CN108066314A - A kind of pectin is the biological microsphere preparation method and application of wall material - Google Patents
A kind of pectin is the biological microsphere preparation method and application of wall material Download PDFInfo
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- 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
- A61K9/5005—Wall or coating material
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Abstract
The present invention relates to one kind using pectin as wall material biological microsphere preparation method and applications.The biological microsphere, preparation process is easy, and envelop rate is high, and cytoactive is good.It is characterized in that using pectin as microballoon wall material, it is added dropwise after cell or other biological activity substance are mixed with wall material in solidify liquid, is formed by curing the microballoon of matters of containing biological activities.The method preparation process is easy, and process is environmentally protective, it can be achieved that high density and activity is encapsulated.This embedding for carrying cell microsphere and can be used for the various actives substances such as cell, albumen, polypeptide, for the application in the fields such as structure and the protein drug transmission of external threedimensional model.
Description
Technical field
It is specifically a kind of to be used to embed using pectin as wall material the present invention relates to bioengineering and biomedicine field
The method for preparing microsphere of bioactive substance and its application, this method simple process, envelop rate is high, bioactivity is good, it can be achieved that
The high density of embedded object, high activity culture.
Research background
Pectin is a kind of heteroglycan being widely present in higher plant cell wall, between molecular mass 5-40 ten thousand.Fruit
Glue extracted to obtain in 1824 by French pharmacists Bracennot, and was named as pectin.Pectic backbone is with D- galas
The backbone that uronic acid (D-Galacturonic Acids, D-Gal-A) is formed by α-Isosorbide-5-Nitrae-glycosidic bond links, side chain is by mouse
The neutral sugars and D mannoses, L-fucose etc. such as Lee's sugar, arabinose, galactolipin and xylose monose form, as shown in Figure 3
(William G.T Willats, et al.Trends in Food Science＆Technology, 2006,17 (3):97-
104,Beli R,et al.Critical Reviews in Food Science and Nutrition,2005,37(1):
47-73).Pectin is widely used in food and biomedicine field, is the food additive from additive amount limitation that FAO/WHO recommends
Add agent, in field of medicaments, since Pectin calcium can be commonly used for colon locating administrated in colon by pectinase enzymatic hydrolysis, pectin
Systemic vectors material (LS Liu, et al.Biomaterals, 2003,24 (19):3333-3343), meanwhile, pectin is commonly used for
Thickener, stabilizer, suspending agent, emulsifier, prodrug carrier and filmogen etc..Pectin is that a kind of polygalacturonic acid is more
Pectin according to the difference of pectin esterification degree (DE), is divided into high-ester pectin (DE by sugar>And low-ester pectin (DE 50%)<50%).It is low
The net structure hydrogel to form " eggshell " sample can be complexed, it can be achieved that right in esterification pectin with high volence metal ions such as two, trivalents
Cell, pharmaceutical grade protein isoreactivity ingredient encapsulating (Y Fang, et al.Carbothydrate polymers, 2008,72:
334-341).Low this characteristic of esterification is similar to sodium alginate, and the latter is the more of the load cell microsphere that is most widely used at present
One of sugared material.Sodium alginate is derived from the plant polyose of brown alga, by (Isosorbide-5-Nitrae)-α-D-MANNOSE aldehydic acid (M) and (Isosorbide-5-Nitrae)-β-
Two kinds of structural units of L- guluronic acids (G) composition linear polymer (Donati et al.Biomaccromolecules,
2005,6:1031-1040) for compared with calcium alginate, Pectin calcium is considered more advantage, and especially it is to that can destroy seaweed
The ion and chemical constituent sensibility of sour calcium microballoon are lower, therefore pectin calcium gel stability is more preferable, is more suitable for cell and activity
Embedding (Gemeiner.P, the et al.Biotechnol.Appl.Biochem.1991,13 of substance:335-345).
Therefore, to solve the problems, such as that existing biological microsphere carrier material exists, be embedded cell and biological active matter are improved
The Activity and stabill of matter, the present invention develop a kind of preparation method and applications of Pectin calcium biological microsphere.It is prepared by this method
Simple process, process are environmentally protective, it can be achieved that encapsulating to living cells or bioactive substance high density and activity.
The content of the invention
For problem present in current common living cells biological microsphere carrier material application, the present invention provides one kind with
Pectin is the preparation method and applications of the biological microsphere of carrier material.
To achieve the above object, it is biological microspherical carrier material that the present invention, which selects pectin, embeds living cells or other biological
Active material the described method comprises the following steps：
(1) pectin powder is dissolved in the physiological saline that pH is 5.5~7.4, stirring and dissolving is sterile filtered；
(2) it will need to be encapsulated cell and/or other biological activity substance added in pectin solution made from step (1),
Form mixing suspension；
(3) mixing suspension obtained in step (2) is added dropwise in solidify liquid using syringe pump, cures 20-30 points
Clock；
(4) solidify liquid in (3) is discarded, collects the microballoon for obtaining being loaded with cell and/or active material.
Above-mentioned steps (1) pectin is from higher plant cells such as apple, citrus, dragon fruit, gingko episperm, shaddocks
The one or more of the extract of wall, for molecular weight in 40kDa~400kDa, esterification degree DE is less than 50%.
Cell density is 5 × 10 in above-mentioned steps (2) mixing suspension4~5 × 106/ mL, pectin solution made from step (1)
Mass concentration is 2%~6%.
Syringe pump speed is in above-mentioned steps (3), and syringe volume is 5~20mL, and syringe needle 19-24#, syringe needle is with curing
Liquid liquid level difference is 10~15cm.
Solidify liquid is the one or more of the aqueous solutions such as divalent metal calcium salt, barium salt or zinc salt in above-mentioned steps (3),
Concentration is 280~320mmol/L.
Solidification temperature is 20-25 degree conditions in above-mentioned steps (3), and the volume ratio of solidify liquid and pectin solution is：1:2~
1:20。
Above-mentioned microballoon is suitable for cell, the embedding of the bioactive substances such as protein drug or polypeptide.
The biologies such as above-mentioned cell behaviour or liver cell, adrenal medullary cell, the thyroid cell of mammal source
Active material functioning cell, various cell line cells：Such as HepG2 cells, C3A cells, MCF-7 cells and stem cell or
It is more than one or both of various cells of stem cell differentiation；The protein drug is more and peptide includes：Insulin, interferon,
It is more than one or both of monoclonal antibody, growth factor etc..
The present invention has the following advantages：
1. pectin is a kind of natural polysaccharide for deriving from plant, biological safety is high, cheap and easy to get；
2. not introducing additive and organic solvent in preparation process, preparation process is environmentally protective, ensure that cell and biology
The high activity embedding of active material, simple process and is easy to enlarged experiment；
3. Pectin calcium can realize cell, the embedding and/or culture of the high active substances such as protein drug or polypeptide are cultivated 20 days
When cytoactive it is good.
Description of the drawings
The dead coloration result of Fig. 1 .C3A Pectin calciums bead cell work (green represents cell work, and red represents cell death)；
Fig. 2 .C3A Pectin calcium bead cell activity curves；
Fig. 3 is pectin structure schematic diagram.
Specific embodiment
The present invention is described in detail with reference to specific embodiment.Following instance will be helpful to art technology and grind
Study carefully personnel and further understand the present invention, but do not form any limitation of the invention.Anyone is in scope of the invention as claimed
Interior done any type of modification, still within the claims in the present invention protection domain.
Embodiment 1
(1) 4g apple pectins powder (molecular weight 230kDa, DE=28%) is dissolved in 10ml deionized waters, be fully swollen
After stir to being completely dissolved, 0.22um filter membranes are sterile filtered.
(2) by 1 × 107Liver cancer cells C3A be uniformly mixed with the sterile apple pectin solution of 10mL, will mixing suspension move into
In 10mL syringes, No. 19 syringe needles are selected, pump speed 1mL/min, liquid level 10cm is set to be instilled using syringe pump
In 100mL320mM calcium chloride solutions, cure 30min at room temperature, prepare grain size 2000umC3A Pectin calcium microballoons.
(3) solidify liquid is discarded, is collected after sterile serum free medium cleaning, adds the MEM culture mediums containing 10% hyclone
Culture changes liquid, and measured cytoactive and motility rate in the 0th, 2,1,5,9,12,15 day every other day (the result is shown in attached drawings 1,2).
(4) take the C3A Pectin calciums microballoon after cultivating 15 days mutual for the in-vitro screening or drug of antitumor activity
Evaluation of effect.
Embodiment 2
(1) 3g citrus pectins powder (molecular weight 400kDa, DE=32%) is dissolved in 10ml deionized waters, be fully swollen
After stir to being completely dissolved, 0.22um filter membranes are sterile filtered.
(2) by 1 × 107Hepatocellular carcinoma H22 be uniformly mixed with the sterile citrus pectin solution of 10mL, will mixing suspension move
Enter in 10mL syringes, select No. 19 syringe needles, pump speed 2mL/min, liquid level 10cm is set to be instilled using syringe pump
In 150mL320mM calcium chloride solutions, cure 30min at room temperature, prepare grain size 1500-2000um HepG2 Pectin calcium microballoons.
(3) solidify liquid is discarded, is collected after sterile serum free medium cleaning, adds the MEM culture mediums containing 10% hyclone
Culture changes liquid, and measured cytoactive and motility rate in the 0th, 2,1,5,9,12,15 day every other day.
(4) the HepG2 Pectin calciums microballoon after cultivating 12-15 days is taken to be used for the in-vitro evaluation model of drug interaction.
Embodiment 3
(1) 10g citrus pectins powder (molecular weight 150kDa, DE=15%) is dissolved in 20ml deionized waters, it is fully molten
To being completely dissolved, 0.22um filter membranes are sterile filtered for stirring after swollen.
(2) by 1 × 106Melanoma A375 be uniformly mixed with the sterile citrus pectin solution of 20mL, will mixing suspension move
Enter in 10mL syringes, select No. 24 syringe needles, pump speed 0.8mL/min, liquid level 15cm is set to be instilled using syringe pump
In 250mL320mM barium chloride solutions, cure 20min at room temperature, prepare grain size about 1500umA375 Pectin calcium microballoons.
(3) solidify liquid is discarded, is collected after sterile serum free medium cleaning, the DMEM in high glucose containing 10% hyclone is added to train
Support base culture.
(4) the A375 Pectin calciums microballoon of the high cytoactive of culture 12-15 days is taken to be used for melanoma In vitro chemo-drug sensitive test
Or pharmacodynamic evaluation model.
Embodiment 4
(1) 6g citrus pectins powder (molecular weight 150kDa, DE=35%) is dissolved in 20ml deionized waters, be fully swollen
After stir to being completely dissolved, 0.22um filter membranes are sterile filtered.
(2) 10mg fibrin ferments are dissolved in the sterile citrus pectin solution of 10mL, fully after dissolving, mixed liquor is moved into 10mL
In syringe, No. 24 syringe needles are selected, pump speed 3mL/min, liquid level 10cm are set, 300mL280mM is instilled using syringe pump
In liquor zinci chloridi, cure 20min at room temperature, prepare the grain size about 1500um microballoons of Pectin calcium containing fibrin ferment.
(3) solidify liquid is discarded, is collected after sterile water wash.Three parts of 0.5g Pectin calciums microballoon is weighed respectively to be placed in test tube,
Add in 10mL brine.Test tube is placed in 37 DEG C, rotating speed is in 50rpm shaking tables, setting time point (1h, 3h, 8h, 12h, for 24 hours,
Supernatant 1mL 48h) is taken, fluid infusion after every sub-sampling.Measure fibrin ferment release profiles.
(4) microballoon containing fibrin ferment can be rescued as arterial intervention hemostasis suppository for the treatment of internal Parenchyma organ injury
It controls.
Embodiment 5
(1) 8g apple pectins powder (molecular weight 300kDa, DE=25%) is dissolved in 20ml deionized waters, be fully swollen
After stir to being completely dissolved, 0.22um filter membranes are sterile filtered.
(2) 10IU insulin powders are dissolved in the sterile apple pectin solution of 10mL, fully after dissolving, mixed liquor is moved into
In 10mL syringes, No. 19 syringe needles are selected, pump speed 3mL/min, liquid level 10cm is set to be instilled using syringe pump
In 400mL300mM calcium chloride solutions, cure 20min at room temperature, prepare grain size about 2000um insulin-containing Pectin calcium microballoons.
(3) solidify liquid is discarded, is collected after sterile water wash.Vacuum in culture dish is laid in after filter paper adsorption surface moisture to do
Dry 12h up to grain size 300-500um insulin microsphere, vacuum or freeze-drying after to get oral hypoglycaemic microball preparation
Embodiment 6
(1) 4g citrus pectins powder (molecular weight 350kDa, DE=28%) is dissolved in 20ml deionized waters, be fully swollen
After stir to being completely dissolved, 0.22um filter membranes are sterile filtered.
(2) by 1 × 107Sandlwood saccharobacillus mixed with the sterile citrus pectin solution of 10mL, then suspension is moved into
In 10mL syringes, No. 22 syringe needles are selected, pump speed 1mL/min, liquid level 12cm is set to be instilled using syringe pump
In 200mL300mM calcium chloride solutions, cure 20min at room temperature, prepare grain size about 1500-2000um Pectin calciums containing probiotics
Microballoon.
(3) solidify liquid is discarded, is collected after sterile water wash.Microballoon is laid in vacuum in culture dish after adsorption surface moisture
Dry 12h, obtains the probiotics microballoon dry powder of grain size 500-800um.
(4) probiotics microballoon dry powder is packed into No. 1 capsule to get probiotic oral capsule.
(5) the probiotic oral capsule can be used for the attenuation of meal supplement preparation, immunomodulator and clinical chemicotherapy to increase
Quick preparation.
Claims (8)
1. a kind of pectin is the biological microsphere preparation method of wall material, it is characterised in that：It in turn includes the following steps：
(1) pectin powder is dissolved in the physiological saline that pH is 5.5~7.4, stirring and dissolving is sterile filtered；
(2) it will need to be encapsulated cell and/or other biological activity substance added in pectin solution made from step (1), formed
Mix suspension；
(3) mixing suspension obtained in step (2) is added dropwise in solidify liquid using syringe pump, cured 20-30 minutes；
(4) solidify liquid in (3) is discarded, collects the microballoon for obtaining being loaded with cell and/or active material.
2. according to the method described in claim 1, it is characterized in that：Step (1) pectin is from apple, citrus, fire
The extract of one or two or more kinds of higher plant cell walls in Long Guo, gingko episperm, shaddock etc., molecular weight exist
40kDa~400kDa, esterification degree DE are less than 50%.
3. according to the method described in claim 1, it is characterized in that：Cell density is 5 × 10 in step (2) the mixing suspension4
~5 × 106/ mL, pectin solution mass concentration made from step (1) are 2%~6%.
4. according to the method described in claim 1, it is characterized in that：Syringe pump speed is 0.5~3ml/ in the step (3)
Min, syringe volume are 5~20mL, and syringe needle is 19~24#, and syringe needle is 10~15cm with solidify liquid liquid level difference.
5. according to the method described in claim 1, it is characterized in that：Solidify liquid is divalent metal calcium salt, barium in the step (2)
One or two or more kinds of aqueous solutions in salt or zinc salt etc., concentration are 280~320mmol/L.
6. according to the method described in claim 1, it is characterized in that：Solidification temperature is 20~25 degree of conditions in the step (3),
The volume ratio of solidify liquid and pectin solution is：1:2~1:20.
7. according to the method described in claim 1, it is characterized in that：Microballoon is suitable for cell, the biologies such as protein drug or polypeptide
The embedding and/or culture of active material.
8. according to the method described in claim 7, it is characterized in that：The cell is behaved or the liver of mammal source is thin
The bioactive substances functioning cell such as born of the same parents, adrenal medullary cell, thyroid cell, various cell line cells：As HepG2 is thin
One or two or more kinds in born of the same parents, C3A cells, MCF-7 cells etc. and the various cells of stem cell or stem cell differentiation；
The protein drug is more and peptide includes：One kind in insulin, interferon, monoclonal antibody, growth factor etc. or two kinds
More than.
Priority Applications (1)
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| CN111218421A (en) * | 2018-11-25 | 2020-06-02 | 中国科学院大连化学物理研究所 | Adhesion type cell scaffold and preparation and application thereof |
| CN111939310A (en) * | 2020-08-28 | 2020-11-17 | 四川轻化工大学 | Polyvinyl alcohol-pectin embolism microsphere, medicine-carrying embolism microsphere and preparation method |
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| CN111939310A (en) * | 2020-08-28 | 2020-11-17 | 四川轻化工大学 | Polyvinyl alcohol-pectin embolism microsphere, medicine-carrying embolism microsphere and preparation method |
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