CN114099656A - Co-loaded nano preparation based on antigen and active polysaccharide and preparation method and application thereof - Google Patents
Co-loaded nano preparation based on antigen and active polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN114099656A CN114099656A CN202111458751.4A CN202111458751A CN114099656A CN 114099656 A CN114099656 A CN 114099656A CN 202111458751 A CN202111458751 A CN 202111458751A CN 114099656 A CN114099656 A CN 114099656A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- antigen
- preparation
- active polysaccharide
- active
- 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.)
- Pending
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 121
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 113
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 113
- 239000000427 antigen Substances 0.000 title claims abstract description 64
- 102000036639 antigens Human genes 0.000 title claims abstract description 64
- 108091007433 antigens Proteins 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 53
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 56
- 239000003814 drug Substances 0.000 claims abstract description 18
- 238000009169 immunotherapy Methods 0.000 claims abstract description 18
- 239000000203 mixture Substances 0.000 claims abstract description 15
- 229940079593 drug Drugs 0.000 claims abstract description 12
- 238000011068 loading method Methods 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 7
- 239000002539 nanocarrier Substances 0.000 claims abstract description 7
- 108010058846 Ovalbumin Proteins 0.000 claims description 45
- 229940092253 ovalbumin Drugs 0.000 claims description 45
- 241001061264 Astragalus Species 0.000 claims description 36
- 235000006533 astragalus Nutrition 0.000 claims description 36
- 210000004233 talus Anatomy 0.000 claims description 36
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 16
- 239000010408 film Substances 0.000 claims description 14
- 150000003904 phospholipids Chemical class 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 239000006185 dispersion Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 235000012000 cholesterol Nutrition 0.000 claims description 8
- 239000000523 sample Substances 0.000 claims description 7
- 229920001491 Lentinan Polymers 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 241000237502 Ostreidae Species 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 230000000887 hydrating effect Effects 0.000 claims description 6
- 229940115286 lentinan Drugs 0.000 claims description 6
- 235000020636 oyster Nutrition 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 230000010494 opalescence Effects 0.000 claims description 5
- 229930195210 Ophiopogon Natural products 0.000 claims description 4
- 244000248557 Ophiopogon japonicus Species 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000010409 thin film Substances 0.000 claims description 3
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 2
- 241000251511 Holothuroidea Species 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 2
- 244000131316 Panax pseudoginseng Species 0.000 claims description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 240000001949 Taraxacum officinale Species 0.000 claims description 2
- 235000005187 Taraxacum officinale ssp. officinale Nutrition 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 201000004101 esophageal cancer Diseases 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 235000008434 ginseng Nutrition 0.000 claims description 2
- -1 glycyrrhiza polysaccharides Chemical class 0.000 claims description 2
- 238000006703 hydration reaction Methods 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 201000005202 lung cancer Diseases 0.000 claims description 2
- 208000020816 lung neoplasm Diseases 0.000 claims description 2
- 210000002751 lymph Anatomy 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 241000202807 Glycyrrhiza Species 0.000 claims 1
- 239000012876 carrier material Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 23
- 230000009471 action Effects 0.000 abstract description 7
- 238000001727 in vivo Methods 0.000 abstract description 7
- 230000002035 prolonged effect Effects 0.000 abstract description 3
- 239000002671 adjuvant Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 13
- 150000004804 polysaccharides Polymers 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 8
- 238000005538 encapsulation Methods 0.000 description 6
- 230000006872 improvement Effects 0.000 description 6
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000001186 cumulative effect Effects 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 230000036737 immune function Effects 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 208000031648 Body Weight Changes Diseases 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000005809 anti-tumor immunity Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004579 body weight change Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229940127130 immunocytokine Drugs 0.000 description 2
- 230000000091 immunopotentiator Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000004614 tumor growth Effects 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
- 244000061520 Angelica archangelica Species 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000303040 Glycyrrhiza glabra Species 0.000 description 1
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 1
- 235000001287 Guettarda speciosa Nutrition 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 206010027476 Metastases Diseases 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
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000011398 antitumor immunotherapy Methods 0.000 description 1
- 210000003056 antler Anatomy 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000001571 immunoadjuvant effect Effects 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 235000011477 liquorice Nutrition 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
Images
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
- A61K39/0005—Vertebrate antigens
-
- 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
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- 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
Abstract
The invention belongs to the technical field of medicines, and particularly discloses a co-loading nano preparation based on an antigen and active polysaccharide, and a preparation method and application thereof. The co-carried nano preparation comprises the following components: the antigen, the active polysaccharide and the nano carrier material are co-loaded in the nano carrier material, and the active polysaccharide is used as an immune adjuvant and is used in combination with the antigen, so that a more effective tumor immunotherapy effect is achieved, meanwhile, the action time of the antigen and the active polysaccharide in vivo is prolonged, the co-delivery effect of the composition is realized, and the tumor immunotherapy effect is further obviously improved.
Description
Technical Field
The invention relates to the technical field of medicines, and relates to a co-loading nano preparation based on an antigen and active polysaccharide, and a preparation method and application thereof.
Background
Cancer has become one of the major diseases seriously threatening human life and health, and the occurrence, development and prognosis of tumor are closely related to the body's own immune function. The tumor immunotherapy is a therapeutic method for inhibiting and killing tumor cells by activating the activity of the immune system of an organism and enhancing the anti-tumor immune function of the organism. Compared with the traditional tumor treatment method, the tumor immunotherapy has many advantages, mainly including: the tumor cells are killed and killed specifically, no obvious influence is caused on normal cells, and the systemic side effect is small; the body is stimulated to generate a systemic anti-tumor immune effect, and the action range is wide; in addition to inhibiting tumor growth, can also inhibit tumor metastasis and recurrence; the traditional Chinese medicine composition has lasting curative effect and better curative effect on advanced tumors, and is considered to be the most possible therapy for curing cancers.
With the continuous and deep research on tumor-associated antigens and tumor-specific antigens, the tumor antigens are used for immunotherapy to activate immune response, and cytotoxic T cells can be effectively induced to kill tumor cells, so that anti-tumor immunotherapy is started. The active polysaccharide is an effective polysaccharide component extracted from traditional Chinese medicines possibly having an anti-tumor effect by using a modern scientific and technological method, mainly comes from plants, animals, fungi and the like, is mainly used as an immunopotentiator to stimulate the proliferation and activation of immune cells and the generation and release of cytokines through various mechanisms, improves the host immune function and plays an active anti-tumor effect. The Chinese medicinal polysaccharide can not only activate immune cells such as T cells, B cells, macrophages, NK cells, CTL cells, lymphokine-activated killer cells and the like, but also promote the generation of cytokines such as interleukins IL-1, IL-2, TNF-alpha, INF-gamma and the like, regulate the formation of body antibodies and complements and improve the anti-tumor immunity of the body. Therefore, the active polysaccharide can be used as an ideal immune adjuvant to be combined with an antigen to achieve an effective tumor immunotherapy effect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a co-loading nano preparation based on an antigen and active polysaccharide, a preparation method and application thereof, the preparation method is simple and easy to implement, the antigen and the active polysaccharide are co-loaded in a nano material, the active polysaccharide is used as an immunoadjuvant to be combined with the antigen for use, a more effective tumor immunotherapy effect is achieved, meanwhile, the action time of the antigen and the active polysaccharide in vivo is prolonged, the co-delivery effect of a composition is realized, and the tumor immunotherapy effect is further improved.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a co-carried nanometer preparation based on antigen and active polysaccharide comprises the following components: antigens, active polysaccharides and nanocarrier materials.
In a refinement, the antigen is ovalbumin; the active polysaccharide is plant polysaccharide or animal polysaccharide.
The further improvement is that the plant polysaccharide is one or more of astragalus polysaccharide, ginseng polysaccharide, liquorice polysaccharide, dandelion polysaccharide, pachyman, acanthopanax polysaccharide, angelica polysaccharide, lentinan and ophiopogon polysaccharide.
In an improvement, the animal polysaccharide is any one or a mixture of a sea cucumber polysaccharide, an oyster polysaccharide and a pilose antler polysaccharide.
The improvement is that when the nano-carrier material is phospholipid and cholesterol, the nano-carrier is prepared by adopting a film dispersion-hydration drug loading method.
The preparation method of the antigen and active polysaccharide based co-carried nano preparation comprises the following steps:
completely dissolving the antigen and the active polysaccharide by using a buffer solution with the pH of 7.4 to obtain a medicinal solution;
and 3, adding the thin film dispersion obtained in the step 1 into the drug solution obtained in the step 2, hydrating, and carrying out ice-bath ultrasonic treatment by using a 200-400W probe to obtain the nano preparation with the light blue opalescence.
As an improvement, the specific preparation process of the step 1 is as follows: weighing 200 mg of phospholipid, and according to the weight ratio of phospholipid: weighing cholesterol at a weight ratio of 5:1, mixing, dissolving in 20 mL ethanol, placing in 250 mL eggplant-shaped bottle, and evaporating to dryness under reduced pressure at 37 deg.C to form film to obtain film dispersion.
The improvement is that the specific steps of the step 2 are as follows: when the antigen is 5 mg of ovalbumin, the ratio of ovalbumin: when the mass ratio of the active polysaccharide is 1:10, the active polysaccharide is completely dissolved by PBS buffer solution (pH 7.4) with the concentration of 0.02 mol/L, and then the medicine solution is obtained.
The improvement is that the specific steps of step 3 are as follows: and (3) adding the film dispersion obtained in the step (1) into the solution containing the ovalbumin and the active polysaccharide obtained in the step (2), hydrating for 20 min at 37 ℃, and then carrying out ultrasonic treatment for 5 min by using a 200 w probe in an ice bath to obtain the co-carried nano preparation with light blue opalescence.
The co-carried nano preparation is applied to tumor immunotherapy, wherein tumors include but are not limited to lung cancer, esophageal cancer, cervical cancer, pancreatic cancer, breast cancer, kidney cancer, gastric cancer or lymph cancer.
Has the advantages that:
compared with the prior art, the antigen and active polysaccharide composition provided by the invention can activate tumor immune reaction, and the active polysaccharide is used as an immunopotentiator to improve the immune function of an organism by stimulating the generation of immune cells and cytokines, so that an effective tumor immunotherapy effect is achieved by combining the antigen. The antigen and the active polysaccharide are co-loaded in the nano carrier material, so that the co-loading nano preparation has an ideal encapsulation effect, prolongs the action time of the antigen and the active polysaccharide in vivo, realizes the co-delivery effect of the composition, and further obviously improves the tumor immunotherapy effect.
Drawings
FIG. 1 is a cumulative release profile of ovalbumin and astragalus polysaccharide from a co-loaded nano-formulation;
FIG. 2 is a comparison of the levels of the immunocytokines IFN- γ from different polysaccharide groups;
FIG. 3 is a comparison of the levels of immunocytokine IFN- γ in different drug groups;
FIG. 4 is a graph showing the body weight change of each group of tumor-bearing mice;
FIG. 5 is a graph showing the change in tumor volume of each group of tumor-bearing mice.
The specific implementation mode is as follows:
the following examples are intended to further illustrate the present invention, but the present invention is not limited by the following examples.
The antigen ovalbumin and the T lymphoma cell E.G7-OVA used in the embodiment of the invention are conventional commercial test materials;
the molecular weight of the active polysaccharide is 10 KD-1000 KD.
Example 1 preparation of Co-loaded Nanodiulation of antigen ovalbumin and active polysaccharide Astragalus polysaccharides
(1) The amount of phospholipid was weighed to 200 mg, phospholipid: cholesterol is 5:1 (w/w), after mixing, the mixture is dissolved by 20 mL of ethanol, the mixture is placed in a 250 mL eggplant-shaped bottle, and the mixture is decompressed, rotated and evaporated to dryness at 37 ℃ to form a film, so that a film dispersion is obtained;
(2) weighing 5 mg of antigen ovalbumin, wherein the weight ratio of the ovalbumin: the astragalus polysaccharide is 1:10 (w/w), and is completely dissolved by PBS buffer solution (pH 7.4) with the concentration of 0.02 mol/L;
(3) and (3) adding the film dispersion obtained in the step (1) into the solution containing the ovalbumin and the astragalus polysaccharide obtained in the step (2), hydrating for 20 min at 37 ℃, and then carrying out ultrasonic treatment for 5 min by using a 200 w probe in an ice bath to obtain the nano preparation with light blue opalescence.
Example 2 preparation of Co-loaded Nanodiulation of antigen ovalbumin and oyster polysaccharide
(1) The amount of phospholipid was weighed to 200 mg, phospholipid: cholesterol of 5:1 (w/w), dissolving with 20 mL ethanol, placing in 250 mL eggplant-shaped bottle, and evaporating to dryness under reduced pressure at 37 deg.C to form film;
(2) weighing 5 mg of antigen ovalbumin, wherein the weight ratio of the ovalbumin: the oyster polysaccharide is 1:10 (w/w), and is completely dissolved by PBS buffer solution with pH 7.4;
(3) and (3) adding the film dispersion obtained in the step (1) into the solution containing the ovalbumin and the oyster polysaccharide obtained in the step (2), hydrating for 20 min at 37 ℃, and then carrying out ultrasonic treatment for 5 min in an ice bath by using a 200 w probe to obtain the antigen ovalbumin and oyster polysaccharide co-carried nano preparation.
Example 3 determination of particle size, Zeta potential and encapsulation efficiency of ovalbumin and astragalus polysaccharide co-carried nanometer preparation
(1) The fresh Nano-preparation prepared in example 1 was diluted appropriately, and the particle size and the distribution thereof were examined by a Zetasizer-Nano ZS90 particle size and Zeta potential analyzer, and the Zeta potential thereof was measured, and the results are shown in Table 1.
(2) The nano-drug preparation prepared in example 1 was separated and purified by dialysis, methanol was added to fix the volume, ultrasonic disruption was performed, the ovalbumin content in the preparation was measured by coomassie brilliant blue method, the content of astragalus polysaccharide in the preparation was measured by phenol-sulfuric acid method, and the Encapsulation Efficiency (EE) was calculated, and the results are shown in table 1.
TABLE 1 measurement of particle size, Zeta potential and encapsulation efficiency of the Nanoparticulate (mean. + -. standard deviation, n = 3)
As can be seen from table 1, the nano-formulation prepared in example 1 of the present invention has a nano-particle size and a good particle size distribution; and has higher drug encapsulation rate of antigen ovalbumin and astragalus polysaccharide, and is suitable for the encapsulation of the antigen ovalbumin and the astragalus polysaccharide and the subsequent research on the multidrug resistance treatment of tumors.
EXAMPLE 4 examination of in vitro Release behavior of Nanodiesents
Precisely sucking 0.1 mL of the isolated and purified nano preparation prepared in example 1 into a dialysis bag, placing the dialysis bag into 200 mL of PBS release medium (pH 7.4), shaking the dialysis bag in a shaking table at 37 ℃ and 100 r/min, and periodically taking out 200 μ L of the release medium while supplementing fresh release medium with the same volume and temperature. The content of ovalbumin and astragalus polysaccharide in the release medium is respectively measured by adopting a Coomassie brilliant blue method and a phenol-sulfuric acid method, the cumulative release degree is calculated, a cumulative release curve is drawn, the in vitro release behavior is inspected, and the result is shown in figure 1.
Cumulative release (%) = [ C%n*V0+(C1+C2+C3+……+Cn-1)*V]/WGeneral assemblyWherein: cnIs the concentration of the nth sample point, V0For releasing the volume of the medium, V is the volume sampled at each time, WGeneral assemblyIs the total amount of the medicine.
In an in vitro release experiment, under the condition of pH 7.4, the prepared medicinal preparation is slowly released, the accumulated release amounts of antigen ovalbumin and astragalus polysaccharide in 48 hours are 47.3 percent and 44.2 percent respectively, a better medicament slow release effect is shown under physiological conditions, the medicament can be effectively carried to an action part, and the in vivo action time is prolonged.
Example 5 immunization Studies of different drug groups
And (3) carrying the subcutaneous tumor of the mouse T lymphoma cell E.G7-OVA cell into a female C57BL/6 mouse body to establish an animal model. The tumor volume of the mouse to be tumor-bearing was about 50 mm on the fifth day3And (3) grouping: a normal saline control group, an antigen group (5 mg ovalbumin/kg), an astragalus polysaccharide group (50 mg polysaccharide/kg), an antigen and astragalus polysaccharide group (5 mg ovalbumin/kg +50 mg polysaccharide/kg) and an antigen and astragalus polysaccharide contained nano-preparation group (the co-loading nano-preparation of the embodiment 1) are respectively adopted; meanwhile, a lentinan group (50 mg polysaccharide/kg), an ophiopogon polysaccharide group (50 mg polysaccharide/kg), an antigen and lentinan group (5 mg ovalbumin/kg +50 mg polysaccharide/kg) and an antigen and ophiopogon polysaccharide group (5 mg ovalbumin/kg +50 mg polysaccharide/kg) are arranged, and the immune effect of different polysaccharide combinations is examined. Different drug groups are injected into tumor-bearing mice for administration by subcutaneous injection every three days, the administration dose is respectively 5 mg ovalbumin/kg and 50 mg polysaccharide/kg (the administration dose of the co-carried antigen and astragalus polysaccharide nano preparation group is 5 mg ovalbumin/kg and 50 mg astragalus polysaccharide/kg), splenocytes are obtained after the sacrifice on the 14 th day, and the content of the cell factor IFN-gamma in the splenocytes is determined by ELISA.
The cell factor is a protein molecule produced by immunocompetent cells and related cells, mainly plays a role in regulating the immune response of an organism, and IFN-gamma is a vital cell factor in antitumor immunity. The observation of the immune effect of different polysaccharide and antigen combinations shows that (figure 2), the expression of the cell factor IFN-gamma of the antigen and astragalus polysaccharide group is higher than that of the antigen and lentinan group and the antigen and ophiopogonpolysaccharide group, which shows that the astragalus polysaccharide has better immune promoting effect compared with lentinan and ophiopogonpolysaccharide.
The experimental determination result shows that (figure 3) the contents of the cell factors IFN-gamma of the ovalbumin and astragalus polysaccharide group are obviously higher than those of the ovalbumin and astragalus polysaccharide group independently, which shows that the tumor antigen and the astragalus polysaccharide have better tumor immunity effect. Meanwhile, the combined nano preparation group containing the ovalbumin and the astragalus polysaccharide can promote the effect of the drug to be effectively exerted, and has higher expression level of the cell factor IFN-gamma.
EXAMPLE 6 Co-Loading composition Nanodiulation in vivo tumor immunotherapy Studies
(1) Establishment of tumor-bearing mouse model
Taking T lymphoma cells in logarithmic growth phase E.G7-OVA, and adjusting cell suspension concentration to 4 × 10 with sterile cell culture solution6cells/mL. 0.2 mL of cell suspension was injected subcutaneously in the right axilla of female C57BL/6 mice. After inoculation, the tumor size is observed, measured and recorded, and when the requirement is met, the tumor size is used for subsequent experiments.
(2) In vivo antitumor Activity study
The tumor volume of the mouse to be tumor-bearing was about 50 mm on the fifth day3At that time, it started as a tumor model. Tumor-bearing mice were randomly divided into five groups (six per group): respectively, normal saline control group, antigen group, Astragalus polysaccharides group, antigen and Astragalus polysaccharides group, nanometer preparation group containing ovalbumin and Astragalus polysaccharides. Each group is administrated by intravenous injection through a mouse tail every three days for four times, the administration dose of each time is 5 mg of ovalbumin/kg, and 50 mg of astragalus polysaccharide/kg are taken as the basis (the administration dose of the ovalbumin and astragalus polysaccharide co-carried nano preparation group is 5 mg of ovalbumin/kg, and 50 mg of astragalus polysaccharide/kg). The body weight of the mice was weighed every two days, and the length of the tumor was measured and recorded. Tumor volume was determined by the formula: v = 0.5 × length × wide2And (6) performing calculation. The in vivo tumor immunotherapy activity and safety of each group were investigated by analyzing the changes in body weight and tumor volume of each group of mice.
The body weight changes of each group of tumor-bearing mice during the course of the in vivo tumor immunotherapy activity study experiment are shown in fig. 4. The change in body weight of animals was also used as one of the indicators for evaluating safety. The change of the body weight of each group is small, and compared with a normal saline control group, the body weight of each treatment group is increased, which shows the safety of the antigen ovalbumin and active polysaccharide astragalus polysaccharide composition and the prepared co-carried composition nano preparation.
Compared with the rapid increase of the tumor volume of the tumor-bearing mice in the normal saline group, the other treatment groups all showed a certain degree of tumor inhibition, as shown in fig. 5. The growth inhibition rates of the astragalus polysaccharide, the ovalbumin group, the ovalbumin and astragalus polysaccharide combined group to the tumor are respectively 38.2 percent, 49.2 percent and 63.1 percent (P is less than 0.01), the ovalbumin and astragalus polysaccharide co-carried nano preparation group shows the strongest tumor immunotherapy effect, and the tumor growth inhibition rate reaches 72.4 percent (P is less than 0.01).
The combined application of the ovalbumin and the astragalus polysaccharide shows that the antigen has stronger tumor immunotherapy effect than the single application of the antigen; and the antigen ovalbumin and astragalus polysaccharide are carried together with the nano preparation group, and the ovalbumin and the astragalus polysaccharide are carried together to have the strongest anti-tumor effect through the synergistic effect and the slow release to prolong the action time, thereby achieving the more effective tumor immunotherapy effect.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (10)
1. A co-carried nanometer preparation based on antigen and active polysaccharide is characterized by comprising the following components: antigens, active polysaccharides and nanocarrier materials.
2. The co-carried nanometer preparation based on the antigen and the active polysaccharide as claimed in claim 1, wherein the antigen is ovalbumin, and the active polysaccharide is plant polysaccharide or animal polysaccharide.
3. The antigen and active polysaccharide based co-carried nanometer preparation as claimed in claim 1, wherein the plant polysaccharide is one or more of astragalus polysaccharides, ginseng polysaccharides, glycyrrhiza polysaccharides, dandelion polysaccharides, pachyman, acanthopanax polysaccharides, angelicae sinensis polysaccharides, lentinan, and ophiopogon polysaccharides.
4. The antigen and active polysaccharide based co-carried nanometer preparation as claimed in claim 1, wherein the animal polysaccharide is one or more of sea cucumber polysaccharide, oyster polysaccharide, and cartialgenous polysaccharide.
5. The antigen and active polysaccharide based co-carried nanometer preparation as claimed in claim 1, wherein when the nanometer carrier material is phospholipid and cholesterol, the preparation is carried out by thin film dispersion-hydration drug loading method.
6. The method for preparing the antigen and active polysaccharide co-carried nano preparation based on claim 5 is characterized by comprising the following steps: step 1, ultrasonically dissolving phospholipid and cholesterol into an organic solvent, and rotationally evaporating the organic solvent to form a film dispersion; completely dissolving the antigen and the active polysaccharide by using a buffer solution with the pH of 7.4 to obtain a medicinal solution; and 3, adding the thin film dispersion obtained in the step 1 into the drug solution obtained in the step 2, hydrating, and carrying out ice-bath ultrasonic treatment by using a 200-400W probe to obtain the nano preparation with the light blue opalescence.
7. The method for preparing the antigen and active polysaccharide co-carried nanometer preparation according to claim 5, wherein the specific preparation process in the step 1 is as follows: weighing 200 mg of phospholipid, and according to the weight ratio of phospholipid: weighing cholesterol at a weight ratio of 5:1, mixing, dissolving in 20 mL ethanol, placing in 250 mL eggplant-shaped bottle, and evaporating to dryness under reduced pressure at 37 deg.C to form film to obtain film dispersion.
8. The method for preparing the antigen and active polysaccharide co-carried nanometer preparation according to claim 5, wherein the specific steps of the step 2 are as follows: when the antigen is 5 mg of ovalbumin, the ratio of ovalbumin: when the mass ratio of the active polysaccharide is 1:10, the active polysaccharide is completely dissolved by PBS buffer solution with pH 7.4, and then the medicine solution is obtained.
9. The method for preparing the antigen and active polysaccharide co-carried nanometer preparation according to claim 5, wherein the specific steps of the step 3 are as follows: and (3) adding the film dispersion obtained in the step (1) into the solution containing the ovalbumin and the active polysaccharide obtained in the step (2), hydrating for 20 min at 37 ℃, and then carrying out ultrasonic treatment for 5 min in an ice bath by using a 200 w probe to obtain the co-carried nano preparation with light blue opalescence.
10. The use of the co-carried nano-formulation according to claim 1 or claim 5 for immunotherapy of tumors, wherein said tumors include, but are not limited to, lung cancer, esophageal cancer, cervical cancer, pancreatic cancer, breast cancer, kidney cancer, stomach cancer or lymph cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111458751.4A CN114099656A (en) | 2021-12-02 | 2021-12-02 | Co-loaded nano preparation based on antigen and active polysaccharide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111458751.4A CN114099656A (en) | 2021-12-02 | 2021-12-02 | Co-loaded nano preparation based on antigen and active polysaccharide and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114099656A true CN114099656A (en) | 2022-03-01 |
Family
ID=80365719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111458751.4A Pending CN114099656A (en) | 2021-12-02 | 2021-12-02 | Co-loaded nano preparation based on antigen and active polysaccharide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114099656A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6340461B1 (en) * | 1996-12-17 | 2002-01-22 | David Stephen Terman | Superantigen based methods and compositions for treatment of diseases |
| CN103191424A (en) * | 2012-06-20 | 2013-07-10 | 南京农业大学 | Astragalus polysaccharide nanoliposome capable of improving livestock and poultry immunity and preparation method thereof |
| CN105884719A (en) * | 2016-04-14 | 2016-08-24 | 沈阳药科大学 | Preparation and application of taxane prodrug |
| CN106924747A (en) * | 2017-03-17 | 2017-07-07 | 临沂大学 | Imitative lipoprotein structure pharmaceutical carrier of a kind of nanometer and its preparation method and application |
| CN108653211A (en) * | 2018-06-12 | 2018-10-16 | 佛山科学技术学院 | A kind of preparation method of Aps Liposomes |
| CN108969485A (en) * | 2018-07-15 | 2018-12-11 | 启东创潞新材料有限公司 | A kind of preparation method of Aps Liposomes |
| CN110755607A (en) * | 2019-11-19 | 2020-02-07 | 中国科学院过程工程研究所 | Zinc oxide and antigen co-drug-loaded nano vaccine, and preparation method and application thereof |
| CN113476405A (en) * | 2021-08-12 | 2021-10-08 | 临沂大学 | Nanometer medicinal preparation for treating multidrug resistant tumor, and its preparation method and application |
-
2021
- 2021-12-02 CN CN202111458751.4A patent/CN114099656A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6340461B1 (en) * | 1996-12-17 | 2002-01-22 | David Stephen Terman | Superantigen based methods and compositions for treatment of diseases |
| CN103191424A (en) * | 2012-06-20 | 2013-07-10 | 南京农业大学 | Astragalus polysaccharide nanoliposome capable of improving livestock and poultry immunity and preparation method thereof |
| CN105884719A (en) * | 2016-04-14 | 2016-08-24 | 沈阳药科大学 | Preparation and application of taxane prodrug |
| CN106924747A (en) * | 2017-03-17 | 2017-07-07 | 临沂大学 | Imitative lipoprotein structure pharmaceutical carrier of a kind of nanometer and its preparation method and application |
| CN108653211A (en) * | 2018-06-12 | 2018-10-16 | 佛山科学技术学院 | A kind of preparation method of Aps Liposomes |
| CN108969485A (en) * | 2018-07-15 | 2018-12-11 | 启东创潞新材料有限公司 | A kind of preparation method of Aps Liposomes |
| CN110755607A (en) * | 2019-11-19 | 2020-02-07 | 中国科学院过程工程研究所 | Zinc oxide and antigen co-drug-loaded nano vaccine, and preparation method and application thereof |
| CN113476405A (en) * | 2021-08-12 | 2021-10-08 | 临沂大学 | Nanometer medicinal preparation for treating multidrug resistant tumor, and its preparation method and application |
Non-Patent Citations (5)
| Title |
|---|
| RUONAN BO等: "Optimization on conditions of Lycium barbarum polysaccharides liposome by RSM and its effects on the peritoneal macrophages function", CARBOHYDRATE POLYMERS, vol. 117, no. 6, pages 215 - 222 * |
| RUONAN BO等: "Simple nanoliposomes encapsulating Lycium barbarum polysaccharides as adjuvants improve humoral and cellular immunity in mice", INTERNATIONAL JOURNAL OF NANOMEDICINE, vol. 12, pages 6289 - 6301 * |
| YUNPENG FAN等: "Liposome can improve the adjuvanticity of astragalus polysaccharide on the immune response against ovalbumin", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 60, pages 206 - 212, XP028683332, DOI: 10.1016/j.ijbiomac.2013.05.030 * |
| ZHENGUANG LIU等: "Ganoderma lucidum polysaccharides encapsulated in liposome as an adjuvant to promote Th1-bias immune response", CARBOHYDRATE POLYMERS, vol. 142, no. 20, pages 141 - 148 * |
| 范云鹏等: "正交试验优选黄芪多糖脂质体的制备工艺", 中草药, vol. 42, no. 3, pages 470 - 473 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20170196902A1 (en) | Antitumor use of cyclic dinucleotide cgmp | |
| CN100467059C (en) | Lactoferrin in the treatment of malignant neoplasms and other hyperproliferative diseases | |
| Cheng et al. | The effects of polysaccharides from the root of Angelica sinensis on tumor growth and iron metabolism in H22-bearing mice | |
| CN112826808B (en) | Neutral/cation mixed lipid nano preparation of cyclic dinucleotide or analogue thereof and application thereof | |
| CN108892700B (en) | Novel anti-tumor compound and application thereof in preparing anti-tumor medicine | |
| WO2017162055A1 (en) | Application of cyclic dinucleotide cgamp-liposome for resisting tumours | |
| CN105983097B (en) | Anti-tumor preparation and preparation method thereof | |
| US9901602B2 (en) | Ejaculum of animals as medicinal material and uses thereof in medicaments for treatment of diseases such as tumors, depression, etc | |
| CN114099656A (en) | Co-loaded nano preparation based on antigen and active polysaccharide and preparation method and application thereof | |
| CN110448681B (en) | Combined medicine for malignant tumor immunotherapy | |
| CN115261319A (en) | Polypeptide-induced amplified NK (Natural killer) cell and application thereof in cell therapy | |
| CN106540253A (en) | The application of cGAMP and its derivant in anti-tumor vaccine is prepared | |
| CN113151371A (en) | Probiotic extracellular polysaccharide, preparation method and anti-tumor application thereof | |
| TW201127959A (en) | Method of producing immune killer cell | |
| WO2022222388A1 (en) | Use of l-sorbose in preparation of drug for treating tumors | |
| CN109453195B (en) | Pharmaceutical composition for inhibiting tumor cells | |
| CN115364206A (en) | Combined anti-tumor vaccine and preparation method thereof | |
| CN101327317B (en) | Injection for treating cancer and its preparation method | |
| CN106119192A (en) | Compositions and the application in CIK cell is cultivated thereof | |
| CN116687971A (en) | Application of gamma delta T cells in preparation of antitumor drugs | |
| Xiong et al. | Natural polysaccharides exert anti-tumor effects as dendritic cell immune enhancers | |
| CN111450110A (en) | Application of achyranthes bidentata polysaccharides in preparation of medicines for preventing non-small cell lung cancer metastasis | |
| Wang | Application of cytokines in the treatment of breast cancer | |
| CN117717565A (en) | Application of antisense oligonucleotide in preparing medicament for treating esophagus cancer | |
| CN114796458A (en) | Steam equipment multivitamin for adjuvant therapy of tumors and preparation method and application thereof |
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 |