CN113648405B - Oral recombinant helicobacter pylori protein vaccine nanoparticle and preparation method thereof - Google Patents
Oral recombinant helicobacter pylori protein vaccine nanoparticle and preparation method thereof Download PDFInfo
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Abstract
The present invention belongs to the field of medicine preparation technology. The invention relates to recombinant protein vaccine alpha 1, 3-fucosyltransferase nano-particles and a preparation method thereof. The recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticle prepared by the invention not only can improve the absorption of alpha 1, 3-fucosyltransferase in the body intestine, but also can improve the capacity of taking alpha 1, 3-fucosyltransferase by macrophages, and can be used for preventing and treating infection caused by helicobacter pylori.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to oral recombinant helicobacter pylori protein vaccine nanoparticles and a preparation method thereof.
Background
Helicobacter pylori infection can cause gastroduodenal diseases, vaccines are widely adopted worldwide as a preventive and therapeutic method, but vaccines are often required to be administered by injection, patient compliance is poor, and oral administration is the most compliant route of administration for patients. The recombinant protein vaccine alpha 1, 3-fucosyltransferase can help helicobacter pylori to evade host immune monitoring, plays an important role in the field planting and long-term infection of helicobacter pylori, but has the defects of poor permeability, difficult absorption by intestinal tracts, difficult uptake by antigen presenting cells (macrophages and the like) in lymphoid tissues and the like, and limits the wide clinical application of the medicine.
The vaccine nanoparticle has advantages over conventional dosage forms of vaccines. The auxiliary material polyester material polylactic acid-glycolic acid copolymer is a high molecular compound formed by polymerizing lactic acid and glycolic acid, has good degradability, and can be used as a carrier to load antigen to cause mucosal immune reaction. Hydroxypropyl methylcellulose phthalate as an enteric material encapsulates protein drugs is beneficial to help vaccines overcome the harsh gastrointestinal environment. The 2-hydroxypropyl trimethyl ammonium chloride chitosan can open epithelial cells to be tightly connected, helps antigens to pass through an epithelial layer, and has a vaccine adjuvant function. In the course of the study of the present invention, ovalbumin was used as a model antigen because the molecular weight and isoelectric point (43 kDa, 4.71) of ovalbumin were similar to those of alpha 1, 3-fucosyltransferase (53 kDa, 5.25, respectively).
No research report of oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nano-particles is available at present through inquiring patents and documents. The nanoparticle of the invention not only can improve the in vivo intestinal absorption of alpha 1, 3-fucosyltransferase and model antigen, but also can improve the capacity of macrophage to take in alpha 1, 3-fucosyltransferase and model antigen. The invention prepares the oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nano-particles, which can be used for preventing and treating infection caused by helicobacter pylori.
Disclosure of Invention
The invention aims to provide an oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticle formula and a preparation method thereof. The oral recombinant protein vaccine alpha 1,3 fucosyltransferase nano-particles overcome the defects of poor permeability of alpha 1, 3-fucosyltransferase, poor intestinal absorption, difficult uptake by antigen presenting cells (macrophages and the like) in lymphoid tissues and the like, and the nano-particles not only can improve the in vivo intestinal absorption of alpha 1, 3-fucosyltransferase and model antigens, but also can improve the capacity of the macrophages to uptake the alpha 1, 3-fucosyltransferase and the model antigens.
The invention provides an oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticle, which is characterized in that the content of alpha 1, 3-fucosyltransferase in a preparation is 1.2-6.0mg/mL, and the weight ratio of the rest components in the preparation is as follows: 0.2-0.8 part of polylactic acid-glycolic acid copolymer, 0.1-0.4 part of hydroxypropyl methyl cellulose phthalate, 0.01-0.04 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.3-1.2 parts of emulsifier and 50-100 parts of distilled water. The preparation process of the oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticle provided by the invention comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate in one or two or three mixtures of dichloromethane, acetone or trichloromethane to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving a prescribed amount of emulsifying agent in distilled water, dissolving a prescribed amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the emulsifying agent aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 18-36 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
The invention provides an oral recombinant protein vaccine alpha 1, 3-fucosylation transferThe average particle size of the enzyme nanoparticle was 175.70nm, less than 200nm (FIG. 1). Compared with the fluorescein isothiocyanate-marked alpha 1, 3-Fucosyltransferase (FT), the hydroxypropyl methyl cellulose phthalate coated polylactic acid-glycolic acid copolymer with the 2-hydroxypropyl trimethyl ammonium chloride chitosan is added as the skeleton, and the fluorescein isothiocyanate-marked alpha 1, 3-fucosyltransferase nanoparticle (HCFTN) has the drug absorption rate constant (K) in the intestinal tract a ) The increases in the duodenum, jejunum, ileum and colon compared to FT were 1.84, 1.34, 4.64 and 5.02 times, respectively. Compared with fluorescein isothiocyanate-labeled model antigen (FO), the K-type nanoparticle (HCFON) of fluorescein isothiocyanate-labeled model antigen (HCFON) with poly (lactic-co-glycolic acid) coated by hydroxypropyl methyl cellulose phthalate and added with 2-hydroxypropyl trimethyl ammonium chloride chitosan as skeleton a The increases in the duodenum, jejunum, ileum and colon compared to FO were 1.76, 1.29, 4.27 and 5.03 times, respectively. HCFON (HFON) without added 2-hydroxypropyl trimethyl ammonium chloride chitosan in intestinal tract K a The increases in the duodenum, jejunum, ileum and colon compared to FO were 1.21, 1.13, 2.05 and 1.84 fold, respectively (fig. 2A). Drug effective permeability coefficient (P) of HCFTN in intestinal tract eff ) The increases in the duodenum, jejunum, ileum and colon were 2.66, 1.31, 3.89 and 8.21 fold respectively compared to FT. HCFON in intestinal tract P eff The increases in the duodenum, jejunum, ileum and colon were 2.71, 1.39, 4.89 and 21.18 times, respectively, compared to FO. Drug P of HFON in intestinal tract eff The increases in the duodenum, jejunum, ileum and colon compared to FO were 1.41, 1.14, 2.98 and 12.29 fold, respectively (fig. 2B). The result shows that after the 2-hydroxypropyl trimethyl ammonium chloride chitosan is added, the K of the oral recombinant protein vaccine alpha 1, 3-fucosyltransferase and model antigen nanoparticle in the intestinal tract can be obviously increased a And P eff . K of alpha 1, 3-fucosyltransferase and model antigen in the intestinal tract a And P eff The cause of the significant increase was analyzed as follows: (1) 2-hydroxypropyl trimethyl ammonium chloride chitosan and hydroxypropyl methyl cellulose phthalate coated polylactic acid-glycolic acid copolymer are taken as the nano particles of the framework, so that the stability and activity of alpha 1, 3-fucosyltransferase are improved; (2) Hydroxy groupThe propyl methyl cellulose phthalate serving as an enteric material for coating protein medicines is beneficial to helping vaccines to overcome the severe gastrointestinal tract environment; (3) The 2-hydroxypropyl trimethyl ammonium chloride chitosan has gastrointestinal protection effect on protein vaccine, and can be used as vaccine adjuvant to enhance immune response. The chitosan has slow release property, low toxicity, biodegradability and the like, can enhance the humoral and cellular immunity of a host, and is a potential vaccine adjuvant candidate. However, chitosan has limited use due to poor solubility in water, and in preliminary experiments we have tried to prepare chitosan and hydroxypropyl methylcellulose phthalate coated α1, 3-fucosyltransferase polylactic acid-glycolic acid copolymer nanoparticles with chitosan, but the measurement of characteristics such as appearance, particle size, conductivity, etc. indicates that such nanoparticles cannot be successfully prepared. In order to improve the water solubility of chitosan, the chitosan is dissolved in an aqueous phase in polylactic acid-glycolic acid copolymer nanoparticles, the chitosan is subjected to quaternization modification to obtain 2-hydroxypropyl trimethyl ammonium chloride chitosan, and the alpha 1, 3-fucosyltransferase polylactic acid-glycolic acid copolymer nanoparticles coated by the 2-hydroxypropyl trimethyl ammonium chloride chitosan and hydroxypropyl methyl cellulose phthalate are further prepared.
Antigen uptake by antigen presenting cells (e.g., macrophages) is a critical step in eliciting immune responses in the organism, and after incubation of mouse macrophages RAW 264.7 with FO, FT, HFON, HCFON and HCFTN for 12h, 24h, 48h, respectively, the relative cell viability was above 80%, indicating that FO, FT, HFON, HCFON and HCFTN have substantially no effect on macrophage activity within 48h at 20 μg/mL, initially demonstrating that FO, FT, HFON, HCFON and HCFTN are better for macrophage safety (FIG. 3). The uptake of HCFTN by macrophages was significantly higher than that of FT by macrophages, and hcpon by macrophages (fig. 4). Experimental results show that the uptake of both FO and hcon by macrophages at 37 ℃ is concentration dependent over a range of concentrations (figure 5). The ability of oral recombinant protein vaccine nanoparticles to enhance FT and FO uptake by macrophages may be due to: (1) The antigen is embedded in the polymer nano particles coated by the 2-hydroxypropyl trimethyl ammonium chloride chitosan and the enteric material, so that the antigen degradation can be prevented, and the durability of the antigen can be enhanced: (2) The d-glucosamine and N-acetyl-d-glucosamine units of 2-hydroxypropyl trimethyl ammonium chloride chitosan can strongly bind to the C-type sugar recognition domain on the surface of macrophages, thereby increasing the uptake of the nanoparticle preparation by macrophages.
The present invention is different from the oral vaccine delivery nanosystems and preparation processes reported in general research. Oral vaccine delivery nanosystems studies report liposome, bilosome, immune complexes, complex emulsions, polymer particles, adenovirus delivery. The oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticle is a novel polyester nanoparticle, and chitosan has unique properties such as low toxicity, good biocompatibility and biodegradability, can enhance humoral and cellular immunity of a host, however, the application of the chitosan is greatly limited by the poor water solubility of the chitosan. In order to improve the water solubility of chitosan, the chitosan is dissolved in the water phase in polylactic acid-glycolic acid copolymer nano particles, and the chitosan is subjected to quaternization modification to obtain the 2-hydroxypropyl trimethyl ammonium chloride chitosan. The 2-hydroxypropyl trimethyl ammonium chloride chitosan has better water solubility than chitosan, has gastrointestinal protection effect on protein vaccine and can be used as vaccine adjuvant to enhance immune response, and can be better applied to vaccine preparation. There are only a few reports of the use of 2-hydroxypropyl trimethylammonium chloride chitosan in the delivery of veterinary vaccines. For example, 2-hydroxypropyl trimethyl ammonium chloride chitosan improves immunogenicity of porcine parvovirus inactivated vaccine to sow after intramuscular injection administration, and the auxiliary material components contained in the nanoparticle mainly comprise: 2-hydroxypropyl methyl ammonium chloride chitosan (the volume ratio of virus solution to 2-hydroxypropyl methyl ammonium chloride chitosan is 2000:1), beta propiolactone (the volume ratio of virus solution to beta propiolactone is 2000:1) and mineral oil, wherein the preparation method of the nanoparticle comprises the following steps: microemulsion method (Zhou M, et al water-soluble N-2-Hydroxypropyl trimethyl ammonium chloridechitosan enhanced the immunogenicity of inactivated porcine parvovirus vaccinevaccination on sows against porcine parvovirus in reaction. Immunol Lett.2020; 223:26-32.); the attenuated live vaccine for resisting newcastle disease and infectious bronchitis carried by the 2-hydroxypropyl trimethyl ammonium chloride chitosan nanoparticle increases the humoral, cellular and mucosal immune response of pathogen chicken through intranasal administration, and the auxiliary material components contained in the nanoparticle mainly comprise: 2-hydroxypropyl trimethyl ammonium chloride chitosan (1.0 mg/mL) and N, O-carboxymethyl chitosan (0.8 mg/mL), the preparation method of the nanoparticle is as follows: polyelectrolyte complex methods (Zhao K, et al, quantized chitosan nanoparticles loaded with thecombined attenuated live vaccine against newcastle disease and infectious bronchitis elicitimmune response in chicken after intranasal administation. Drug Deliv.2017;24 (1): 1574-1586.); the newcastle disease live vaccine carried by the 2-hydroxypropyl methyl ammonium chloride chitosan nanoparticle enhances pathogen chicken cells, body fluid and mucosa immune response through oral administration, and the auxiliary material components contained in the nanoparticle mainly comprise: 2-hydroxypropyl methyl ammonium chloride chitosan (1.2 mg/mL), phosphate buffer (PBS, pH 7.2), span-80 and sodium tripolyphosphate solution (1.5 mg/mL), the preparation method of the nanoparticle is as follows: ionic crosslinking (Zhao K, et al biological evaluation of N-2-hydroxypropyl trimethyl ammonium chloride chitosan as a carrier for the delivery of livenewcastle disease vaccinee. Carbohydrate Polym.2016; 149:28-39). At present, no research on delivering human vaccines by using 2-hydroxypropyl trimethyl ammonium chloride chitosan as a vaccine adjuvant and a carrier material is seen. At present, no report of delivering oral recombinant helicobacter pylori protein vaccine by using 2-hydroxypropyl trimethyl ammonium chloride chitosan as a vaccine adjuvant and a carrier material is yet seen. According to the invention, polylactic acid-glycolic acid copolymer is used as a carrier material for the first time, 2-hydroxypropyl trimethyl ammonium chloride chitosan is used as a vaccine adjuvant and a carrier material, hydroxypropyl methylcellulose phthalate is used as an enteric material to prepare the oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticle, and the nanoparticle can not only improve the in vivo intestinal absorption of alpha 1, 3-fucosyltransferase and model antigen, but also improve the capacity of taking the alpha 1, 3-fucosyltransferase and the model antigen by macrophages. The oral recombinant helicobacter pylori protein vaccine nanoparticle can be used for preventing and treating infection caused by helicobacter pylori.
Drawings
FIG. 1 is a particle size measurement test of oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nanoparticles prepared by the present invention.
Test conditions: particle size was measured using a malvern laser particle sizer.
Study results: the average particle size of the oral recombinant protein vaccine alpha 1, 3-fucosyltransferase nano-particles is 175.70nm and less than 200nm.
FIG. 2 shows the absorption rate constants K of FO, FT, HFON, HCFON and HCFTN prepared according to the present invention a (Panel A) and effective permeation coefficient P eff (Panel B) measurement test.
Test conditions: 15 SD male rats (no water supply after 12h of fasting before administration) were anesthetized with 7% chloral hydrate, and the rat plates were fixed to expose the abdominal cavity. The duodenal segment, jejunal segment, ileal segment, and colonic segment are found in sequence. Taking 10cm of each intestinal segment, respectively inserting tubes at two ends of the intestinal segment for ligation, and washing the intestinal content with 37 ℃ physiological saline. The blank Krebs-Ringer circulation liquid is balanced for 10min at a flow rate of 0.4mL/min and then is replaced by a medicine-containing circulation liquid (83.33 mug/mL), the circulation liquid is collected after 1h (0.2 mL/min), the residual liquid medicine in the intestinal tract is collected, and the length and the inner diameter of each intestinal tract are measured after the test is finished. Fluorescence spectrophotometry is used for respectively detecting the content of the alpha 1, 3-fucosyltransferase marked by the fluorescein isothiocyanate and the content of the model antigen: diluting the effluent with 5% SDS 0.1/M NaOH for 5 times, oscillating for 10min, sampling to detect fluorescence intensity, and calculating K a And P eff 。
Study results: compared with FO, HCFON has K in intestinal tract a The K of HFON in the intestinal tract is increased by 1.76, 1.29, 4.27 and 5.03 times in the duodenum, jejunum, ileum and colon respectively a The improvement in the duodenum, jejunum, ileum and colon was 1.21, 1.13, 2.05 and 1.84 fold, respectively. Compared with FT, HCFTN in the K of intestinal tract a The improvement in the duodenum, jejunum, ileum and colon was 1.84, 1.34, 4.64 and 5.02 times, respectively. Compared with FO, HCFON is in intestinal tract P eff The K of HFON in the intestinal tract is increased by 2.71, 1.39, 4.89 and 21.18 times in the duodenum, jejunum, ileum and colon respectively a The improvement in the duodenum, jejunum, ileum and colon was 1.41, 1.14, 2.98 and 12.29 fold, respectively. P of HCFTN in intestinal tract eff With FT phaseThe improvement was 2.66, 1.31, 3.89 and 8.21 times higher in the duodenum, jejunum, ileum and colon, respectively.
FIG. 3 shows the assay of the activity of FO, FT, HFON, HCFON and HCFTN on macrophages prepared in accordance with the present invention.
Test conditions: the cell density was set at 3X 10 4 The macrophages/mL were seeded into 96-well plates at 100. Mu.L per well. After the cells are cultured in a cell culture box until the cells adhere to the wall, 200 mu L of a blank culture medium is added into cells of a control group, alpha 1, 3-fucosyltransferase marked by fluorescein isothiocyanate and model antigen nanoparticles are respectively added into cells of an experimental group, the concentration gradient of the alpha 1, 3-fucosyltransferase marked by fluorescein isothiocyanate and the model antigen nanoparticles coated by hydroxypropyl methylcellulose phthalate is 2.5, 5, 10, 20 and 40 mu g/mL of a drug-containing culture medium is 200 mu L, a blank group without cells is additionally arranged, and 3 multiple holes are arranged in each group. The cells were cultured in the incubator for a predetermined period of time (12 h, 24h and 48 h) and the 96-well plate was removed, and the cell morphology was recorded by photographing. MTT 20. Mu.L was added to each well, incubated in a biochemical incubator at 37℃in the absence of light for 4 hours, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and after shaking in the absence of light, A570 was measured with an ELISA reader, zeroed in the blank, and the cell activity was calculated.
Study results: the relative activity of cells in 48h is over 80%, which shows that FO, FT, HFON, HCFON and HCFTN have no influence on the activity of macrophages in 48h when the concentration is 20 mug/mL, and the research results initially show that FO, FT, HFON, HCFON and HCFTN have better safety on the macrophages.
FIG. 4 shows the uptake assay of FO, FT, HFON, HCFON and HCFTN produced by macrophages according to the present invention.
Test conditions: the cell density was set at 5X 10 5 The macrophages/mL were seeded in 96-well plates at 100. Mu.L per well and incubated for 12h to adhere. The culture medium is discarded, 200 mu L of blank culture medium is added into a control group, 200 mu L of culture medium containing alpha 1, 3-fucosyltransferase marked by fluorescein isothiocyanate and model antigen, hydroxypropyl methylcellulose phthalate coated alpha 1, 3-fucosyltransferase marked by fluorescein isothiocyanate or model antigen nanoparticle (with the concentration of 20 mu g/mL) is respectively added into an experimental group, a blank group without cells is additionally arranged,3 duplicate wells were placed in each group, and after incubation at 37℃for 12h, the supernatant was discarded and washed three times with PBS. 50. Mu.L of RIPA cells were added to each well and fluorescence intensities (EX: 495nm, EM: 520 nm) were measured using a fluorescence microplate reader, and uptake of model antigen labeled with fluorescein isothiocyanate and model antigen nanoparticle labeled with hydroxypropylmethyl cellulose phthalate by macrophages, and uptake of alpha 1, 3-fucosyltransferase labeled with fluorescein isothiocyanate and alpha 1, 3-fucosyltransferase nanoparticle labeled with hydroxypropylmethyl cellulose phthalate by macrophages were compared, respectively.
Study results: at a concentration of 20 μg/mL, the fluorescence intensity in hcon-treated macrophages was 18.16 times (P < 0.01) the fluorescence intensity in FO-treated macrophages. The fluorescence intensity in HFON-treated macrophages was 8.79 times the fluorescence intensity in FO-treated macrophages. The fluorescence intensity in HCFTN-treated macrophages was 18.23 times that in FT-treated macrophages. The uptake of hcfo by macrophages is significantly higher than that of FO by macrophages, and HCTFN by macrophages is significantly higher than that of FT.
FIG. 5 is a graph showing the effect of concentrations of FO and HCFO produced by the present invention on macrophage uptake at 37 ℃.
Test conditions: the cell density was set at 5X 10 5 The macrophages/mL were seeded in 96-well plates at 100. Mu.L per well and incubated for 12h to adhere. After the medium was discarded, 200. Mu.L of a blank medium was added to the control group, 200. Mu.L of a medium containing free FO and HFON (concentration gradients of 2.5, 5, 10, 20, 40. Mu.g/mL) was added to the experimental group, and a blank group containing no cells was additionally provided, each group was provided with 3 wells, and after incubation at 37℃for 12 hours, the supernatant was discarded and washed 3 times with PBS. 50. Mu.L of RIPA was added to each well to lyse the cells, and the fluorescence intensity was measured by a fluorescence microplate reader (EX: 495nm, EM:520 nm), and the effect of different concentrations on macrophage uptake was investigated.
Study results: ingestion of both FO and hcon by macrophages is positively correlated with concentration. At concentrations of 5-40 μg/mL, the fluorescence intensity of uptake of HFON by macrophages is significantly greater than that of FO by macrophages (< P < 0.01). Experimental results show that in a certain concentration range, the ingestion of FO and HCFON by macrophages at 37 ℃ is concentration dependent.
Detailed Description
In order to further illustrate the invention and its advantages, the following specific examples are given with the understanding that these examples are intended to be illustrative only and are not to be construed as limiting the scope of the invention.
Example 1:
the content of alpha 1, 3-fucosyltransferase is 2.40mg/mL, and the weight ratio of the rest components in the formula is as follows: 0.40 part of polylactic acid-glycolic acid copolymer (the monomer ratio of lactic acid to glycolic acid is 75:25, hereinafter referred to as L: G is 75:25), 0.20 part of hydroxypropyl methyl cellulose phthalate HP55, 0.02 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.60 part of pluronic F-68 and 90 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (the monomer ratio of lactic acid to glycolic acid is 75:25) and hydroxypropyl methyl cellulose phthalate (model HP 55) in methylene dichloride to form solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of pluronic (model F-68) in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 18 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 2:
the content of alpha 1, 3-fucosyltransferase is 3.90mg/mL, and the weight composition ratio of the rest components in the formula is as follows: polylactic acid-glycolic acid copolymer (L: G50:50) 0.65 parts, hydroxypropyl methylcellulose phthalate HP50 0.325 parts, 2-hydroxypropyl trimethyl ammonium chloride chitosan 0.0325 parts, pluronic F-68 0.975 parts, distilled water 146.25 parts.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate HP50 in chloroform to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving a prescription amount of pluronic F-68 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 20 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 3:
The content of alpha 1,3 fucosyltransferase is 1.20mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.20 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.10 part of hydroxypropyl methyl cellulose phthalate HP55, 0.01 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.30 part of pluronic F-127 and 45 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate HP55 in acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of pluronic F-127 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 22 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 4:
the content of alpha 1, 3-fucosyltransferase is 3.30mg/mL, and the weight composition ratio of the rest components in the formula is as follows: polylactic acid-glycolic acid copolymer (L: G is 75:25) 0.55 parts, hydroxypropyl methylcellulose phthalate HP50 0.275 parts, 2-hydroxypropyl trimethyl ammonium chloride chitosan 0.0275 parts, pluronic F-127 0.825 parts, distilled water 123.75 parts.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methyl cellulose phthalate HP50 in a mixture of dichloromethane and chloroform to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of pluronic F-127 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 24 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 5:
the content of alpha 1, 3-fucosyltransferase is 3.60mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.60 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.30 part of hydroxypropyl methyl cellulose phthalate HP50, 0.03 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.45 part of pluronic F-68, 0.45 part of pluronic F-127 and 135 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 50:50) and hydroxypropyl methyl cellulose phthalate HP50 in a mixture of dichloromethane and acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving the prescription amount of pluronic F-68 and pluronic F-127 in distilled water, dissolving the prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 26 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 6:
the content of alpha 1, 3-fucosyltransferase is 3.60mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.30 part of polylactic acid-glycolic acid copolymer (L: G is 25:75), 0.30 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.10 part of hydroxypropyl methylcellulose phthalate HP50, 0.20 part of hydroxypropyl methylcellulose phthalate HP55, 0.03 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.45 part of pluronic F-68, 0.45 part of pluronic F-127 and 135 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 25:75), polylactic acid-glycolic acid copolymer (L: G is 50:50), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in a mixture of chloroform and acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving the prescription amount of pluronic F-68 and pluronic F-127 in distilled water, dissolving the prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 28 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 7:
the content of alpha 1, 3-fucosyltransferase is 4.20mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.30 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.40 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.10 part of hydroxypropyl methylcellulose phthalate HP50, 0.25 part of hydroxypropyl methylcellulose phthalate HP55, 0.035 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.45 part of pluronic F-68, 0.60 part of pluronic F-127 and 157.5 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 75:25), polylactic acid-glycolic acid copolymer (L: G is 50:50), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in methylene dichloride, a mixture of chloroform and acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of pluronic F-68 and pluronic F-127 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 30 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 8:
the content of alpha 1, 3-fucosyltransferase is 2.70mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.10 part of polylactic acid-glycolic acid copolymer (L: G is 25:75), 0.10 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.25 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.125 part of hydroxypropyl methylcellulose phthalate HP50, 0.10 part of hydroxypropyl methylcellulose phthalate HP55, 0.0225 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.255 part of pluronic F-68, 0.420 part of pluronic F-127 and 101.25 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 25:75), polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in methylene dichloride, a mixture of chloroform and acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving the prescription amount of pluronic F-68 and pluronic F-127 in distilled water, dissolving the prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 32 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 9:
the content of alpha 1, 3-fucosyltransferase is 4.50mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.20 part of polylactic acid-glycolic acid copolymer (L: G is 25:75), 0.20 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.35 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.125 part of hydroxypropyl methylcellulose phthalate HP50, 0.25 part of hydroxypropyl methylcellulose phthalate HP55, 0.0375 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.125 part of pluronic F-68, 1 part of pluronic F-127 and 168.75 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 25:75), polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in methylene dichloride to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving the prescription amount of pluronic F-68 and pluronic F-127 in distilled water, dissolving the prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 34 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 10:
the content of the alpha 1, 3-fucosyltransferase is 2.10mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.35 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.175 part of hydroxypropyl methyl cellulose phthalate HP50, 0.0175 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.525 part of polyvinyl alcohol 1750 and 78.75 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 50:50) and hydroxypropyl methyl cellulose phthalate HP50 in chloroform to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving a prescription amount of polyvinyl alcohol 1750 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 36 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 11:
the content of the alpha 1, 3-fucosyltransferase is 1.50mg/mL, and the weight composition ratio of the components in the formula is as follows: 0.25 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.125 part of hydroxypropyl methyl cellulose phthalate HP55, 0.0125 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.375 part of polyvinyl alcohol 1788 and 56.25 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methyl cellulose phthalate HP55 in methylene dichloride to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of polyvinyl alcohol 1788 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 18 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 12:
the content of alpha 1, 3-fucosyltransferase is 4.80mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.40 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.40 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.40 part of hydroxypropyl methyl cellulose phthalate HP55, 0.04 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 1.20 parts of polyvinyl alcohol 1750 and 180 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methylcellulose phthalate HP55 in methylene dichloride to form solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of polyvinyl alcohol 1750 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 20 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 13:
the content of the alpha 1, 3-fucosyltransferase is 1.50mg/mL, and the weight composition ratio of the components in the formula is as follows: 0.25 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.025 part of hydroxypropyl methyl cellulose phthalate HP50, 0.1 part of hydroxypropyl methyl cellulose phthalate HP55, 0.0125 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.375 part of polyvinyl alcohol 1750 and 56.25 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 75:25), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of polyvinyl alcohol 1750 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 22 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 14:
the content of the alpha 1, 3-fucosyltransferase is 1.50mg/mL, and the weight composition ratio of the components in the formula is as follows: 0.25 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.125 part of hydroxypropyl methyl cellulose phthalate HP55, 0.0125 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.375 part of polyvinyl alcohol 1788 and 56.25 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methyl cellulose phthalate HP55 in methylene dichloride to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of polyvinyl alcohol 1788 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 24 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 15:
the content of the alpha 1, 3-fucosyltransferase is 2.10mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.15 part of polylactic acid-glycolic acid copolymer (L: G is 25:75), 0.10 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.10 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.075 part of hydroxypropyl methylcellulose phthalate HP50 and 0.1 part of hydroxypropyl methylcellulose phthalate HP55, 0.0175 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, and 0.525 part of polyvinyl alcohol 1788 and 78.75 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 25:75), polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in a mixture of dichloromethanol and trichloromethane to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving a prescription amount of polyvinyl alcohol 1788 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 26 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 16:
the content of alpha 1, 3-fucosyltransferase is 3.30mg/mL, and the weight composition ratio of the rest components in the formula is as follows: 0.15 part of polylactic acid-glycolic acid copolymer (L: G is 25:75), 0.15 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.25 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.275 part of hydroxypropyl methyl cellulose phthalate HP50, 0.0275 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.225 part of polyvinyl alcohol 1750, 0.60 part of polyvinyl alcohol 1788 and 123.75 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 25:75), polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methylcellulose phthalate HP50 in methylene dichloride, a mixture of chloroform and acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving prescription amount of polyvinyl alcohol 1750 and polyvinyl alcohol 1788 in distilled water, dissolving prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting emulsion B into ice bath, slowly adding the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and performing ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 28 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 17:
the content of alpha 1, 3-fucosyltransferase is 2.40mg/mL, and the weight ratio of the rest components in the formula is as follows: 0.40 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.20 part of hydroxypropyl methyl cellulose phthalate HP55, 0.02 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.30 part of pluronic F-68, 0.30 part of polyvinyl alcohol 1750 and 90 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methyl cellulose phthalate HP55 in methylene dichloride to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B; (2) Dissolving a prescription amount of pluronic F-68 and polyvinyl alcohol 1750 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the pluronic aqueous solution, the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 30 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 18:
the content of alpha 1, 3-fucosyltransferase is 2.40mg/mL, and the weight ratio of the rest components in the formula is as follows: 0.40 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.10 part of hydroxypropyl methyl cellulose phthalate HP50, 0.10 part of hydroxypropyl methyl cellulose phthalate HP55, 0.02 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.30 part of pluronic F-127, 0.30 part of polyvinyl alcohol 1788 and 90 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 50:50), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in chloroform to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving a prescription amount of pluronic F-127 and polyvinyl alcohol 1788 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into an ice bath, slowly adding the pluronic aqueous solution, the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 18 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 19:
the content of alpha 1, 3-fucosyltransferase is 3.90mg/mL, and the weight ratio of the rest components in the formula is as follows: 0.35 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.30 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.325 part of hydroxypropyl methylcellulose phthalate HP50, 0.0325 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.375 part of pluronic F-68, 0.30 part of pluronic F-127, 0.30 part of polyvinyl alcohol 1750 and 146.25 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25) and hydroxypropyl methylcellulose phthalate HP50 in acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and performing ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving the prescription amount of pluronic F-68, pluronic F-127 and polyvinyl alcohol 1750 in distilled water, dissolving the prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution, the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain emulsion C; (3) Stirring the emulsion C for 20 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Example 20:
the content of alpha 1, 3-fucosyltransferase is 2.70mg/mL, and the weight ratio of the rest components in the formula is as follows: 0.10 part of polylactic acid-glycolic acid copolymer (L: G is 25:75), 0.10 part of polylactic acid-glycolic acid copolymer (L: G is 50:50), 0.25 part of polylactic acid-glycolic acid copolymer (L: G is 75:25), 0.125 part of hydroxypropyl methylcellulose phthalate (model HP 50), 550.10 parts of hydroxypropyl methylcellulose phthalate HP, 0.0225 part of 2-hydroxypropyl trimethyl ammonium chloride chitosan, 0.025 part of pluronic (model F-68), 0.350 part of pluronic F-127, 0.20 part of polyvinyl alcohol 17588, 0.10 part of polyvinyl alcohol and 101.25 parts of distilled water.
The preparation method comprises the following steps: (1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer (L: G is 25:75), polylactic acid-glycolic acid copolymer (L: G is 50:50), polylactic acid-glycolic acid copolymer (L: G is 75:25), hydroxypropyl methylcellulose phthalate HP50 and hydroxypropyl methylcellulose phthalate HP55 in methylene dichloride, a mixture of chloroform and acetone to form a solution A, dissolving a prescribed amount of alpha 1, 3-fucosyltransferase in distilled water, adding the solution A, mixing, and carrying out ultrasonic treatment in an ice bath to obtain an emulsion B; (2) Dissolving a prescription amount of pluronic F-68, pluronic F-127, polyvinyl alcohol 1750 and polyvinyl alcohol 1788 in distilled water, dissolving a prescription amount of 2-hydroxypropyl trimethyl ammonium chloride chitosan in distilled water, rapidly putting the emulsion B into ice bath, slowly adding the pluronic aqueous solution, the polyvinyl alcohol aqueous solution and the 2-hydroxypropyl trimethyl ammonium chloride chitosan aqueous solution, and carrying out ultrasonic treatment in the ice bath to obtain an emulsion C; (3) Stirring the emulsion C for 24 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
Claims (3)
1. An oral recombinant helicobacter pylori protein vaccine nanoparticle is characterized in that the nanoparticle contains alpha 1, 3-fucosyltransferase with the content of 1.20-6.00mg/mL, and comprises the following components in parts by weight:
The polylactic acid-glycolic acid copolymer is prepared by the following steps of: 25, a polymer to monomer ratio of 50:50, a copolymer to monomer ratio of 25:75 or a mixture of two or three of the copolymers;
the hydroxypropyl methyl cellulose phthalate is one or a mixture of two of HP55 and HP 50;
the emulsifier is one or a mixture of a plurality of pluronic F-68, pluronic F-127, polyvinyl alcohol 1750 and polyvinyl alcohol 1788.
2. The method for preparing oral recombinant helicobacter pylori protein vaccine nanoparticles according to claim 1, characterized in that the method comprises the following steps:
(1) Dissolving a prescribed amount of polylactic acid-glycolic acid copolymer and hydroxypropyl methyl cellulose phthalate in a solvent to obtain a solution A, wherein the solvent is one or a mixture of two or three of dichloromethane, acetone and chloroform; adding the alpha 1, 3-fucosyltransferase solution with the prescription amount into the solution A, uniformly mixing, and performing ultrasonic treatment in an ice bath to obtain emulsion B;
(2) Rapidly putting the emulsion B into ice bath, slowly adding the emulsifier and the aqueous solution of the 2-hydroxypropyl trimethyl ammonium chloride chitosan, and performing ultrasonic treatment in the ice bath to obtain emulsion C;
(3) Stirring the emulsion C for 18-36 hours to volatilize and remove the organic solvent, thus obtaining the oral recombinant helicobacter pylori protein vaccine nanoparticle.
3. An oral recombinant helicobacter pylori protein vaccine nanoparticle according to claim 1, characterized in that the average particle size is less than 200nm.
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| CN202110954931.5A CN113648405B (en) | 2021-08-19 | 2021-08-19 | Oral recombinant helicobacter pylori protein vaccine nanoparticle and preparation method thereof |
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| CN202110954931.5A CN113648405B (en) | 2021-08-19 | 2021-08-19 | Oral recombinant helicobacter pylori protein vaccine nanoparticle and preparation method thereof |
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