 0.01-0.1M, preferably 0.01M, adding the mixture into sodium hydroxide solution with the molar concentration of 0.01-0.1M, preferably 0.15M, stirring for 10-20min, preferably 15min, 12000, centrifuging for 1-10min, preferably 5min, shaking and mixing uniformly with carbonated water at 30 ℃ for 20-35min, preferably 30min, transferring the mixture to a dialysis bag for dialysis for 1-10h, preferably 5h, transferring the mixture to a closed container at 30 ℃ and 400rpm, preferably 280rpm, shaking and suspending the mixture for 0.5-3h, preferably 1h, transferring the mixture to a polytetrafluoroethylene hydrothermal kettle at 100 ℃ and 130 ℃, preferably 120 ℃ and keeping the temperature for 10-20h, preferably 15h, and naturally cooling to obtain the dihydroxy composite metal oxide.

Preferably, the preparation of the dihydroxy composite metal oxide comprises the following specific steps:

10mL of a mixture of 0.03M magnesium nitrate and 0.01M aluminum nitrate was quickly added to 40mL of a 0.15M NaOH solution, and after quickly stirring at room temperature for 15min, the mixture was centrifuged at 12000g for 5 min. The obtained product is resuspended by using carbonated water, then is shaken and suspended for 30min at the temperature of 30 ℃, the obtained suspension is transferred to a dialysis bag, and is dialyzed for 5h at room temperature by using the carbonated water with the volume of 100 times of the suspension in a closed container. Then the suspension is transferred into a closed container, is shaken and suspended for 1h at 30 ℃ and 280rpm, is transferred into a 100mL polytetrafluoroethylene hydrothermal kettle (the sample loading volume is 50 percent), is kept at constant temperature for 15h at 120 ℃, and is naturally cooled.

Preferably, the final concentration of the small interfering RNA described in step (2) is 0.01-1. mu.g/. mu.L, such as 0.01. mu.g/. mu.L, 0.02. mu.g/. mu.L, 0.0.3. mu.g/. mu.L, 0.05. mu.g/. mu.L, 0.06. mu.g/. mu.L, 0.08. mu.g/. mu.L, 0.1. mu.g/. mu.L, 0.12. mu.g/. mu.L, 0.15. mu.g/. mu.L, 0.2. mu.g/. mu.L, 0.3. mu.g/. mu.L, 0.4. mu.g/. mu.L, 0.5. mu.g/. mu.L, 0.6. mu.g/. mu.L, 0.7. mu.g/. mu.L, 0.8. mu.g/. mu.L, 0.9. mu.g/. mu.L or 1. mu.g/. mu.L, preferably 0.05-0.5. mu.g/. mu..

Preferably, the temperature of the standing in the step (2) is 30-45 ℃, preferably 35-40 ℃, for example 30 ℃, 31 ℃, 32 ℃, 33 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 45 ℃, and more preferably 37 ℃, and the specific values between the above values are limited by the space and the conciseness, and the invention is not exhaustive list of the specific values included in the range.

Preferably, the standing time in the step (2) is 10-30min, such as 10min, 11min, 12min, 13min, 15min, 16min, 18min, 20min, 22min, 23min, 25min, 26min, 28min, 30min, preferably 15-25min, and more preferably 20min, and the specific values between the above values are limited by the space and for the sake of brevity, the invention is not exhaustive.

Preferably, the epitope peptide of step (3) has a final concentration of 0.1-10. mu.g/. mu.L, such as 0.1. mu.g/. mu.L, 0.2. mu.g/. mu.L, 0.3. mu.g/. mu.L, 0.5. mu.g/. mu.L, 0.6. mu.g/. mu.L, 0.8. mu.g/. mu.L, 0.9. mu.g/. mu.L, 1. mu.g/. mu.L, 1.2. mu.g/. mu.L, 1.5. mu.g/. mu.L, 2. mu.g/. mu.L, 3. mu.g/. mu.L, 4. mu.g/. mu.L, 5. mu.g/. mu.L, 6. mu.g/. mu.L, 7. mu.g/. mu.L, 8. mu.g/. mu.L, 9. mu.g/. mu.L or 10. mu.g/. mu.L, preferably 0.5. mu.g, and the particular values between the above, are not intended to be exhaustive or to limit the invention to the precise values encompassed within the scope, for reasons of brevity and clarity.

Preferably, the temperature of the standing in the step (3) is 30-45 ℃, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 45 ℃, preferably 35-40 ℃, and more preferably 37 ℃, and the specific values between the above values are limited by the space and the conciseness, and the invention is not exhaustive list of the specific values included in the range.

Preferably, the standing time in step (3) is 20-40min, such as 10min, 11min, 12min, 13min, 15min, 16min, 18min, 20min, 22min, 23min, 25min, 26min, 28min, 30min, 31min, 33min, 35min, 36min, 38min, 40min, preferably 25-35min, and further preferably 30min, and the specific point values between the above values are limited to space and are not exhaustive, and the specific point values included in the range are not exhaustive in the present invention for simplicity.

Preferably, the rotation speed of the centrifugation in the step (3) is 10000-.

Preferably, the centrifugation time in step (3) is 1-10min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min, preferably 3-8min, and more preferably 5min, and the specific points between the above values are not exhaustive, and for brevity, the invention does not provide an exhaustive list of the specific points included in the range.

In a third aspect, the present invention provides a vaccine comprising a nanoparticle according to the first aspect.

Preferably, the vaccine further comprises a pharmaceutically acceptable carrier and a water-in-oil emulsion.

In a fourth aspect, the present invention provides the use of a nanoparticle according to the first aspect or a vaccine according to the third aspect in the manufacture of a medicament for targeting a tumour.

Preferably, the tumor is melanoma and/or breast cancer.

Compared with the prior art, the invention has the following beneficial effects:

(1) the nanoparticle can co-deliver hydrotalcite nanoparticles of indoleamine 2,3-dioxygenase small interfering RNA and related antigen peptide, the particle has the functions of targeting DC cells, breaking immune tolerance, activating immune system and causing stronger cellular immune response, and compared with the independent use of vaccine or IDO-siRNA based on tumor antigen specific epitope, the nanoparticle has the advantages that the inhibition effect on the growth of melanoma and breast cancer is obviously enhanced;

(2) the invention is a compound tumor immunotherapy vaccine, compare with single immunotherapy mode such as tumor antigen specificity epitope vaccine, dendritic cell vaccine, cytokine vaccine, the expression of IDO gene in the silent dendritic cell of the invention on one hand, inhibit dendritic cell to immune tolerant cell differentiation caused by IDO gene overexpression, thus promote T cell proliferation and activation; on the other hand, the escape capability of the vaccine in the endosome promotes the tumor antigen epitope to enter cytoplasm to be combined with MHC class I molecules, and the generation of Th1 immune response is stimulated.

Drawings

FIG. 1 is an electrophoresis gel chart of the invention for determining IDO-siRNA entering LDH interlamination by agarose gel electrophoresis test, wherein, from left to right, 1 pore channel is free IDO siRNA, 2 pore channel is LDH, 3-8 pore channels are nanoparticles with different mass ratios of IDO siRNA and LDH, and the mass ratios are 1:1, 1:5, 1:10, 1:20, 1:30 and 1:40 respectively;

FIG. 2 is a graph showing the results of the determination of the ability of LDH/IDO-siRNA to adsorb Trp2 in the 2, 2-biquinoline-4, 4-dicarboxylic acid disodium assay of the present invention;

FIG. 3 is a graph showing the results of the determination of the movement path and localization of Trp2/LDH/IDO-siRNA vaccine particles in DC cells;

FIG. 4 is a graph showing the results of Trp2/LDH/IDO-siRNA nanoparticle vaccine of the present invention down-regulating IDO mRNA level in DC cells;

FIG. 5 is a graph showing the expression result of the Trp2/LDH/IDO-siRNA nanoparticle vaccine of the present invention for down-regulating IDO protein in DC cells, wherein FIG. 5(A) shows the protein level of IDO expression detected by Western-blot, and FIG. 5(B) shows the optical density of IDO analyzed by Odyssey LICOR SA two-color infrared laser imaging system;

FIG. 6 is a graph showing the results of Trp2/LDH/IDO-siRNA particle vaccine of the present invention enhancing mouse cellular immunity, wherein FIG. 6(A) is CD8 measured by flow cytometry analysis⁺/IFN-γ⁺Cell ratio, FIG. 6(B) IFN-. gamma.levels in cell culture supernatants determined by IFN-. gamma.ELISA kit;

FIG. 7 is a graph showing the results of Trp2/LDH/IDO-siRNA nanoparticle vaccine of the present invention inhibiting the growth of mouse melanoma.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1: preparation of nanoparticle vaccines

Preparing LDH: 10mL of a mixture of magnesium nitrate (0.03M) and aluminum nitrate (0.01M) was quickly added to 40mL of a NaOH (0.15M) solution, and after quickly stirring at room temperature for 15min, the mixture was centrifuged at 12000g for 5 min. The obtained product is resuspended by using carbonated water, then is shaken and suspended for 30min at the temperature of 30 ℃, the obtained suspension is transferred to a dialysis bag, and is dialyzed for 5h at room temperature by using the carbonated water with the volume of 100 times of the suspension in a closed container. Then the suspension is transferred into a closed container, is shaken and suspended for 1h at 30 ℃ and 280rpm, is transferred into a 100mL polytetrafluoroethylene hydrothermal kettle (the sample loading volume is 50 percent), is kept at constant temperature for 15h at 120 ℃, and is naturally cooled.

Preparation of LDH/IDO-siRNA: mixing the prepared hydrotalcite with IDO siRNA, wherein a sense strand (SEQ ID NO.1) of the IDO siRNA: 5'-GGGCUUCUUCCUCGUCUCUTT-3', antisense strand (SEQ ID NO. 2): 5'-AGAGACGAGGAA GAAGCCCTT-3', diluting with decarbonized acid water until the final concentration of IDO siRNA is 0.1 μ g/μ L, vortexing and shaking to mix them uniformly, standing at 37 deg.C for 20min to obtain nanoparticles containing IDO siRNA intercalation with chemical formula of LDH/IDO-siRNA.

Preparation of Trp 2/LDH/IDO-siRNA: after the prepared LDH/IDO-siRNA and a certain amount of Trp2 are mixed uniformly, the amino acid sequence (SEQ ID NO.3) of Trp2 is: SVYDFFVWL, diluting with decarbonized acid water until the final concentration of hydrotalcite is 1 mug/mug L, standing at 37 ℃ for 30min, and then centrifuging at 12000g speed for 5min to obtain Trp 2/LDH/IDO-siRNA.

The vaccines in which epitope peptides can be replaced by Melan-A/MART-1 epitope peptides, gp100 epitope peptides and chicken ovalbumin for epitope peptides have similar effects to those of Trp2, and all have therapeutic effects on melanoma, and the following examples are described with reference to Trp 2/LDH/IDO-siRNA.

The epitope peptide can be replaced by HER-2/neu tumor-associated antigen, MUC1 antigen peptide, and the vaccine replacing the epitope peptide has growth inhibition effect on breast cancer, except that the effect of the vaccine for the breast cancer is similar to that of Trp2, and the following example is explained by Trp 2/LDH/IDO-siRNA.

Example 2: determination of the ability of IDO-siRNA to insert into LDH in vaccines

Agarose gel electrophoresis was performed according to conventional methods, and in the gel electrophoresis pattern, free IDO siRNA migrated to the bottom of the gel, and IDO siRNA that entered between LDH layers remained in the loading wells. The method for calculating the capability of the IDO-siRNA intercalation entering the LDH interlamination is as follows:

the intercalation ability of IDO siRNA possessed by hydrotalcite is calculated according to the following formula:

the test results are shown in FIG. 1, wherein the mass of free IDO siRNA in 1 channel is 1 μ g, the mass of LDH in 2 channels is 40 μ g, and the mass of IDO siRNA in 3-8 channels is 1 μ g, and the LDH mass is added according to different mass ratios.

The result shows that when the LDH is intercalated with the IDO siRNA by using the method described by the invention, the IDO siRNA can be completely intercalated when the mass ratio of the hydrotalcite to the IDO siRNA is 10:1, namely the intercalation capability of the IDO siRNA is the highest and is 10%. With the increase of the mass ratio of the hydrotalcite to the IDO siRNA, the IDO siRNA can be completely intercalated into hydrotalcite interlayers, but the intercalation capability is gradually reduced and is between 0 and 10 percent.

Example 3: determination of the ability of LDH/IDO-siRNA to adsorb Trp2

The ability of LDH/IDO-siRNA to adsorb Trp2 was determined using the conventional disodium 2, 2-biquinoline-4, 4-dicarboxylate assay, and the ability of LDH/IDO-siRNA to adsorb Trp2 was calculated according to the following equation:

description of the drawings: mg (magnesium)₃The mass of the Al-IDO siRNA-LDH is calculated by the mass of hydrotalcite

The test results are shown in fig. 2:

when the mass of the LDH/IDO-siRNA and the Trp2 is 8:1, the adsorption capacity of Trp2 is 8.5%;

when the mass of the LDH/IDO-siRNA and the Trp2 is 8:2, the adsorption capacity of Trp2 is 13.9%;

when the mass of the LDH/IDO-siRNA and the Trp2 is 8:3, the adsorption capacity of Trp2 is 15.5%;

when the mass of the LDH/IDO-siRNA and the Trp2 is 8:4, the adsorption capacity of Trp2 is 17.8%;

when the mass of the LDH/IDO-siRNA and the Trp2 is 8:6, the adsorption capacity of Trp2 is 30.9%;

when the mass of the LDH/IDO-siRNA and Trp2 is 8:10, the adsorption capacity of Trp2 is 52.0%;

when the mass of the LDH/IDO-siRNA and Trp2 is 8:20, the adsorption capacity of Trp2 is 122.0%.

As can be seen,

in the examples described below, the vaccine particles used in the present invention had an amount of IDO-siRNA intercalated into LDH of 6% and an amount of Trp2 adsorbed onto LDH of 20%.

Example 4: trp2/LDH/IDO-siRNA can be phagocytized by DC cells and escape from lysosome

The test method comprises the following steps: 1X 105 DC2.4 cells were seeded on a petri dish and cultured at 37 ℃ in an environment of 5% CO2 concentration for 24h, and then Trp2/LDH/IDO-siRNA nanoparticles using FAM fluorescent molecule-labeled IDO-siRNA were added at a final concentration of 20. mu.g/mL for 24h of CO-culture. Selecting three time points of 4h,12h and 24h, adding Lyso tracker red to stain lysosomes, immediately fixing cells by using 4% paraformaldehyde, and staining cell nuclei by using DAPI. The cells treated by the above method were observed under a confocal laser microscope.

The results of the tests are shown in figure 3,

blank: lysosome staining (Red fluorescence, the same below) with Lyso Tracker Red and cell nucleus staining (blue fluorescence, the same below) with DAPI, lysosome (Red fluorescence) being more densely distributed around cell nucleus (blue);

4 h: trp2/LDH/IDO-siRNA (green fluorescence, the same below) is mainly adsorbed on cell membranes to form a compact green fluorescent layer, and only a small amount of nanoparticles are phagocytosed into cells and co-localized with lysosomes (red fluorescence) to form yellow fluorescence;

12 h: most of Trp2/LDH/IDO-siRNA was phagocytosed into cells and co-localized with lysosomes (yellow fluorescence);

24 h: most of Trp2/LDH/IDO-siRNA escaped from lysosomes, diffused into cytoplasm and enriched around nucleus.

Example 5Trp2/LDH/IDO-siRNA vaccine downregulated DC cell mRNA levels

2 x 10 to⁶Individual mouse BMDC cells were plated in 12-well cell culture plates and Trp2/LDH/IDO-siRNA nanoparticles were added to a final concentration of 20. mu.g/mL at 37 ℃ with 5% CO₂Culturing for 16h in a concentration environment. Total cellular RNA was extracted as per the RNeasy Lipid Tissue Mini Kit (Qiagen) instructions. The total RNA was reverse transcribed into cDNA using PrimeScripTM RT-PCR (TaKaRa) kit. IDO gene expression was detected using SYBR Select Master Mix (Applied Biosystems) fluorescent probe and 7500-fast real-time quantitative PCR instrument (Applied Biosystems), GAPDH was used as an internal control. The primer sequence is as follows: IDO: 5'-GGGCTTTGCTCTACCACATCCACT-3' and 5'-ACATCGTCATCCCCTCGGTTCC-3', GAPDH: 5'-TGATGACATCAAGAAGGTGGTGAA-3', and 5'-TGGGATGGAAATTGTGAGGGAGAT-3'.

The results are shown in figure 4, Trp2/LDH/IDO-siRNA nanoparticle vaccine was able to down-regulate IDO mRNA levels in significant mouse BMDC cells.

Example 6Trp2/LDH/IDO-siRNA downregulation of DC cell IDO protein levels

2 x 10 to⁶Mouse BMDC cells were plated in 12-well cell culture plates and Trp2/LDH/IDO-s were addedThe iRNA nanoparticles were brought to a final concentration of 20. mu.g/mL at 37 ℃ with 5% CO₂Culturing for 16h in a concentration environment. Cells were lysed using RIPA lysate, proteins were separated using a 12% polyacrylamide gel, and then the proteins were electroporated onto nitrocellulose membranes. Blocking was performed with 5% skim milk at room temperature for 2h, adding IDO antibody (1:200, Millipore, Billerica, MA) and α -tubulin antibody (1:1000, Sigma, Germany) as primary antibodies, incubating overnight at 4 deg.C, and hybridizing with horseradish peroxidase-labeled goat anti-mouse antibody as secondary antibody. Optical density was analyzed using an Odyssey LICOR SA two-color infrared laser imaging system (LICOR, USA).

The results are shown in FIGS. 5(A) -5(B), in which the levels of IDO protein in the DC cells treated with Trp2/LDH/IDO-siRNA were significantly reduced compared to the control cells.

Example 7Trp2/LDH/IDO-siRNA nanoparticle vaccine enhances the cellular immune response in mice

Each of the immunized mice inoculated with B16-F10 tumor cells was surgically splenomed after euthanasia on day 20, ground, and filtered with a 70- μm nylon cell filter (BD Biosciences, USA) to obtain spleen cells, followed by 5% CO at 37 deg.C₂Culturing for 48h in the environment. Spleen cells were labeled with mouse monoclonal antibodies CD3(PE/Cy7-labelled), CD8(Pacific blue TM-labelled) or CD4(FITC-labelled), IFN-. gamma. (APC-labelled) and IL-4(PE-labelled), and analyzed for CD8 in spleen cells by BD Arial III flow cytometer⁺/IFN-γ⁺Cell ratio. IFN- γ levels in cell culture supernatants were analyzed using an IFN- γ ELISA kit (Neobioscience, China).

The results are shown in FIG. 6(A), and Trp2/LDH/IDO-siRNA nanoparticle vaccine can significantly improve CD8 in mice⁺/IFN-γ⁺The cell proportion reaches 0.55 percent. Control group LDH, free Trp2, free Trp2+ IDO-siRNA, LDH/IDO-siRNA, Trp2/LDH mice in spleen cells CD8⁺/IFN-γ⁺The cell ratios were 0.2%, 0.29%, 0.21%, 0.34% and 0.49%, respectively.

FIG. 6(B) shows that in each immunized group of mice, spleen cells of mice using the Trp2/LDH/IDO-siRNA nanoparticle vaccine expressed IFN- γ at a level of 1148 pg/mL. Control LDH, free Trp2, free TThe spleen cells of the rp2+ IDO-siRNA, LDH/IDO-siRNA and Trp2/LDH mice express IFN-gamma levels of 438pg/mL, 345pg/mL, 680pg/mL, 789pg/mL and 756pg/mL respectively. The level of IFN-gamma expression of spleen cells of mice treated by the Trp2/LDH/IDO-siRNA nanoparticle vaccine is obviously higher than that of mice in a control group (II)^*P<0.05)。

Example 8Trp2/LDH/IDO-siRNA nanoparticle vaccine significantly inhibited melanoma growth in mice

Female C57/BL mice (Wafunkang Bio, China) aged 8 weeks old were inoculated subcutaneously in the thoracic cavity with 2X 10B 16-F10 melanoma cells⁶Each cell (resuspended in 50. mu.L of 10mM HEPES solution) was inoculated on day 0. After 4 days of feeding, the growth condition of the melanoma on the thoracic epidermis of the mice is observed, and the mice with good tumor growth uniformity (standard that the black spots grow without dispersion and the shape is close to a circle) are randomly selected to be divided into 6 test groups, and each group comprises 6 mice. On day 4, groups of mice were injected with either control or drug in groups. The administration content and the dose are respectively as follows: (1) 500. mu.g LDH, (2) 100. mu.g free Trp2, (3) a mixture of 100. mu.g free Trp2 and 30. mu.g free IDO-siRNA, (4) LDH/IDO-siRNA containing 30. mu.g IDO-siRNA and 500. mu.g LDH, (5) Trp2/LDH containing 100. mu.g Trp2 and 500. mu.g LDH, (6) Trp2/LDH/IDO-siRNA containing 100. mu.g Trp2, 500. mu.g LDH and 30. mu.g IDO-siRNA. The drug was prepared using 200. mu.L of HEPES (10mM) solution and injected subcutaneously into the groin on both sides of the mouse at two points by 100. mu.L each. From day 10, the tumor volume of the mice was measured every 2 days using an electronic vernier caliper (the length of the long side and the length of the wide side of the tumor were measured, respectively, according to the formula: tumor volume ═ length of long side × length of wide side²And calculating to obtain the tumor volume).

The results are shown in FIG. 7, and the inhibitory capacities of Trp2/LDH/IDO-siRNA, LDH/IDO-siRNA with intercalated IDO-siRNA alone, and Trp2/LDH with adsorbed Trp2 alone on melanoma growth were 75%, 33% and 48%, respectively, compared with LDH without drug. The results show that the Trp2/LDH/IDO-siRNA nanoparticles co-delivering IDO-siRNA and Trp2 have the capability of obviously inhibiting the growth of melanoma, and the effect is obviously better than that of LDH nanoparticles loaded with IDO-siRNA or Trp2 (the product is shown in the specification) (the product is a mixture of the LDH nanoparticles and the LDH nanoparticles)^*P<0.05)。

The applicant states that the present invention is illustrated by the above examples to the drug-loaded polymer nano-micelle, the preparation method and the application thereof, but the present invention is not limited by the above examples, that is, the present invention is not meant to be implemented only by relying on the above examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

1. A nanoparticle is characterized in that a layered double-hydroxyl composite metal oxide is used as a carrier, and the carrier is loaded with small interfering RNA and epitope peptide, wherein the small interfering RNA is inserted into the carrier, and the epitope peptide is adsorbed on the surface of the carrier;

the small interfering RNA is indoleamine 2,3-dioxygenase small interfering RNA; the sense strand of the small interfering RNA is shown as SEQ ID NO.1, and the antisense strand of the small interfering RNA is shown as SEQ ID NO. 2;

the epitope peptide is any one or the combination of at least two of tyrosinase epitope peptide, Melan-A/MART-1 epitope peptide, gp100 epitope peptide, chicken egg albumin, HER-2/neu tumor-associated antigen or MUC1 antigen peptide.

2. The nanoparticle according to claim 1, wherein the amino acid sequence of the tyrosinase epitope peptide is shown in SEQ ID No. 3.

3. A method for preparing nanoparticles according to claim 1 or 2, comprising the steps of:

(1) preparing a dihydroxy composite metal oxide;

4. The method according to claim 3, wherein the step (1) of preparing the dihydroxy composite metal oxide comprises the following steps:

mixing magnesium nitrate with the molar concentration of 0.01-0.1M and aluminum nitrate with the molar concentration of 0.01-0.1M, adding the mixture into a sodium hydroxide solution with the molar concentration of 0.01-0.1M, stirring for 10-20min, centrifuging for 1-10min at 12000, shaking and uniformly mixing for 20-35min at 30 ℃ with carbonated water, transferring the mixture to a dialysis bag for dialysis for 1-10h, transferring the mixture to a closed container for dialysis for 1-10h, shaking and suspending at 100-400rpm for 0.5-3h, transferring the mixture to a polytetrafluoroethylene hydrothermal kettle for 100-130 ℃ constant temperature for 10-20h, and naturally cooling to obtain the dihydroxy composite metal oxide.

5. The method according to claim 4, wherein the magnesium nitrate is present at a molar concentration of 0.03M.

6. The method according to claim 4, wherein the molar concentration of the aluminum nitrate is 0.01M.

7. The method according to claim 4, wherein the molar concentration of the sodium hydroxide solution is 0.15M.

8. The method according to claim 4, wherein the stirring time is 15 min.

9. The method of claim 4, wherein the centrifugation time is 5 min.

10. The preparation method of claim 4, wherein the shaking and blending time is 30 min.

11. The method of claim 4, wherein the dialysis time is 5 hours.

12. The preparation method according to claim 4, wherein the rotation speed of the suspension by shaking is 280 rpm.

13. The preparation method according to claim 4, wherein the suspension time with shaking is 1 h.

14. The method according to claim 4, wherein the constant temperature is 120 ℃.

15. The method according to claim 4, wherein the constant temperature is maintained for 15 hours.

16. The method according to claim 3, wherein the final concentration of the small interfering RNA of step (2) is 0.01-1. mu.g/. mu.L.

17. The method according to claim 16, wherein the final concentration of the small interfering RNA of step (2) is 0.05-0.5. mu.g/. mu.L.

18. The method according to claim 17, wherein the final concentration of the small interfering RNA of step (2) is 0.08-0.3. mu.g/. mu.L.

19. The method of claim 18, wherein the final concentration of the small interfering RNA of step (2) is 0.1. mu.g/. mu.L.

20. The method according to claim 3, wherein the temperature of the standing in the step (2) is 30 to 45 ℃.

21. The method of claim 20, wherein the temperature of the standing in the step (2) is 35 to 40 ℃.

22. The method of claim 21, wherein the temperature of the standing in the step (2) is 37 ℃.

23. The method according to claim 3, wherein the standing time in the step (2) is 10 to 30 min.

24. The method according to claim 23, wherein the standing time in the step (2) is 15 to 25 min.

25. The method according to claim 24, wherein the standing time in the step (2) is 20 min.

26. The method according to claim 3, wherein the final concentration of the epitope peptide of step (3) is 0.1 to 10. mu.g/. mu.L.

27. The method according to claim 26, wherein the final concentration of the epitope peptide of step (3) is 0.5 to 5. mu.g/. mu.L.

28. The method according to claim 27, wherein the final concentration of the epitope peptide of step (3) is 0.8 to 3. mu.g/. mu.L.

29. The method according to claim 28, wherein the final concentration of the epitope peptide of step (3) is 1. mu.g/. mu.L.

30. The method according to claim 3, wherein the temperature of the standing in the step (3) is 30 to 45 ℃.

31. The method of claim 30, wherein the temperature of the standing in the step (3) is 35 to 40 ℃.

32. The method of claim 31, wherein the temperature of the standing in step (3) is 37 ℃.

33. The method according to claim 3, wherein the standing time in the step (3) is 20 to 40 min.

34. The method of claim 33, wherein the standing time in the step (3) is 25 to 35 min.

35. The method according to claim 34, wherein the standing time in the step (3) is 30 min.

36. The method according to claim 3, wherein the centrifugation in step (3) is carried out under conditions of 10000-15000 g.

37. The method as claimed in claim 36, wherein the centrifugation in step (3) is carried out under conditions of 11000-13000 g.

38. The method according to claim 37, wherein the centrifugation in step (3) is carried out under 12000 g.

39. The method according to claim 3, wherein the time for the centrifugation in the step (3) is 1 to 10 min.

40. The method of claim 39, wherein the centrifugation of step (3) is carried out for 3-8 min.

41. The method of claim 40, wherein the centrifugation of step (3) is performed for 5 min.

42. A vaccine comprising the nanoparticle of claim 1 or 2.

43. The vaccine of claim 42, further comprising a pharmaceutically acceptable carrier and a water-in-oil emulsion.

44. Use of a nanoparticle according to claim 1 or 2 or a vaccine according to claim 42 or 43 for the preparation of a tumour targeting medicament.

45. The use of claim 44, wherein the tumor is melanoma and/or breast cancer.