CN106692981A - Preparation method and application of cationization lentinan - Google Patents
Preparation method and application of cationization lentinan Download PDFInfo
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- CN106692981A CN106692981A CN201710015438.0A CN201710015438A CN106692981A CN 106692981 A CN106692981 A CN 106692981A CN 201710015438 A CN201710015438 A CN 201710015438A CN 106692981 A CN106692981 A CN 106692981A
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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Abstract
The invention relates to a preparation method and application of cationization lentinan. The cationization lentinan can be used as a gene vector delivery system, plays an immunoregulation effect, and effectively solves the application problem in tumor treatment. The invention adopts a technical scheme: the lentinan is connected with low-molecular-weight polyethyleneimine by virtue of a pH sensitive chemical bond, the molecular weight of the lentinan is 20 to 800 kD, the molecular weight of the polyethyleneimine is 600 to 1200 D, and a molar ratio of the polyethyleneimine to the lentinan is 1 : (3 to 10). The preparation process is simple, the sources of raw materials are wide, the cost is low, and the development and application prospect is promising; the prepared cationization lentinan is uniform in shape, the particle size is about 200 nm and is uniform in distribution, the structure is stable, the biological compatibility is good, nucleic acid can be effectively loaded and delivered to tumor cells, and an antitumor effect combining gene therapy and immunotherapy is realized.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of cationization with antitumor activity and delivery of nucleic acids function
Lentinan preparation method and its application of immunologic competence is played while as genophore.
Background technology
Tumour is one of common disease of threat human health and life, and its morbidity and mortality is in rising trend, state
The following 20 years newly-increased tumor cases in the whole world of border DKFZ prediction can rise to annual 22000000, tumor patient death
To rise to annual 13000000 (IARC Science Public Lyon, 2014,1: 1365).Tumour has turned into the whole world
The extremely important public health disease of concern.At present, most of chemotherapeutics of oncotherapy is clinically used for, it is main by suppression
Cell propagation processed and tumour growth play its effect, also kill normal cell while killing tumor cell, the doctor of its costliness
Treatment expense and larger toxic and side effect have had a strong impact on the quality of life of patient, have become patient and that scholar is faced is common
Problem, thus people have been look for the tumor therapeuticing method of optimization.
Chinese medicine increasingly shows very big advantage in terms of oncotherapy, it has also become the Yi great Te of China's therapeutic field of tumor
Color.Polysaccharide is that traditional Chinese medicine research is most deep, one of widest anti-tumor active ingredient, even more the master of the antitumor physiological action of Chinese medicine
One of material is wanted, is referred to as " BRM ".Chemotherapeutics from direct killing tumour cell is different, and herbal polysaccharide is removed
Having directly suppress outside tumour cell effect, more importantly stimulates reticuloendothelial system by its immunoregulation effect, sharp
Panimmunity cell living, improves specific antigen immune response ability of the host to tumour cell, and this is also preventing and treating tumor recurrence
With the essential condition of transfer.China of State of Zhao waits (Acta Pharmaceutica Sinica, 2003,38:37) report, Chinese yam polysaccharide to Lewis lung cancer and
B16 melanomas have optimal inhibitory action.Lee is small to determine grade (Hua Zhong Agriculture University's journal, 2002,21:261) experiment card
Bright, grifolan not only direct killing cancer cell but also can improve the immunity of body.Xie Zunjiang etc. (dissection journal, 2002,
33:538) find panaxan in itself or induction produce cell factor can strengthen IL-2 to NK cells and tumor-infiltrated lymph
The stimulation of cell (TIL), there is extremely strong killing and inhibitory action to tumour.The research of Chinese medicine antitumor mechanism is swollen for treatment
Knurl provides theoretical foundation, while also opening new way.
Lentinan (Lentinan, LNT) is to be separated from the mushroom of traditional integration of drinking and medicinal herbs, extract the medical active for obtaining
Composition.The polysaccharide of the antitumor activities that take the lead in being extracted from mushroom such as Chihara in 1969, active component is its branched structure
In β-(1 → 3)-D- glucans, the glucosyl group of its main chain is formed by connecting by β-(1 → 3) glycosidic bond, the glucose of side chain
Base is connected and formed by β-(1 → 6) glycosidic bond being distributed, and a large amount of active group-OH are contained in molecular structure.Clinically, by mushroom
Polysaccharide can be used to be unable to the auxiliary treatment (Carbohydrate of surgery excision or transfer and relapse tumour with chemotherapeutics combination
Polymers, 2012, 88: 966-972).For example, it can activate NLRP3 inflammatories body and ASK1/ with taxol combination
P38MAPK signal paths induction A549 Apoptosis (Critical Reviews In Food Science and
nutrition, 2015, 10: 132).Lentinan/S-1 therapeutic alliance patients with gastric cancer, as a result primary tumor be obviously reduced, and
Abdominal CT is not detected by lymphatic metastasis (Carbohydrate Polymers, 2016,137: 52).Research discovery, mushroom
Polysaccharide does not have direct repression to tumour cell in itself, but activates T cell, promotes the generation of lymphocyte activating factor (LAF),
Various ThFs are discharged, strengthens host's Peritoneal macrophage function, be finally reached killing tumor cell.
The generation of tumour is directly or indirectly related all with gene to development, can directly be corrected from gene level treatment tumour
The gene relevant with tumor invasion with repairing, effectively reaches the purpose for the treatment of tumour.And the success or not of gene therapy tumour exists
It is heavily dependent on safe and efficient gene delivery vector.With polysaccharide as skeleton, obtained through polyethyleneimine etc. is modified
Nano-carrier not only has the advantages that good biocompatibility, while Efficient Compression nucleic acid and can also transfect to cell (Advanced
Drug Delivery Reviews, 2013, 65: 1123-1147).Therefore, polysaccharide nano-carrier has turned into current gene
Transmit one of the study hotspot of carrier (Zhengzhou University's journal, 2014,4: 457-460).Contain in lentinan molecular structure
A large amount of active groups, are capable of achieving the function of gene delivery, and remain lentinan by carrying out cationization modification to it
Bioactivity, but so far there are no to relevant cationization lentinan as gene delivery vector while play again and be immunized
The open report of adjustment effect.
The content of the invention
For above-mentioned situation, to solve the defect of prior art, the purpose of the present invention is just to provide a kind of cationization perfume
The preparation method of mushroom polysaccharide, as gene carrier delivery system and can play immunoregulation effect, effectively in solution oncotherapy
Application problem.
The technical scheme that the present invention is solved is, by pH sensitive chemicals key by the polyethylene of lentinan and low-molecular-weight
Imines is connected, and lentinan molecular weight is 20-800kD, and polyethyleneimine molecular weight is 600-1200D, polyethyleneimine and perfume
The mol ratio of mushroom polysaccharide is 1:3-10.Specifically preparation method is：
1）50-150mg lentinans are dissolved in 8-10ml deionized waters or dimethyl sulfoxide (DMSO), in 23-27 DEG C, nitrogen protection
It is lower to react 4-48h with 100-450mg activating reagent A lucifuges, reaction solution is dialysed 2-3d, or 50 DEG C with 100-450mg work
Change reagent B reaction 4-24h, obtain the lentinan of activation；Described activating reagent A is potassium metaperiodate, ethylenediamine, contains 3-5% second
The phosphate buffer of diamines, carbonyl dimidazoles, triethylamine one or more, activating reagent B is maleic anhydride, amber
Acid anhydrides, 4- dimethylamino pyridines, 1- ethyls (3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate, N- hydroxysuccinimidyls acyl are sub-
Amine one or more；
2）100-300mg polyethyleneimines are dissolved in the hydrochloric acid or dimethyl sulfoxide (DMSO) that 2-6ml volumetric concentrations are 3.7%, then
It is grafted on the lentinan of activation under nitrogen protection, or directly by 100-300mg grafting polyethylene imines to activation
On lentinan, at 23-27 DEG C, under nitrogen protection, lucifuge reaction 24-72h, dialysed 2-3d in ultra-pure water, and freeze-drying is obtained
The amine-modified lentinan of polyethyleneimine, that is, be cationized lentinan.
Preparation technology of the invention is easy, and raw material sources are relatively broad, with low cost, great development and application prospect, system
The standby cationization lentinan form for obtaining is homogeneous, and particle diameter is evenly distributed in 200 nm or so, Stability Analysis of Structures, bio-compatible
Property it is good, high-efficient carrier nucleic acid and tumour cell can be delivered to, realize gene therapy with the united antitumor work of immunization therapy
With.
Specific embodiment
Specific embodiment of the invention is described in further detail with reference to embodiments.In following embodiments, such as
Without specified otherwise, institute is conventional method, material therefor, reagent etc. using experimental technique can be purchased from biological or chemical company
Buy.
Embodiment 1
In specific implementation, the cationization lentinan can be the present invention, 1）100 mg lentinans are dissolved in 10 mL
In ionized water, 150mg potassium metaperiodates are added while stirring, at 25 DEG C, the lower lucifuge reaction 48h of nitrogen protection, then instill and contain 200
The 5mL phosphate buffers of μ L ethylenediamines, in completion of dropping in 1h, the lower lucifuge of nitrogen protection stirs 36 h, by reaction solution in super
Dialyse 3d in pure water, obtains the lentinan reaction solution of activation；
2）Again to being added dropwise over the 2mL volumetric concentrations containing 250mg polyethyleneimines and be in the lentinan reaction solution of activation
3.7% hydrochloric acid, at 25 DEG C, under nitrogen protection, lucifuge stirs 72 h, and dialyse 2d in ultra-pure water, and freeze-drying obtains polyethylene
The lentinan of imines modification, that is, be cationized lentinan.
Embodiment 2
In specific implementation, the cation lentinan is also possible that 1 to the present invention）100mg lentinans are dissolved in 8mL diformazans
In base sulfoxide, 30mg carbonyl dimidazoles and 100 μ L triethylamines are subsequently adding, at 26 DEG C, the lower lucifuge stirring 4h of nitrogen protection obtains work
The lentinan reaction solution of change；
2）Again to activation lentinan reaction solution in be added dropwise over the 6mL dimethyl sulfoxide (DMSO)s containing 100mg polyethyleneimines
（DMSO）Solution, at 23 DEG C, under nitrogen protection, lucifuge stirring 24h, dialyse 2d in ultra-pure water, and freeze-drying obtains polyethylene
The lentinan of imines modification, that is, be cationized lentinan.
Embodiment 3
In specific implementation, the cation lentinan is also possible that 1 to the present invention）100 mg lentinans are dissolved in 10 mL bis-
In methyl sulfoxide, 10mg 4- dimethylamino pyridines and 400mg maleic anhydrides are sequentially added, 24h is stirred at 50 DEG C, then add successively
Enter 10mg 1- ethyls (3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate and 5mg N-hydroxy-succinamide stirring reactions
4h, obtains the lentinan reaction solution of activation；
2）Again to 300mg polyethyleneimines are added in the lentinan reaction solution of activation, under nitrogen protection, lucifuge stirs 48h, in
Dialyse 2d in ultra-pure water, freeze-drying, obtains the amine-modified lentinan of polyethyleneimine, that is, be cationized lentinan.
Embodiment 4
In specific implementation, the cationization lentinan is also possible that 1 to the present invention）100 mg lentinans are dissolved in 10mL
In dimethyl sulfoxide, 10mg 4- dimethylamino pyridines and 400mg succinyl oxides are sequentially added, 24h is stirred at 50 DEG C, then add successively
Enter 10mg l- ethyls (3 one dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and the stirring of 50mgN- hydroxysuccinimides is anti-
4h is answered, the lentinan reaction solution of activation is obtained；
2）Again to 300mg polyethyleneimines are added in the lentinan reaction solution of activation, under nitrogen protection, lucifuge stirring 48h will
Reaction solution is dialysed 3d in ultra-pure water, and freeze-drying obtains the amine-modified lentinan of polyethyleneimine, that is, the mushroom that is cationized is more
Sugar.
Cationization lentinan prepared by the present invention can obtain preferable result through repeatedly testing repeatedly, reach full
The technique effect of meaning, relevant testing data is as follows：
Experiment one：The cationization lentinan of different quality is dissolved in 20 μ L deionized waters, 2 μ g plasmid eGFP, room are added
Temperature is incubated 30 min, carries out the detection of 0.7 % agarose gel electrophoresis.Experimental result shows, when addition cationization lentinan
It is 16 with plasmid eGFP mass ratioes:When 1, plasmid eGFP bands are trapped in duct completely, and cation lentinan is worked as in this explanation
Nucleic acid can completely be loaded.By different quality than cationization lentinan and plasmid eGFP compounds carry out particle diameter and current potential
Detection, as lentinan ratio increases, composite-grain diameter can taper into, and illustrate plasmid eGFP completely by polysaccharide with reference to simultaneously
Form fine and close compound.Then continue increase when mass ratio increases current potential, to current potential during certain value 0 or so, illustrate when it is positive from
Sub- lentinan is interacted by positive and negative charge and loads nucleic acid completely, and this is consistent with agarose gel electrophoresis result.
Experiment two：Hepatocellular carcinoma H22 is in the culture medium containing 10 % hyclones and 37 DEG C, 5% CO2Under the conditions of often
Rule culture.According to 1.5 × 105Individual/hole is inoculated in the h of 6 orifice plate culture 16, and the siRNA (siPD-L1) that FAM is marked is added
In culture medium, after 4 h of transfection, flow cytometer determines intake, experimental result display cation lentinan load siPD-L1
Intake be 96.2%, test result indicate that cation lentinan can be by delivery of nucleic acids to intracellular.
Experiment three：Breast cancer cell MCF-7 is in the culture medium containing 10 % hyclones and 37 DEG C, 5% CO2Under the conditions of
Cellar culture, according to 5 × 10316 h are cultivated in individual/hole in being inoculated in 96 orifice plates, and the cation lentinan of different quality is added
To in culture medium, bred with tetramethyl azo mile salt colorimetric method for determining cell after culture 24h.Experimental result shows, in 0-640 μ
In the range of g/mL, with the increase of cation lentinan content, cell survival rate is increased slightly, result of the test show sun from
Sub- lentinan has good biocompatibility.
Experiment four：Take the kunming mice 15 of female, every group 5 random point three groups：Blank control group (NC), lentinan
Group (LNT), cationization lentinan group (LNT-PEI), respectively by tail vein injection saline, lentinan solution
（Dosage is 12 mg/kg）, cationization lentinan solution（Dosage is 12 mg/kg）.Every 2 d is administered once, and gives altogether
Medicine 7 times.Mouse animation, detection Mouse Weight change are observed in experimentation.Last time is administered second day after terminating
Mouse is put to death, each group mice organs are taken, and peritoneal macrophage and splenocyte carry out anti tumor activity in vitro investigation.Result shows
Show, compared with NC groups, the changes of weight and organ index of LNT groups and LNT-PEI group mouse are without notable difference.It is external anti-swollen
Tumor activity investigates result and shows that LNT groups and LNT-PEI group splenocyte inhibiting rates are respectively 20.74%, 25.57%, and macrophage is thin
Born of the same parents' inhibiting rate is respectively 10.17%, 18.12%.RT-PCR results show, compared with NC groups the spleen of LNT groups and LNT-PEI groups,
The IL-2 of thymus gland, TNF-α, the mrna expression amount of IFN-γ increased.Illustrate that cationization lentinan maintains mushroom
The immunologic competence of polysaccharide itself, can still play biological regulation effect in vivo.
In above-mentioned experiment, used cell line, plasmid, siRNA is：
1st, cell line：Hepatocellular carcinoma H22, breast cancer cell MCF-7 is purchased from Chinese Academy of Sciences's cell bank.
2nd, plasmid：Enhanced green fluorescence protein expression plasmid eGFP (the GenBank numbers of logging in X83959), purchased from BD
Biosciences Clontech companies.
3rd, siRNA（siPD-L1）：
Positive-sense strand, 5 '-UUCUCCGAACGUGUCACGUTT-3 '；
Antisense strand, 5 '-ACGUGACACGUUCGGAGAATT-3 '.
Application of the subject cationic lentinan as gene delivery vector in tumor prevention with treatment, also includes
External, internal antineoplastic biological assessment is carried out after cationization lentinan is mixed with Antioncogene medicine.
The Antioncogene medicine has：The DNA for being mounted with therapeutic gene is, the virus for being mounted with therapeutic gene
One or more in carrier DNA, ASON, siRNA.
Described tumour cell is the various solid tumor cells in mouse source or people source, including breast cancer cell, ovarian cancer cell,
Nasopharyngeal carcinoma cell, Human Tongue Carcinoma Lines, esophageal cancer cell, lung carcinoma cell, stomach cancer cell, HCC, pancreatic cancer cell, prostate cancer
In cell, kidney cancer cell, penis cancer cell, emerald green ball cancer cell, skin cancer cell, leukaemia, malignant melanoma cell
One kind.
Described tumour behaviour organ surface or the internal various solid tumors for occurring, including breast cancer, oophoroma, nasopharynx
Cancer, tongue cancer, the cancer of the esophagus, lung cancer, stomach cancer, liver cancer, cancer of pancreas, prostate cancer, kidney, carcinoma of penis, emerald green ball cancer, cutaneum carcinoma, white blood
One kind in disease, malignant mela noma.
Shown by above-mentioned, cationization lentinan preparation method of the invention obtains more full through repeated tests
The technique effect of meaning.The present invention while as nucleic acid delivery vector by by grafting polyethylene imine to lentinan, keeping
Lentinan immunocompetence, the cation lentinan synthesis material wide material sources in the present invention, preparation condition is readily satisfied,
Preparation cost is low, has both preferable water solubility, the physics and chemical property of stabilization, stronger nucleic acid load capacity, cation
The immunologic competence of lentinan itself is kept while changing lentinan as a kind of good gene delivery vector, is opened
Antineoplastic new approach, new method are combined in gene therapy with immunization therapy, with huge medical value and social benefit.
Claims (8)
1. a kind of cationization lentinan, it is characterised in that by pH sensitive chemicals key by lentinan and low-molecular-weight
Polyethyleneimine connection, lentinan molecular weight be 20-800kD, polyethyleneimine molecular weight be 600-1200D, polyethylene
Imines is 1 with the mol ratio of lentinan:3-10.
2. it is according to claim 1 cationization lentinan preparation method, it is characterised in that 1）50-150mg is fragrant
Mushroom polysaccharide is dissolved in 8-10ml deionized waters or dimethyl sulfoxide (DMSO), and at 23-27 DEG C, nitrogen protection is lower and 100-450mg is activated
Reagent A lucifuge reacts 4-48h, and reaction solution is dialysed 2-3d, or reacts 4-24h with 100-450mg activating reagents B at 50 DEG C,
The lentinan that must be activated；Described activating reagent A is potassium metaperiodate, ethylenediamine, the phosphate-buffered containing 3-5% ethylenediamines
Liquid, carbonyl dimidazoles, triethylamine one or more, activating reagent B be maleic anhydride, succinyl oxide, 4- dimethylamino
Pyridine, 1- ethyls (3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate, N-hydroxy-succinamide one or two with
On；
2）100-300mg polyethyleneimines are dissolved in the hydrochloric acid or dimethyl sulfoxide (DMSO) that 2-6ml volumetric concentrations are 3.7%, then
It is grafted on the lentinan of activation under nitrogen protection, or directly by 100-300mg grafting polyethylene imines to activation
On lentinan, at 23-27 DEG C, under nitrogen protection, lucifuge reaction 24-72h, dialysed 2-3d in ultra-pure water, and freeze-drying is obtained
The amine-modified lentinan of polyethyleneimine, that is, be cationized lentinan.
3. it is according to claim 2 cationization lentinan preparation method, it is characterised in that 1）By 100 mg mushrooms
Polysaccharide is dissolved in 10mL deionized waters, and 150mg potassium metaperiodates are added while stirring, in 25 DEG C, the lower lucifuge reaction of nitrogen protection
48h, then the 5mL phosphate buffers containing 200 μ L ethylenediamines are instilled, in completion of dropping in 1h, the lower lucifuge stirring of nitrogen protection
36 h, reaction solution is dialysed 3d in ultra-pure water, obtains the lentinan reaction solution of activation；
2）Again to being added dropwise over the 2mL volumetric concentrations containing 250mg polyethyleneimines and be in the lentinan reaction solution of activation
3.7% hydrochloric acid, at 25 DEG C, under nitrogen protection, lucifuge stirs 72 h, and dialyse 2d in ultra-pure water, and freeze-drying obtains polyethylene
The lentinan of imines modification, that is, be cationized lentinan.
4. it is according to claim 2 cationization lentinan preparation method, it is characterised in that 1）By 100mg mushrooms
Polysaccharide is dissolved in 8mL dimethyl sulfoxide (DMSO)s, is subsequently adding 30mg carbonyl dimidazoles and 100 μ L triethylamines, at 26 DEG C, under nitrogen protection
Lucifuge stirs 4h, obtains the lentinan reaction solution of activation；
2）It is molten to the 6mL dimethyl sulfoxide (DMSO)s containing 100mg polyethyleneimines are added dropwise in the lentinan reaction solution of activation again
Liquid, at 23 DEG C, under nitrogen protection, lucifuge stirring 24h, dialyse 2d in ultra-pure water, and freeze-drying obtains polyethyleneimine amine-modified
Lentinan, that is, be cationized lentinan.
5. it is according to claim 2 cationization lentinan preparation method, it is characterised in that 1）By 100 mg mushrooms
Polysaccharide is dissolved in 10 mL dimethyl sulfoxide (DMSO)s, sequentially adds 10mg 4- dimethylamino pyridines and 400mg maleic anhydrides, is stirred at 50 DEG C
24h is mixed, 10mg 1- ethyls (3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate and 5mg N- hydroxysuccinimidyls is sequentially added
Acid imide stirring reaction 4h, obtains the lentinan reaction solution of activation；
2）Again to 300mg polyethyleneimines are added in the lentinan reaction solution of activation, under nitrogen protection, lucifuge stirs 48h, in
Dialyse 2d in ultra-pure water, freeze-drying, obtains the amine-modified lentinan of polyethyleneimine, that is, be cationized lentinan.
6. it is according to claim 2 cationization lentinan preparation method, it is characterised in that 1）By 100 mg mushrooms
Polysaccharide is dissolved in 10mL dimethyl sulfoxides, sequentially adds 10mg 4- dimethylamino pyridines and 400mg succinyl oxides, is stirred at 50 DEG C
24h, sequentially adds 10mg l- ethyls (3 one dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and 50mgN- hydroxysuccinimidyls
Acid imide stirring reaction 4h, obtains the lentinan reaction solution of activation；
2）Again to 300mg polyethyleneimines are added in the lentinan reaction solution of activation, under nitrogen protection, lucifuge stirring 48h will
Reaction solution is dialysed 3d in ultra-pure water, and freeze-drying obtains the amine-modified lentinan of polyethyleneimine, that is, the mushroom that is cationized is more
Sugar.
7. the cationization lentinan described in claim 1 as nucleic acid delivery vector in anti-tumor medicine is prepared should
With.
8. the cationization lentinan that prepared by preparation method described in any one of claim 2-6 is as nucleic acid delivery vector in system
Application in standby anti-tumor medicine.
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| CN111647096A (en) * | 2020-05-20 | 2020-09-11 | 昆明理工大学 | Neutral maca polysaccharide and extraction method and application thereof |
| CN113501889A (en) * | 2021-07-06 | 2021-10-15 | 郑州大学 | Preparation method and application of pseudo-ginseng polysaccharide cationic derivative |
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