CN111544586A - Application of nanoparticles with negative charges as immunomodulators - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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Abstract
The invention belongs to the marine biotechnology, and particularly relates to application of nanoparticles (chitosan derivatives) with negative charges as an immune regulator (activating DCS cell NF-kB protein expression). By a polyelectrolyte complex method, chitosan negative charge derivatives with different molecular weights and different substitution sites and chitosan positive charge derivatives with different molecular weights are magnetically stirred at room temperature to obtain the chitosan derivative nanoparticles with negative charges, the particle size of which is 192.7nm-334.2nm and the potential of which is (-13.2) mV- (-28.2) mV. The chitosan derivative nanoparticles with negative charges on the surface are prepared, and the immunocompetence of the nanoparticles is measured. Provides a certain method and theoretical guidance for the research of chitosan as an immunologic adjuvant in recent years.
Description
Technical Field
The invention belongs to the marine biotechnology, and particularly relates to application of nanoparticles (chitosan derivatives) with negative charges as an immune regulator (activating DCS cell NF-kB protein expression).
Background
The research of the nanoparticles is one of the research hotspots in recent years, and the utilization of the nanoparticles as a drug carrier is an important research direction of various drugs in medicine at present. The electrical property of the nanoparticles has important influence on the properties of the nanoparticles, and the effect and the mechanism of the nanoparticles are determined to a great extent. In previous studies, nanoparticles with positive charges were demonstrated to have superior immune effects. However, the application of the nanoparticles with negative charges as the immunoadjuvant has not been studied yet. The action effect and the action mechanism of the compound are further proved.
Chitosan is difficult to dissolve in water, and particularly, under the condition of large molecular weight, the chitosan is subjected to derivatization to improve the water solubility of the chitosan, so that the research on the chitosan is more, but the research on the preparation of nanoparticles by taking the chitosan as a raw material after derivatization is not complete. Nanoparticles used as immune carriers or vaccine adjuvants in previous researches are mainly positive charge nanoparticles, mainly because the nanoparticles are prepared by using cross-linking agents (such as glutaraldehyde and sodium tripolyphosphate) and the like in the traditional method, and the positively charged nanoparticles are prepared by adding a small amount of negatively charged cross-linking agents into chitosan with positive charge. The safety of the nanoparticles is necessarily reduced by adding excessive cross-linking agent, so that the application range of the nanoparticles is greatly reduced. The preparation of negatively charged nanoparticles has been limited.
Disclosure of Invention
The invention takes the problems into consideration and provides the application of the nanoparticle with negative charge in regulating the immunological activity (activating the expression of NF-kB protein of DCS cells).
In order to achieve the purpose, the technical scheme adopted by the experiment is as follows:
a preparation method of nanoparticles with negative charges comprises the steps of magnetically stirring chitosan negative charge derivatives with different molecular weights and different substitution sites and chitosan positive charge derivatives with different molecular weights for 20-40min at room temperature at 500r-700r/min by a polyelectrolyte composite method to obtain chitosan derivative nanoparticles with negative charges, wherein the particle size of the chitosan derivative nanoparticles is 192.7nm-334.2nm, and the potential of the chitosan derivative nanoparticles is (-13.2) mV- (-28.2) mV, filtering and storing at 4 ℃; the chitosan negative charge derivatives with different molecular weights and different substitution sites and the chitosan positive charge derivatives with different molecular weights are mixed according to the mass ratio of (2-5): 1-3.
The chitosan negative charge derivatives with different molecular weights and different substitution sites are chitosan sulfate with different molecular weights and different substitution sites; the chitosan positive charge derivatives with different molecular weights are chitosan quaternary ammonium salts with different molecular weights;
wherein, the chitosan quaternary ammonium salt and the chitosan sulfate with similar molecular weight are used as the same group for preparing the nano-particles.
The chitosan sulfate with different molecular weights and different substitution sites has the molecular weight of 200kDa or 50kDa and has different substitution sites:
the substitution sites are chitosan sulfate at C2,3, 6-position, C6-position, C3, 6-position, C3-position, C2-position, C2, 3-position or C2, 6-position;
the chitosan quaternary ammonium salt with different molecular weights is 2, 3-epoxypropyl trimethyl ammonium chloride chitosan with the molecular weight of 50kDa or 200 kDa.
The application of the nanoparticle with negative charge in the adjuvant which can activate the expression of DCS cell NF-kB protein after wrapping antigen is used for adjusting the immunocompetence.
The nanoparticle with negative charges wrapping the antigen has the effect of activating the expression of DCS cell NF-kB protein; wherein, the mass ratio of the negative charge chitosan derivative nanoparticles to the antigen is 0.5-2: 1.
the preparation for regulating the immunological activity comprises an antigen and an adjuvant, wherein the mass ratio of the adjuvant to the antigen is 0.5-2: 1; the adjuvant is the negatively charged nanoparticle prepared according to claim 1.
The positive charge derivative and the antigen are mixed evenly, then the negative charge derivative is added, and the antigen is wrapped in the negative charge derivative through electrostatic adsorption, so as to prepare the nano material wrapping the antigen, namely the preparation for regulating the immunocompetence.
The antigen is common standard antigen OVA and inactivated virus or inactivated virus protein and the like.
Furthermore, the chitosan derivative nanoparticles are used for measuring the immunocompetence of dendritic cells. The maximum concentration of the nanoparticles which are not toxic to cells is determined to be 100 mug/mL through cytotoxicity measurement of the uncoated antigen nanoparticles. Through the determination of immune factor expression quantity and cell immune factor secretion quantity, C2,3, 6-substituted sulfate chitosan with molecular weight of 50kDa and 200kDa and C6-substituted sulfate chitosan with molecular weight of 50kDa and 50kDa are preferably selected as negative ion chitosan derivatives, and quaternary ammonium salt chitosan is selected as positive ion chitosan derivatives, at the moment, the sulfate chitosan concentration is 1.5mg/mL and the quaternary ammonium salt concentration is 1.0mg/mL under the condition of preparing the nanoparticles, and the nanoparticles have the best immune effect.
The invention has the advantages that:
1. the invention uses the derivatives of chitosan with positive and negative charges to prepare the nanoparticles without introducing a cross-linking agent, the method removes the toxic and side effects of the cross-linking agent, ensures the safety of the nanoparticles in organisms, and the nanoparticles have biological safety under proper concentration through the detection of cytotoxicity test.
2. The nanoparticles prepared by the invention have negative charges, are different from the prior nanoparticles which are mostly used for immune adjuvants and have positive charges, and show higher immunological activity; furthermore, the surface of the nanoparticle is provided with negative charges, so that the nanoparticle is more beneficial to being combined with cells and entering the cells, so that substances such as encapsulated antigens or target proteins are released, and meanwhile, the nanoparticle is used for verifying the immune effect of mouse DCS cells, and the result shows that most of the nanoparticles can promote the expression and secretion of four cell factors of mice, thereby proving that the prepared nanoparticle has a certain immune effect.
3. The invention determines the influence of the nanoparticles on the protein level of p65 in a DCS cell NF-kB protein family by a Western Blot method (Western Blot test), and shows that the nanoparticles are beneficial to activating and promoting the expression of NF-kB protein, presumably activates dendritic cells by a TLR 4-NF-kB channel, and has good guiding significance in the aspects of preparing high-efficiency immune adjuvant without side reaction and researching subsequent mechanisms thereof.
Drawings
FIGS. 1A and 1B are HPLC charts showing the results of measuring the molecular weight of chitosan having different molecular weights obtained in example 1 of the present invention; wherein A is 200kDa and B is 50 kDa.
FIG. 2 is an infrared spectrum of chitosan quaternary ammonium salt (HACC) and chitosan sulfate derivatives at different sites obtained in example 3 of the present invention.
Fig. 3 is a graph showing the potential (a) and particle size (B) characteristics of chitosan derivative nanoparticles obtained in example 4 of the present invention.
Fig. 4 is a scanning electron microscope image of the chitosan derivative nanoparticles obtained in example 4 of the present invention.
FIG. 5 is a graph showing that the nanoparticles obtained in example 6 of the present invention promote the secretion of NF- κ B protein in DC cells.
Detailed Description
The present invention is further described with reference to the drawings attached to the specification, and the scope of the present invention is not limited to the following examples.
The invention takes chitosan as a basis to prepare the nanoparticles and measure the immunocompetence of the nanoparticles, and finally the chitosan derivative nanoparticles with the function of the immunologic adjuvant are obtained.
The specific method comprises the following steps: preparing nanoparticles from two derivatives with chitosan negative charges and two quaternary ammonium salt derivatives with molecular weights at different substitution sites by a polyelectrolyte composite method to obtain nanoparticles with negative charges; meanwhile, substances capable of causing an organism immune reaction are wrapped in the obtained nanoparticles with negative charges through a cell experiment to obtain the chitosan derivative nanoparticles with the optimal immune activity, the substances prepared from the raw materials under the conditions have no toxic or side effect, and a mouse dendritic cell experiment proves that the gene expression quantity of four immune factors, namely dendritic cells IL-6, TNF-alpha, IL-1 beta and IFN-gamma, can be improved, and the secretion quantity of the four cytokines is increased. The influence of the nanoparticles on the secretion of NF-kB protein of DCS cells is determined by a Western Blot method, which shows that the nanoparticles are beneficial to activating and promoting the expression of the NF-kB protein, dendritic cells can be activated by a TLR 4-NF-kB channel, and a certain method and guidance are provided for the research of taking chitosan derivatives as immune adjuvants in recent years.
EXAMPLE 1 preparation of chitosans of different molecular weights
Taking 6g of raw material chitosan with the molecular weight of 1820kDa and adding 98mL of H2O, 2mL of acetic acid, stirring at 45 ℃ at a speed of 200r/min, adding 0.5g of chitosanase (chitosan degrading enzyme provided by Shandong Weikang biological medicine science and technology Co., Ltd.) after stirring for 1h, and measuring the molecular weight of the chitosan after 1 h. The chitosan with the molecular weight of 200kDa is obtained by purchasing, and the molecular weight of the sample obtained by degradation and the molecular weight of the purchased sample are measured by high performance liquid chromatography, and the obtained result is shown in figure 1.
EXAMPLE 2 preparation of Chitosan Quaternary ammonium salt
5g of chitosan of different molecular weights from the examples described above (chitosan with a molecular weight of 57kDa was used in this example) and 10g of 2, 3-epoxypropyltrimethylammonium chloride were taken. 70mL of distilled water is added into the mixture, and the mixture is stirred in a water bath at the temperature of 80 ℃ under the condition of the rotating speed of 200r/min, and the reaction time is 24 hours. And dialyzing the obtained reaction solution for 72h by using distilled water in a dialysis bag, and freeze-drying in a freeze dryer at the temperature of minus 80 ℃ to obtain a sample a, namely the chitosan quaternary ammonium salt with the molecular weight of 54 kDa.
The chitosan quaternary ammonium salt with the molecular weight of 194kDa can be prepared by adopting chitosan with the molecular weight of 200kDa and 2, 3-epoxypropyltrimethylammonium chloride according to the process.
EXAMPLE 3 preparation of Chitosan sulfate esters at different substitution sites
1) Preparation of chitosan sulfate with different molecular weights at C2,3, 6-position
2g of chitosan of different molecular weights prepared in the previous examples (chitosan of 57kDa was used in this example), 50mL of formamide solvent was added, the mixture was stirred well, 5mL of formic acid and 50mL of sulfonating agent DMF & SO were added3Stirring and reacting for 1.5h at 50 ℃ water bath, then precipitating the reaction solution by using 3 times volume of absolute ethyl alcohol, and precipitating in a refrigerated cabinet at 4 ℃ for about 30min to obtain a precipitate, namely a crude product of chitosan sulfate. Will be at the topFiltering the precipitate, dissolving the filter cake with distilled water, neutralizing with 2N NaOH solution, dialyzing, concentrating the dialyzate, and freeze-drying to obtain light yellow sample b, i.e. C2,3, 6-chitosan sulfate with different molecular weights (the molecular weight of the C2,3, 6-chitosan sulfate obtained in this example is 51kDa, and the infrared characterization is shown in figure 2);
in the same way, chitosan with molecular weight of 200kDa is modified according to the method described above to obtain C2,3, 6-position chitosan sulfate with molecular weight of 199 kDa.
2) Preparation of C3, 6-chitosan sulfate with different molecular weights
4g of chitosan with different molecular weights prepared in the above examples (chitosan with a molecular weight of 57kDa is adopted in the example), 100mL of formamide, 5g of phthalic anhydride and 3mL of ethylene glycol are uniformly mixed, and then reacted at 90 ℃ for 2.5 hours, after the reaction is finished, the temperature is reduced to 55 ℃, and then slowly dropped, 80mL of a sulfonation reagent is reacted for 2.5 hours, after the reaction is finished, the mixture is poured into ice water and neutralized by 2N NaOH to obtain a transparent solution, and the transparent solution is dialyzed by distilled water, concentrated and freeze-dried to obtain a 2-phthaloyl imidoyl chitosan sulfate product. And removing a phthalic anhydride protecting group from the 2-phthalimide chitosan sulfate, fully dissolving 3g of 2-phthalimide chitosan sulfate in 100mL of water, adding 20mL of hydrazine hydrate, reacting at 70 ℃ for 4 hours, adding 100mL of distilled water after the reaction is finished, concentrating, repeating the reaction for four times, dialyzing with distilled water, concentrating, and freeze-drying to obtain a white cotton-shaped solid d. Thus obtaining the C3, 6-chitosan sulfate with different molecular weights (the example obtains the C3, 6-chitosan sulfate with the molecular weight of 50kDa, the infrared characterization is shown in figure 2)
In the same manner, chitosan with molecular weight of 200kDa was modified in the manner described above to obtain chitosan sulfate with molecular weight of 201kDaC3, 6-position.
3) Preparation of C3-chitosan sulfate with different molecular weights
Taking 2g of C3, 6-position chitosan sulfate (the molecular weight of the chitosan sulfate is 50kDa C3, 6-position) obtained in the step 2) with different molecular weights, dissolving the mixture with 50mL of water, adding 160mL of N-methyl-2-pyrrolidone, uniformly mixing, reacting at 90 ℃ for 6h, adjusting the pH value to 9 with NaOH after the reaction is finished, dialyzing, concentrating, and freeze-drying to obtain a light yellow powdery product C; thus obtaining the C3-chitosan sulfate with different molecular weights (the C3-chitosan sulfate with the molecular weight of 50kDa is obtained in the example, and the infrared characterization is shown in figure 2);
in the same manner, the C3, 6-position chitosan sulfate having a molecular weight of 201kDa obtained in step 2) of example 2 was used, and the product obtained by modifying the chitosan sulfate in the manner described above was C3-position chitosan sulfate having a molecular weight of 201 kDa.
4) Preparation of C6-chitosan sulfate with different molecular weights
Firstly, preparing C2, 3-position chitosan copper chelate with different molecular weights: 3g of chitosan (with a molecular weight of 57kDa in this example) prepared in the above examples was added to 80mL of 1% formic acid to dissolve it in a viscous state, and 10mL of distilled water was taken to dissolve 4.65g of CuSO4·5H2O was slowly added dropwise to the above viscous solution, and stirred at room temperature for 3 hours. Then adjusting the pH value to 6.0-6.5 by ammonia water, and continuously stirring for 3 hours at room temperature. After stirring, precipitating the chelate by using a mixed solvent of absolute ethyl alcohol and acetone in a volume ratio of 1:1, standing for a little time, carrying out suction filtration, drying and crushing for later use.
Then sulfonation reaction is carried out, 2g of chitosan copper chelate is taken, 50mL of formamide and 30mL of sulfonating agent DMF & SO are added3Stirring and reacting at 55 ℃ for 1.5h, adding 3 times volume of absolute ethyl alcohol into the reaction solution, precipitating, and standing in a refrigerated cabinet at 4 ℃ for about 30min to obtain white flocculent precipitate. And (3) carrying out suction filtration on the precipitate, dissolving a filter cake with distilled water to obtain a blue solution, neutralizing the blue solution with a 2N NaOH solution, dialyzing, concentrating the dialyzate, and freeze-drying to obtain a sulfated product of the blue chitosan copper chelate. Finally, decoppering treatment is carried out, the sulfonated product of the chitosan copper chelate is dissolved by distilled water and passes through a strongly acidic styrene cation exchange resin column, the color of the solution is changed from blue to yellow, Cu is removed, 2N NaOH is used for neutralization, concentration, dialysis and freeze drying are carried out, thus obtaining sample e, namely C6-chitosan sulfate with different molecular weights (the obtained component in the example isThe molecular weight is 51kDa C6-position chitosan sulfate, and the infrared characterization is shown in figure 2).
In the same manner, chitosan having a molecular weight of 200kDa in example 1 was used, and the resulting sulfate ester of chitosan having a molecular weight of 198kDa, C6-position, was modified in the manner described above. Meanwhile, according to the records of the prior art, the sulfate ester of chitosan at C2-position, the sulfate ester of chitosan at C2, 3-position and the sulfate ester of chitosan at C2, 6-position can be obtained, and meanwhile, the protection method and the protective agent of the group are adjusted in each preparation process to protect and derive different sites. (Holme et al, 1997; Nishimura et al, 1998; Jayakumar et al, 2007) using the chitosans of different molecular weights obtained in example 1, the above-mentioned different substitution positions of chitosan sulfate esters of different molecular weights were obtained by the same derivatization.
Example 4 preparation of nanoparticles of chitosan derivative having negative charge
Reagent: 1.0mg/mL of chitosan quaternary ammonium salt solution with different molecular weights, 1.5mg/mL of C2,3, 6-sulfate chitosan solution with different molecular weights and 2.0mg/mL of standard antigen OVA solution m;
taking 3mL of chitosan quaternary ammonium salt solution with different molecular weights in a 25 mL beaker, and placing the beaker in a magnetic stirrer at the rotating speed of 300 r/min. 2mL of antigen m solution is added dropwise, and after stirring for 10min, 3mL of C2,3, 6-sulfate chitosan solution with different molecular weights are added dropwise. Namely the mass ratio of positive charge derivatives to negative charge derivatives is 2: 3. Stirring is continued for 30 min. Filtering to obtain a nanoparticle solution, storing at 4 ℃ to obtain nanoparticles wrapping the antigen chitosan derivative, and determining the particle size range of the nanoparticles as follows: 190.2nm-334.2nm, potential range (1.05) mV- (-28.2) mV (see Table 1)
According to the recording method, the C2,3, 6-sulfate chitosan solutions with different molecular weights are replaced by sulfate chitosan with different substitution sites with different Molecular Weights (MW), so that the chitosan derivative nanoparticles with different molecular weights and different substitution sites for coating the antigen can be obtained, and the physical and chemical property characterization of the nanoparticles is shown in Table 1.
TABLE 1 characterization of chitosan derivative nanoparticles with different molecular weights and different substitution sites for envelope antigen
Note: in the table, the molecular weights of the chitosan positive charge derivative, namely the quaternary ammonium salt chitosan and the chitosan sulfate are the same.
Example 5 determination of the immunoreactivity of negatively charged Chitosan derivative nanoparticles
1) Cytotoxicity assay, blank nanoparticles (i.e., chitosan derivative nanoparticles that do not encapsulate antigen) and the nanoparticles coated with the antigen chitosan derivative obtained in the above examples (nanoparticles prepared above, nanoparticles with lower charge were discarded). Selecting nanoparticles with moderate and stable charge) and testing the toxicity of the nanoparticles on DC cells according to a CCK-8 method, as shown in tables 3 and 4.
The blank nanoparticles (i.e., chitosan derivative nanoparticles that do not encapsulate an antigen) were prepared according to the procedure described in example 4 above, wherein the mass ratio of the negative chitosan charge derivatives with different molecular weights and different substitution sites to the positive chitosan charge derivatives with different molecular weights was 3: 2.
Table 3 toxicity testing of blank nanoparticles on DC cells
Note: in the table, the molecular weights of the chitosan positive charge derivative, namely the quaternary ammonium salt chitosan and the chitosan sulfate are the same.
TABLE 4 toxicity test of encapsulated antigen nanoparticles on DC cells
Note: in the table, the molecular weights of the chitosan positive charge derivative, namely the quaternary ammonium salt chitosan and the chitosan sulfate are the same.
As can be seen from the above tables 3 and 4, most of the blank nanoparticles are not toxic to cells within 100 μ g/mL, and the encapsulated antigen nanoparticles are also not toxic to cells within 100 μ g/mL; the subsequent immunological activity measurement can select 100 microgram/mL concentration for blank nanometer particle and 100 microgram/mL concentration for packed antigen nanometer particle.
2) Measuring the expression quantity of cell factors, namely testing the influence of chitosan derivative nanoparticles with positive and negative charges, which are prepared in the embodiment and have the mass ratio of negative and positive charges, on the expression quantities of four immune factors IL-6, TNF-alpha, IL-1 beta and IFN-gamma of DC cells by fluorescence quantitative PCR, wherein the chitosan derivative nanoparticles with negative charge and two molecular weights wrap antigens, and the experimental results are shown in Table 5; wherein the mass ratio of the chitosan negative charge derivatives with different molecular weights and different substitution sites to the chitosan positive charge derivatives with different molecular weights is 3: 2.
The fluorescent quantitative PCR: (firstly, an RNA extraction kit is used for extracting total RNA, a NanoDrop 2000 ultramicro ultraviolet \ visible spectrophotometer is used for measuring the RNA content and the RNA quality after the extraction is finished, and then subsequent RNA reversion and quantitative test are carried out, wherein the test PCR test conditions comprise Stage 2, 5s at 95 ℃, 34s at 60 ℃, Stage 3, 15s at 95.)
TABLE 5 influence of two molecular weights and different substitution sites of chitosan derivatives to obtain antigen-coated chitosan derivative nanoparticles on DC cell gene expression level
Note: in the table, the molecular weights of the chitosan positive charge derivative, namely the quaternary ammonium salt chitosan and the chitosan sulfate are the same.
Example 6 measurement of protein level of negatively charged Chitosan derivative nanoparticles p65
The DC cells were cultured in DMEM so that the cell concentration in the culture system was 1 × 105 Setting 6 groups of cells/mL in total, and then replacing culture media of each group when the cells of each group are attached to the wall stably, wherein the culture media replaced by each group are respectivelyDMEM is used as a negative control group, OVA is used as a positive control group, the DMEM culture medium is used as an experimental group, the C2.3.6SCS-HACC with 200kDa, the C2.3.6SCS-HACC with 50kDa, the C3.6 SCS-HACC with 50kDa and the C6 SCS-HACC nano-particles with negative charge coated antigen (OVA) prepared by the preparation are respectively added into the DMEM culture medium, the concentration of the final nano-particles in each system is 100 mu g/mL, and the total proteins of each group are respectively extracted for subsequent determination after 24h of culture.
The steps for extracting total protein were as follows: adding RIPA lysate, protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and a small spoon of glass beads into each tube, fully shaking, centrifuging at 12000rpm at 4 ℃ for 15min, taking the supernatant as a protein extracting solution, and measuring the protein concentration in the supernatant by using a BCA protein concentration measuring kit. The detection steps are as follows: the extracted protein was boiled and denatured, and then spotted with 40. mu.g of nuclear protein per well, followed by SDS-PAGE electrophoresis. The protein samples on the gel were then transferred to PVDF membrane, then the membrane was washed once with 1 × TBS and blocked with 3% BSA for 1h at room temperature. And washing the membrane for 3 times by using TTBS, respectively incubating with a primary antibody and a secondary antibody marked by horseradish catalase, adding luminous liquid prepared by luminol and hydrogen peroxide according to a certain proportion after washing the membrane, and then exposing and photographing.
The experimental result is shown in fig. 5, which shows that the negative charge nanoparticles prepared from the chitosan derivative can promote the expression of p65, while p65 is an important member of NF- κ B, and the secretion of p65 protein is improved compared with that of the control group under the same time condition. The prepared negative charge nanoparticles can promote nuclear transcription factor NF-kB to enter the nucleus, as shown in figure 5. Therefore, the results prove that the chitosan derivative negative charge nanoparticles can activate a PI3K/Akt pathway of cells and promote a transcription factor NF-kB to enter a core, thereby increasing the expression of cytokines such as TNF-alpha and the like.
Claims (7)
1. The preparation method of the nanoparticle with negative charge is characterized by comprising the following steps: through a polyelectrolyte complex method, chitosan negative charge derivatives with different molecular weights and different substitution sites and chitosan positive charge derivatives with different molecular weights are magnetically stirred for 20-40min at room temperature at 500r-700r/min to obtain chitosan derivative nanoparticles with negative charges, the particle size of which is 192.7nm-334.2nm and the potential of (-13.2) mV- (-28.2) mV, and the chitosan derivative nanoparticles are filtered and stored at 4 ℃; the chitosan negative charge derivatives with different molecular weights and different substitution sites and the chitosan positive charge derivatives with different molecular weights are mixed according to the mass ratio of (2-5): 1-3.
2. The preparation of negatively charged nanoparticles according to claim 1, characterized in that: the chitosan negative charge derivatives with different molecular weights and different substitution sites are chitosan sulfate with different molecular weights and different substitution sites; the chitosan positive charge derivatives with different molecular weights are chitosan quaternary ammonium salts with different molecular weights.
3. The method of claim 2, wherein the nanoparticle of chitosan derivative with negative charge comprises: the chitosan sulfate with different molecular weights and different substitution sites has the molecular weight of 200kDa or 50kDa and has different substitution sites:
the substitution sites are chitosan sulfate at C2,3, 6-position, C6-position, C3, 6-position, C3-position, C2-position, C2, 3-position or C2, 6-position;
the chitosan quaternary ammonium salt with different molecular weights is 2, 3-epoxypropyl trimethyl ammonium chloride chitosan with the molecular weight of 50kDa or 200 kDa.
4. Use of negatively charged nanoparticles prepared by the method of claim 1 for modulating immune activity, wherein: the application of the nanoparticle (wrapped antigen) with negative charge prepared by the method of claim 1 as an adjuvant for activating the expression of DCS cell NF-kB protein.
5. Use according to claim 4, characterized in that: the nanoparticle with negative charges wrapping the antigen has the effect of activating the expression of DCS cell NF-kB protein; wherein, the mass ratio of the negative charge chitosan derivative nanoparticles to the antigen is 0.5-2: 1.
6. a formulation for modulating immune activity, comprising: the preparation comprises an antigen and an adjuvant, wherein the mass ratio of the adjuvant to the antigen is 0.5-2: 1; the adjuvant is the negatively charged nanoparticle prepared according to claim 1.
7. The formulation for modulating immune activity of claim 6, wherein: the chitosan derivative with positive charge of the nanoparticle with negative charge prepared in the method of claim 1 is mixed with antigen uniformly, then the derivative with negative charge is added, and antigen is wrapped in the mixture through electrostatic adsorption, so as to prepare the nano material for wrapping antigen, namely the preparation for regulating immunological activity.
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