CN114470188B - 一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 - Google Patents
一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 Download PDFInfo
- Publication number
- CN114470188B CN114470188B CN202210309968.7A CN202210309968A CN114470188B CN 114470188 B CN114470188 B CN 114470188B CN 202210309968 A CN202210309968 A CN 202210309968A CN 114470188 B CN114470188 B CN 114470188B
- Authority
- CN
- China
- Prior art keywords
- ucms
- mesoporous silica
- lbp
- ultra
- polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 150000004676 glycans Chemical class 0.000 title claims abstract description 22
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 21
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 21
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 235000017784 Mespilus germanica Nutrition 0.000 title claims abstract description 9
- 235000000560 Mimusops elengi Nutrition 0.000 title claims abstract description 9
- 235000007837 Vangueria infausta Nutrition 0.000 title claims abstract description 9
- 240000002624 Mespilus germanica Species 0.000 title 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000008367 deionised water Substances 0.000 claims abstract description 12
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 12
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000003756 stirring Methods 0.000 claims abstract description 9
- 244000182216 Mimusops elengi Species 0.000 claims abstract description 8
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims abstract description 5
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims abstract description 5
- 238000000227 grinding Methods 0.000 claims abstract description 5
- 239000002244 precipitate Substances 0.000 claims abstract description 5
- 239000000047 product Substances 0.000 claims abstract description 5
- 244000241838 Lycium barbarum Species 0.000 claims abstract description 4
- 235000015459 Lycium barbarum Nutrition 0.000 claims abstract description 4
- 235000015468 Lycium chinense Nutrition 0.000 claims abstract description 4
- 238000001354 calcination Methods 0.000 claims abstract description 4
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 230000001571 immunoadjuvant effect Effects 0.000 claims abstract description 4
- 239000000568 immunological adjuvant Substances 0.000 claims abstract description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 3
- 238000005303 weighing Methods 0.000 claims abstract description 3
- 239000003814 drug Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims 1
- 230000036039 immunity Effects 0.000 abstract description 6
- 238000002156 mixing Methods 0.000 abstract description 5
- 238000012377 drug delivery Methods 0.000 abstract description 3
- 210000002540 macrophage Anatomy 0.000 description 18
- 230000000242 pagocytic effect Effects 0.000 description 12
- 239000012071 phase Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 5
- 102000004127 Cytokines Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 4
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 229910002808 Si–O–Si Inorganic materials 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010022078 Injection site inflammation Diseases 0.000 description 1
- 206010022086 Injection site pain Diseases 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-N N,N-Diethylethanamine Substances CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 229910004298 SiO 2 Inorganic materials 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000008518 lycium barbarum polysaccharide Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 231100000098 severe acute toxicity Toxicity 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229940043263 traditional drug Drugs 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K39/385—Haptens or antigens, bound to carriers
-
- 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
-
- 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
-
- 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
- A61K2039/55583—Polysaccharides
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途，包括如下步骤：(1)将CTAC溶液、TEA和去离子水混合均匀制成水相；(2)取氯苯和TEOS混合均匀制成油相；(3)将油相滴加入水相后水浴，匀速搅拌，离心收集沉淀，清洗，箱干，产物研磨后煅烧，得UCMS；(4)称取LBP和UCMS，分散于去离子水中，超声处理后常温搅拌，得LBP‑UCMS溶液。本发明首次通过结合枸杞多糖和药物递送载体(超大介孔二氧化硅)两者的优势构建新型免疫佐剂，体现交叉学科的优势。与常规介孔二氧化硅相比，超大介孔二氧化硅可以装载更多的多糖发挥更佳的免疫作用。
Description
技术领域
本发明涉及免疫佐剂制备方法及用途，特别涉及一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途。
背景技术
枸杞，甘、平，是一味药食同源的中药材。归肝、肺、肾经；滋肾，润肺，补肝，明目。枸杞中含有的枸杞多糖(LBP)具有促进免疫、抗衰老、抗肿瘤、清除自由基、抗疲劳、抗辐射、保肝、生殖功能保护和改善等多种作用。然而LBP临床应用中存在代谢快，靶向性差和用量大等缺点。因此，亟需寻求有效的方法解决这一问题。
药物递送系统是目前研究的热点。介孔二氧化硅(MSNs)就是一种很有希望克服传统佐剂缺点的新型纳米递送载体(诸如注射部位炎症、疼痛以及严重的急性毒性等)。它拥有高比表面积、大孔体积、生物相容性好、可功能化修饰等良好的性质，比传统的药物输送系统(如聚合物纳米颗粒，脂质体等)更灵活、更坚固。此外，MSNs制造工艺相对简单，成本显著降低，这对于满足未来的临床需求和商业化非常重要。目前介孔二氧化硅已被广泛作为亚单位疫苗的靶向递送载体，而超大介孔二氧化硅(UCMS)可以更容易吸附负载抗原、只需简单混合吸附即可，也可装载更多的抗原并抵抗胃肠液的破坏。
巨噬细胞是机体防御病原体的第一道防线，它既是一类重要的抗原提呈细胞，也是重要的炎症和免疫效应细胞，具有识别、吞噬、抗感染和免疫调节等多种免疫功能，在特异性免疫和非特异性免疫中起关键作用。鉴于巨噬细胞在机体免疫系统中的重要地位，
发明内容
发明目的：本发明目的是提供一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法。
技术方案：所述的枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法，包括如下步骤：
(1)将CTAC溶液、TEA和去离子水混合均匀制成水相；
(2)取氯苯和TEOS混合均匀制成油相；
(3)将油相滴加入水相后水浴，匀速搅拌，离心收集沉淀，清洗，箱干，产物研磨后煅烧，得UCMS；
(4)称取LBP和UCMS，分散于去离子水中，超声处理后常温搅拌，得LBP-UCMS溶液。
进一步地，所述步骤(3)采用无水乙醇、去离子水清洗。
进一步地，所述步骤(1)中将4.8mL 25wt％CTAC溶液、0.04g TEA和7.2mL去离子水混合均匀制成水相。
进一步地，所述步骤(2)中将3.5mL氯苯和0.5mL TEOS混合均匀制成油相。
所述方法制备得到的枸杞多糖超大介孔二氧化硅纳米佐剂在中药多糖新型免疫佐剂的研制及动物疫病药物中的用途。
本发明采用两相界面合成法制备UCMS并物理搅拌装载LBP，成功制备出LBP-UCMS。本发明以RAW264.7为研究的靶细胞，通过评估巨噬细胞的吞噬能力、细胞因子的mRNA含量和表面共刺激分子的表达量，测定LBP-UCMS对RAW264.7细胞的免疫调节作用。制备出的UCMS可装载更多的多糖，发挥更佳的免疫效果。产物LBP-UCMS可显著提高巨噬细胞分泌细胞因子的含量和表面标记分子的表达量，且更有效地刺激巨噬细胞，增强巨噬细胞的吞噬能力，将为中药多糖新型免疫佐剂的研制及动物疫病的防控提供参考。
有益效果：本发明与现有技术相比，具有如下优势：
1.首次通过结合枸杞多糖和疫苗递送载体(超大介孔二氧化硅)两者的优势构建新型免疫佐剂，体现交叉学科的优势。
2.与常规介孔二氧化硅相比，超大介孔二氧化硅可以装载更多的多糖发挥更佳的免疫作用。
附图说明
图1LBP、UCMS、LBP-UCMS固体外观图；
图2UCMS、LBP-UCMS的透射电镜图；
图3LBP、UCMS、LBP-UCMS红外光谱图；
图4LBP-UCMS对RAW264.7细胞因子分泌的影响；
图5LBP-UCMS对RAW264.7表面共刺激分子的影响；
图6LBP-UCMS对RAW264.7吞噬能力的影响。
具体实施方式
实施例1：制备UCMS并物理装载LBP
准确将4.8mL 25wt％CTAC溶液、0.04g TEA和7.2mL去离子水混合均匀制成水相。其次，准确量取3.5mL氯苯和0.5mL TEOS混合均匀制成油相。将油相缓慢滴加入水相中后60℃水浴，500r/min下匀速搅拌12h。高速离心收集沉淀后，用无水乙醇、去离子水各清洗3遍，置于60℃烘箱中风干。得到的产物研磨后于马弗炉550℃煅烧5h，得UCMS。准确称取20mgLBP和5mg UCMS，分散于2mL去离子水中。超声处理后常温搅拌12h，得LBP-UCMS溶液。
实施例2：表征测定
(1)透射电镜结果分析
制备的LBP-UCMS固体粉末如图1所示。透射电子显微镜(TEM)观察到LBP-UCMS的形态如图2所示。在电子高倍镜下观察可看到UCMS尺寸匀一，粒径在80-100nm左右，有褶皱状孔道直通内部，皱襞较薄，为物理吸附装载多糖提供更多的空间。LBP-UCMS纳米粒也呈球形，均匀分布，且UCMS的介孔间隙被填满，说明LBP成功被装载于UCMS的介孔间隙内。
(2)粒径、Zeta电位分析
如表1所示，通过动态光散射仪(DLS)测得UCMS在装载LBP后，粒径有所增大。LBP-UCMS的平均粒径为493.4±14.17nm(n＝3)，与通过TEM拍摄的纳米粒大小基本一致，且PDI均在0.1-0.35范围内。LBP、UCMS和LBP-UCMS的电势均为负数，而LBP-UCMS的Zeta电势绝对值比LBP、UCMS的值更大。
表1 LBP-UCMS的申势、粒径和PDI(n＝3)
(3)傅里叶红外光谱结果分析
样品LBP、UCMS和LBP-UCMS与KBr粉末混合研磨压片，在4000～400cm-1波长范围内进行红外扫描后，其红外光谱结果见图3。LBP在800-1200cm-1、1450-1800cm-1、2900-3000cm-1和3200-3600cm-1处有明显的红外吸收峰。LBP在3401cm-1处有一个典型的羟基(-OH)峰，2929cm-1处的峰值是C-H键拉伸振动产生的。1641cm-1处出现的峰值对应C＝C键和C＝O基团的吸收。UCMS在3435cm-1的峰值是H-O-H键伸缩振动产生的；804cm-1和1086cm-1出现的峰值分别对应的是Si-O-Si键对称伸缩振动和Si-O-Si键非对称伸缩振动产生的。LBP-UCMS中包含LBP、UCMS共同的红外特征峰。上述结果说明UCMS纳米材料是由SiO2组成的，且LBP-UCMS成功物理装载LBP。
实施例3：性能测试
(1)LBP-UCMS对RAW264.7细胞因子分泌的影响
植物多糖能够活化巨噬细胞，并通过促进巨噬细胞增殖、提高吞噬能力以及产生TNF-α、IL-6、和IL-1β等细胞因子来发挥免疫调节作用，从而提高免疫能力。qRT-PCR结果如图4所示，LBP-UCMS(250μg/mL)刺激巨噬细胞分泌的TNF-α含量与Control组差异显著(P＜0.001)，但与LBP组相比，略高于LBP组但无显著差异。LBP-UCMS(250μg/mL)刺激巨噬细胞分泌IL-1β、IL-6的含量显著高于LBP组(P＜0.0001)。表明与LBP单独刺激相比，LBP-UCMS可更多地刺激巨噬细胞产生相应的炎性因子。
(2)LBP-UCMS对RAW264.7表面共刺激分子的影响
外源性抗原经吞噬或吞饮作用，将抗原分解为抗原肽，并与内质网合成的MHC-II类分子结合形成抗原肽-MHC-II类分子复合物。该复合物表达于抗原呈递细胞表面，同时增加共刺激分子(如CD86、CD80)表达，激活特异性T和B淋巴细胞效应机制(适应性免疫)。通过流式细胞术检测细胞表面刺激分子结合的荧光量来反映其表达量的多少。结果如图5所示，浓度分别为125μg/mL、250μg/mL的LBP-UCMS作用细胞24h后，MHC-II、CD80、CD86的表达量都显著高于LBP组和细胞对照组。250μg/mL的LBP-UCMS组的MHC-II的平均表达量达到65.18％，而同浓度下的LBP组MHC-II的平均表达量仅为27.6％，并低于125μg/mL LBP-UCMS组的平均表达量。LBP-UCMS(250μg/mL)组CD80+的平均表达量达到48.91％，而同浓度的LBP组CD80+的平均表达量为16.84％，250μg/mL LBP-UCMS组CD80+的平均表达量是LBP组的2.9倍。LBP-UCMS(250μg/mL)组CD86+的平均表达量为54.05％，而同浓度的LBP组CD86+的平均表达量为21.77％，LBP-UCMS(250μg/mL)组CD86+的平均表达量是LBP组的2.48倍。试验结果表明LBP经过UCMS装载后对小鼠巨噬细胞表面分子的表达有显著的促进作用。
(3)LBP-UCMS对RAW264.7吞噬能力的影响
吞噬能力是反应巨噬细胞免疫防御能力的一个重要指标，通过吞噬作用，杀死病原体或参与炎症反应来引导抵抗病原体感染，本试验通过激光共聚焦检测巨噬细胞吞噬荧光量(FITC-葡聚糖)的多少来反映巨噬细胞的吞噬功能。如图6所示，与Control组相比，LPS组的FITC-葡聚糖分布更多，吞噬能力较强，但发生形态改变的细胞较多。在125μg/mL、250μg/mL浓度LBP-UCMS的作用下，巨噬细胞内分布的绿色荧光明显多于Control、LBP、UCMS组，吞噬能力明显增强。而125μg/mL的LBP-UCMS吞噬能力略高于250μg/mL浓度。说明UCMS装载LBP后，能够更有效的刺激巨噬细胞，增强巨噬细胞的吞噬能力。
Claims (2)
1.一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法，其特征在于：包括如下步骤：
（1）将4.8 mL 25 wt% CTAC溶液、0.04 g TEA和7.2 mL去离子水混合均匀制成水相；
（2）将3.5 mL氯苯和0.5 mL TEOS混合均匀制成油相；
（3）将油相滴加入水相后水浴，匀速搅拌，离心收集沉淀，采用无水乙醇、去离子水清洗，烘干，产物研磨后煅烧，得超大介孔二氧化硅；
（4）称取枸杞多糖和超大介孔二氧化硅，分散于去离子水中，超声处理后常温搅拌，得枸杞多糖超大介孔二氧化硅纳米佐剂溶液。
2.权利要求1所述方法制备得到的枸杞多糖超大介孔二氧化硅纳米佐剂在制备中药多糖免疫佐剂或动物疫病药物中的用途。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210309968.7A CN114470188B (zh) | 2022-03-28 | 2022-03-28 | 一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210309968.7A CN114470188B (zh) | 2022-03-28 | 2022-03-28 | 一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114470188A CN114470188A (zh) | 2022-05-13 |
| CN114470188B true CN114470188B (zh) | 2024-01-23 |
Family
ID=81488249
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210309968.7A Active CN114470188B (zh) | 2022-03-28 | 2022-03-28 | 一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114470188B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107157933A (zh) * | 2017-05-04 | 2017-09-15 | 同济大学 | 一种蛋白自组装新型纳米疫苗及其制备方法 |
| CN110004173A (zh) * | 2019-04-08 | 2019-07-12 | 天津吉诺沃生物科技有限公司 | 一种获得非转基因耐储存鲜食枸杞的方法 |
| CN110974947A (zh) * | 2019-12-31 | 2020-04-10 | 上海长海医院 | 一种具有抗菌及止血功能的纳米介孔二氧化硅止血粉的制备方法 |
| CN113371722A (zh) * | 2021-06-02 | 2021-09-10 | 湖北大学 | 一种可降解小尺寸中空介孔二氧化硅纳米颗粒的制备方法 |
| CN113768903A (zh) * | 2021-09-10 | 2021-12-10 | 浙江海洋大学 | 一种褐藻寡糖修饰的氨基化介孔二氧化硅纳米颗粒 |
| CN113975235A (zh) * | 2021-10-11 | 2022-01-28 | 浙江海洋大学 | 一种递送岩藻黄素的褐藻寡糖-介孔二氧化硅纳米复合物 |
-
2022
- 2022-03-28 CN CN202210309968.7A patent/CN114470188B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107157933A (zh) * | 2017-05-04 | 2017-09-15 | 同济大学 | 一种蛋白自组装新型纳米疫苗及其制备方法 |
| CN110004173A (zh) * | 2019-04-08 | 2019-07-12 | 天津吉诺沃生物科技有限公司 | 一种获得非转基因耐储存鲜食枸杞的方法 |
| CN110974947A (zh) * | 2019-12-31 | 2020-04-10 | 上海长海医院 | 一种具有抗菌及止血功能的纳米介孔二氧化硅止血粉的制备方法 |
| CN113371722A (zh) * | 2021-06-02 | 2021-09-10 | 湖北大学 | 一种可降解小尺寸中空介孔二氧化硅纳米颗粒的制备方法 |
| CN113768903A (zh) * | 2021-09-10 | 2021-12-10 | 浙江海洋大学 | 一种褐藻寡糖修饰的氨基化介孔二氧化硅纳米颗粒 |
| CN113975235A (zh) * | 2021-10-11 | 2022-01-28 | 浙江海洋大学 | 一种递送岩藻黄素的褐藻寡糖-介孔二氧化硅纳米复合物 |
Non-Patent Citations (1)
| Title |
|---|
| Chun Xu等.Core-Cone Structured Monodispersed Mesoporous Silica Nanoparticles with Ultra-large Cavity for Protein Delivery.small.2015,第11卷(第44期),摘要，2.结果与讨论，4.实验部分. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114470188A (zh) | 2022-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105126115B (zh) | 二氧化硅纳米颗粒及其用于疫苗接种的用途 | |
| Luo et al. | Designing selenium polysaccharides-based nanoparticles to improve immune activity of Hericium erinaceus | |
| Zhang et al. | The immunoenhancement effects of polyethylenimine-modified Chinese yam polysaccharide-encapsulated PLGA nanoparticles as an adjuvant | |
| CN104000742A (zh) | 一种防晒液及焕颜cc防晒霜 | |
| CN114470188B (zh) | 一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 | |
| Zhai et al. | Protective Effect of chitosan oligosaccharides against cyclophosphamide‐induced immunosuppression and irradiation injury in mice | |
| CN113577086B (zh) | 异戊酰螺旋霉素类化合物或其组合物在制备治疗免疫失调的药物中的应用 | |
| CN112089834B (zh) | 基于氧化石墨烯的茯苓多糖纳米佐剂及佐剂/抗原共递送疫苗的制备与应用 | |
| CN101991850A (zh) | 纳米氢氧化铝佐剂的制备工艺 | |
| Xu et al. | pH-responsive Astragalus polysaccharide-loaded PLGA nanoparticles as an adjuvant system to improve immune responses | |
| LU503719B1 (en) | Preparation method and application of lycium barbarum polysaccharide super-large mesoporous silica nano-adjuvant | |
| CN114010799A (zh) | 一种光热协同化疗的靶向可降解纳米药物载体及其制备方法 | |
| CN101067004A (zh) | 一种艾叶多糖及其用途 | |
| CN115068602B (zh) | 一种表面改性氧化铝的复合疫苗佐剂、制备方法及应用 | |
| TWI439279B (zh) | 治療過敏疾病之方法 | |
| CN101653580B (zh) | 一种治疗胃病的药物及其制备方法 | |
| CN114522247B (zh) | 一种复合纳米粒及其制备方法和用途 | |
| CN106109442B (zh) | 一种山药多糖聚乳酸羟基乙酸纳米粒及其制备方法与应用 | |
| CN114652823B (zh) | 一种白术多糖-纳米氧化锌复合物及其制备方法和用途 | |
| CN102727887B (zh) | 壳聚糖作为免疫佐剂在制备小鼠过敏性哮喘模型中的用途 | |
| CN111406948B (zh) | 一种蜈蚣藻多糖-纳米硒的制备方法及其应用 | |
| CN110200980B (zh) | 一种荷载青蒿素的柑橘果胶的口服纳米粒子 | |
| Li et al. | Acanthopanax senticosus polysaccharide-loaded calcium carbonate nanoparticle as an adjuvant to enhance porcine parvovirus vaccine immune responses | |
| CN1057915C (zh) | 免疫佐剂 | |
| CN110433144A (zh) | 川芎油微囊及制法、川芎油微囊凝胶贴剂及制法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |