CN106399228B - A kind of cell crosslinker material for three-dimensional liver cell ball culture - Google Patents
A kind of cell crosslinker material for three-dimensional liver cell ball culture Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
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- C12N2513/00—3D culture
Abstract
The invention discloses a kind of cell crosslinker materials for three-dimensional liver cell ball culture.The liver cell crosslinker material of synthesis provided by the invention are as follows: ALG-GA-N (CH3)2With SCTS-GA-N (CH3)2.The crosslinker material is using sodium alginate (ALG) or sulfated chitosan as carrier, by the enoxolone (GA-N (CH after hydrophilic modifying3)2) it by ethylenediamine is that linking arm is modified to directly modifying on sodium alginate (ALG) or by modified enoxolone to being prepared on sulfated chitosan.The present invention is by the experimental studies such as co-culturing with cell, it was demonstrated that can be by by ALG-GA-N (CH3)2Or SCTS-GA-N (CH3)2It can promote cell aggregation with liver cell simple blend and form three-dimensional liver cell ball, which has better physiological function.Therefore, cell adhesion material provided by the invention is possibly used for the building of three-dimensional cell model, has good application prospect in organizational project and Field of Drug Discovery.
Description
Technical field
The present invention relates to a kind of liver cell crosslinker material, culture of the material for external three-dimensional liver cell belongs to group
Knit engineering field.
Technical background
Currently, the Primary Hepatic mortality of carcinoma in the whole world occupies third position in malignant tumour, the disease incidence of China's liver cancer and dead
It dies rate and accounts for more than half of whole world (L.A.Torre, F.Bray, R.L.Siegel, J.Ferlay, J.Lortet-
Tieulent,A.Jemal,Global cancer statistics,2012,CA.Cancer J.Clin.65(2015)87–
108.doi:10.3322/caac.21262.).The chemicotherapy scheme of progress and specification recently as surgical technic pushes away
Extensively, the clinical levels of liver cancer have large increase, but 5 years survival rates of liver cancer are not effectively improved.It traces it to its cause,
Other than the high invasion of liver cancer and malignant characteristics easy to recur, the limitation of clinical drug therapy is also to restrict liver cancer treatment effect
An important factor for.The main reason for leading to above-mentioned phenomenon is that common tumor cell in vitro model is difficult in existing medicament sifting motion system
With the growth pattern and behavioral characteristic of tumor tissues in analogue body, therefore lead to clinical test results and preliminary in vitro experiments result
It mutually goes farther out.The outer tumor models of clinical precursor are typically established on traditional 2D cell culture basis, and this cultural method is difficult
To simulate in-vivo tumour three-dimensional growth mode, it is not easy to form the local microenvironment for promoting tumour growth, therefore can not also reflect strictly according to the facts
The biological behaviour of in-vivo tumour and reactivity to drug.
Document confirmation, Three-dimensional cell culture (three-dimensional the cell culture, 3D) in cancer research
Method, which can make up two-dimentional cell culture, can not simulate the defect of living environment in cell body, and can realize the body of tumor stem cell
Outer separation and amplification (Fitzgerald K A, Malhotra M, Curtin C M, et al.Life in 3D is never
flat:3D models to optimise drug delivery[J].Journal of controlled release:
official journal of the Controlled Release Society,2015,215,39-54).With two-dimensional list
Confluent monolayer cells culture is compared, and the form and function of the cell of dimensional culture are more similar to the intracorporal tissue of biology and organ.Therefore,
Three-dimensional cell culture as In vitro cell model can better true extracellular physiological environment in analogue body, in medicament research and development
The success rate that can effectively improve medicament research and development in the process reduces cost (Elliott, the Nelita T. of new drug development；Yuan,
Fan.A review of three-dimensional in vitro tissue models for drug discovery
and transport studies[J].Journal of pharmaceutical sciences,2011,100(1):59-
74).Simultaneously as the characteristics of being similar to organism inner cell physiological function of dimensional culture cell, is expected to be used to mix people
Building (Mironov V, Kasyanov V, Drake C, Markwald RR, the organ printing:From of work organ
bioprinter to organ biofabrication line[J].Current opinion in Biotechnology,
2011,22(5):667-673)。
Traditional Three-dimensional cell culture method has: cytoskeleton culture, microfluid method and water-gel method (Jun Yanga M
G,HirohikoIsec,Chong-SuChod,Toshihiro Akaikea,c,.Galactosylated alginate as a
scaffold for hepatocytes entrapment[J].Biomaterials,2002,23(471-479；Xu Z,Gao
Y,Hao Y,et al.Application ofa microfluidic chip-based 3D co-culture to test
drug sensitivity for individualized treatment oflung cancer[J].Biomaterials,
2013,34(16):4109-4117；Wang Y,Wang J.Mixed hydrogel bead-based tumor spheroid
formation and anticancer drug testing[J].The Analyst,2014,139(10):2449-2458)。
Although cytoskeleton cultivation can turn out the cell with three-dimensional structure cell activity, in terms of it is thin compared with 2D
Born of the same parents' culture shows significantly to improve, but can not be accurately controlled each variable in cell cultivation process；Microfluid method into
Row Three-dimensional cell culture, although the co-cultivation that high-throughput culture and multiple groups are knitted may be implemented, microfluid cultivation is needed
Experimental provision that will be more fine, leads to higher cost, limits microfluid method always and carries out answering extensively for Three-dimensional cell culture
With；For hydrogel cultivation in the preparation of material, step is many and diverse, and experimental flexibility is poor.The traditional three-dimensional cell training of three of the above
There are the differences of batch for feeding method, lack repeatability, are unable to satisfy in experiment and obtain to science, repeatable experimental result
It is required that and cell ball manufacturing cycle it is longer, affect the extensive use of three-dimensional cell model in an experiment.It is three-dimensional thin to accelerate
The building of born of the same parents' culture environment, cell Crosslink bond type have experimental implementation simple and easy as a kind of new Three-dimensional cell culture method
The advantages that, it is expected to become simple and effective three-dimensional hepatocyte cultures method.The dentritic cell crosslinking agent of forefathers' report, which has, to be promoted
Into the ability of cell aggregation, but this method needs to be chemically modified cell membrane, this can generate damage to cell, meanwhile,
The synthesis of dendritic macromole is complicated, make this method (Deqiang Zhao, Siew-Min Ong, Zhilian Yue,
Dendrimer hydrazides as multivalent transient inter-cellular linkers[J]
.Biomaterials, 2008,29,3693-3702) there is the limitation used；In addition also having researchers to pass through will be with cell
There is film func-tional ligand (such as oil base, the RGD, ligands specific) modification of interaction to be used for bridge at linear PEG chain both ends
Even cell (Michiko Ito, Tetsushi Taguchi；Enhanced insulin secretion of physically
crosslinked pancreaticβ-cells by using a poly(ethylene glycol)derivative with
oleyl groups[J].ActaBiomaterialia,2009,5,2945-2952；Zhi Rao,Makoto Sasaki,
Tetsushi Taguchi；Development of amphiphilic,enzymatically-degradable PEG-
peptide conjugate as cell crosslinker for spheroid formation[J].Colloids and
Surfaces B:Biointerfaces, 2013,101,223-227).But the system has the disadvantage in that linear PEG chain both-end
Necessary fully functionalization, reaction condition control are difficult；Crosslinking agent and cell-unit point act on, and binding ability is poor；The function at both ends
Can group act on that the same cell probability is larger, to reduce the bridging rate of cell.
Summary of the invention
Goal of the invention: it is stringent to solve crosslinking agent structure control in existing Three-dimensional cell culture technology, and synthesis condition is harsh, hands over
The problems such as connection agent and cell combination ability are weak, and cell crosslinking rate is low.It designs a kind of based on simple and effective three-dimensional hepatocyte model
Construction method, both can efficiently construct Three-dimensional cell culture environment, large batch of three-dimensional cell model can also be carried out
Preparation；A kind of cell crosslinker material for three-dimensional hepatocyte cultures is provided.Three-dimensional cell training is carried out using cell crosslinking agent
Support be a kind of ideal Three-dimensional cell culture method, furthered intercellular distance by cell crosslinking agent, so that cell
Aggregation form three-dimensional cell culture environment.
The design will be modified with the Enoxolone derivative of good liver cell compatibility to sodium alginate side group, so that often
There are multiple ligands on crosslinking agent chain efficiently solves list so that crosslinking agent and iuntercellular form multiple spot interaction
Site crosslinking agent bridging rate is low, the hypodynamic problem of cell combination.In addition, this method does not need the modification amount of functional group yet
Stringent control is carried out, the function of cell ball can be also adjusted by adjusting ligand species, modification amount etc..Therefore it prepares first herein
The sodium alginate of enoxolone modification, and obtain using it for cell crosslinking agent and by simple blend method with well
The liver cell ball of physiological function.
Technical solution of the present invention
A kind of cell crosslinker material for three-dimensional liver cell ball culture, the cell crosslinker material is with polysaccharide
Sodium alginate (ALG) or sulfated chitosan (SCTS) are carrier, by the enoxolone (GA-N (CH after hydrophilic modifying3)2) modify and arrive
It is prepared on sodium alginate (ALG) or sulfated chitosan (SCTS), expression formula is ALG-GA-N (CH3)2Or SCTS-
GA-N(CH3)2, structure is respectively as follows:
Specifically: ligand enoxolone (GA) progress that will be interacted with liver plasma membrane is hydrophilically modified, i.e., logical
It crosses and introduces tertiary amine group on hydroxyl as water soluble group to GA progress hydrophilic modifying, by esterification in enoxolone
It is reintroduced back to carboxyl on hydroxyl, in order to modify on sodium alginate (ALG) and sulfated chitosan (SCTS), finally utilizes second two
Amine is linking arm by the amidation process of carboxyl by the enoxolone (GA-N (CH after hydrophilic modifying3)2) modify and arrive sodium alginate
On sugar chain, it is prepared for the sodium alginate of Enoxolone derivative modification, is ALG-GA-N (CH3)2, degree of substitution DS=2%-12%；
Chitosan (Chitosan) and sodium alginate (Sodium alginate) are all natural polysaccharides.On chitosan chain
Alkylation, acylation, carboxy methylation, sulfonation, nitrification, halogen can occur for the presence of amino group, under given conditions, chitosan
Change, oxidation, reduction, condensation and complexing etc. are reacted, and the various chitosan derivatives with different performance can be generated.Therefore this hair
Amido on bright carboxyl and sulfated chitosan by modified enoxolone carries out amidation process directly will modified enoxolone
It modifies on the sugar chain of sulfated chitosan, is prepared for the sulfated chitosan SCTS-GA-N (CH of Enoxolone derivative modification3)2,
Degree of substitution is DS=2%-10%, and the building of three-dimensional liver cell equally may be implemented.
Cell crosslinker material ALG-GA-N (CH of the present invention3)2The specific steps of preparation method see embodiment 1；
SCTS-GA-N(CH3)2The specific steps of preparation method see embodiment 2.
The advantages and positive effects of the present invention:
The present invention is compared with prior art has the following characteristics that the present invention with amino modified enoxolone (GA-N (CH3)2)
It as liver cell affinity ligand, not only remains its Liver targeting ability but also significantly improves its hydrophily, be conducive to cell friendship
Join the practical application of agent；By GA-N (CH3)2It is introduced into sodium alginate main chain, by can be effective with liver cell simple blend
Promote the aggregation of cell, accelerates the preparation of three-dimensional liver cell ball；The cell crosslinking agent is without accurate control GA-N (CH3)2Substitution
The molecular weight of degree and crosslinking agent, compared with existing cell crosslinking agent in practical applications with greater advantage.The present invention can
There can be good application prospect in field of tissue engineering technology as a kind of cell crosslinking agent for Three-dimensional cell culture.
It is this ligand modified to macromolecular chain by what can be interacted with liver plasma membrane relative to existing cell crosslinking agent
Crosslinking agent in side group provides binding site for cell, and does not need to carry out stringent limitation to the quantity of func-tional ligand,
And since multiple ligands can be modified on a macromolecular chain, effectively enhance the interaction of crosslinking agent and cell.And
By cellular uptake experiments have shown that modified GA has preferable selectivity to liver cell；It is known that by MTT test result
Liver cell crosslinker material prepared by the present invention has preferable biocompatibility；Live/dead cell dyeing experiment can prove
The activity of cell inside cell ball.Therefore simply by HepG2 cell and ALG-GA-N (CH3)2It suspends and co-cultures, can effectively promote
Into the aggregation of cell, accelerate the building of three-dimensional liver cell ball.
Detailed description of the invention
Fig. 1 is the hydrophilic modifying composite diagram of GA.
Fig. 2 is the composite diagram of synthetic cell crosslinking agent.
Fig. 3 is the synthesis schematic diagram of sulfated chitosan.
Fig. 4 is SCTS-GA-N (CH3)2DE synthesizes schematic diagram.
Fig. 5 is the streaming comparative result figure of HepG2 cell Yu Hela cell.
Fig. 6 is ALG-GA-N (CH3)2MTT test result figure.
Fig. 7 is cellular morphology figure after cell co-cultures for 24 hours, and A, B, C, D are ALG-GA-N (CH3)2Sample, E, F are ALG pairs
According to group.
Fig. 8 is the AO/EB coloration result figure for cultivating cell.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Any feature disclosed in this specification unless specifically stated can be equivalent or with similar purpose by other
Alternative features are replaced.That is, unless specifically stated, each feature is an example in a series of equivalent or similar feature
Son.
Embodiment 1, liver cell crosslinking agent ALG-GA-N (CH3)2Preparation
Step 1, GA hydrophilic modifying:
GA-N(CH3)2Synthesis
5.3g DCC and 3.4g HOBt are dissolved in methylene chloride, stir 30min at room temperature.10g is added into solution
GA (compound 1) stirs 20min.2.3mLN, N- dimethyl-ethylenediamine are added later, room temperature condition stirs 12h.Reaction solution benefit
Chromatographic column (volume ratio: CH is used after removing solvent with revolving instrument2Cl2:MeOH:NH3H2O=15:1:1 it) purifies, later by product
Be dissolved in ethyl acetate, 2h stood at 0 DEG C, filter, filtrate is evaporated, be dried in vacuo after obtain white powder product i.e. chemical combination
Object 2 (10.6g, 92%).
Step 2, liver cell crosslinking agent ALG-GA-N (CH3)2(DS=12%) preparation:
1st, scu-GA-N (CH3)2Synthesis
By 3.2g compound GA-N (CH3)2(compound 2) is dissolved in 30mL pyridine, sequentially adds 4.7g succinic anhydride,
7.7g DMAP heats 12h at 90 DEG C, obtains dark thick liquid, a certain amount of methylene chloride dilution is first added, then using dilute
Salt acid elution will be eluted using chromatographic column after the concentration of obtained dichloromethane solution, use CH first2Cl2:MeOH:NH3H2O=
The eluent of 15:1:1 removes DMAP and unreacted compound 2, uses CH later2Cl2:MeOH:NH3H2O=15:5:1 elution
Obtain brown product i.e. compound 3 (2.4g, 65%).
2nd, N-GA-N (CH3)2Synthesis
By 2.7g compound scu-GA-N (CH3)2(compound 3) is dissolved in the methylene chloride of 20mL, controls temperature -10
Under the conditions of DEG C, 1.05g DCC is added into solution and reacts 30min, addition 0.57g NHS molar ratio is M compound 3:
12h is stirred at room temperature in MDCC:MNHS=1:1.2:1.2；It is filtered to remove DCU, by filtrate added drop-wise to 30mL, contains 70% ethylenediamine
Dichloromethane solution in, 12h is stirred at room temperature, reaction system washed once with dilute hydrochloric acid solution, mention after concentration using chromatographic column
Pure CH2Cl2:MeOH:NH3H2O=15:1:1 obtains white powder product i.e. N-GA-N (CH3)2(compound 4) (1.0g,
40%).
3rd, ALG-GA-N (CH3)2Synthesis
The HCl that 30mL concentration is 0.6M is added in 30mL ethyl alcohol, then 2.0gALG-Na is added in mixed solution,
It is stirred overnight at 4 DEG C；Suction filtration obtains ALG-H, successively uses ethyl alcohol, and acetone washing is dried in vacuum overnight at 40 DEG C；After drying
Alginic acid is dispersed in 100mL distilled water, and it is completely molten to polymer that tetrabutylammonium alkali (TBAOH) is added dropwise dropwise under agitation
Solution, adjusting pH are 7.0-10.0, and dialysis is lyophilized and obtains the ALG-TBA of white；1.0gALG-TBA is dissolved in DMF, ice bath item
The chloro- 1- picoline (CMPI) of 0.3g 2- is added under part, in N2It is stirred to react 1h under environment, removes ice bath, 0.8g chemical combination is added
Simultaneously suitable triethylamine is added dropwise in object 4, and normal-temperature reaction is for 24 hours.30.0mL, the NaCl solution of 2.5mol/L is added；Gained liquid is existed
It precipitates, filters in ethyl alcohol, washing is redissolved in distilled water, is transferred to bag filter (molecular cut off Mw=3500) dialysis two in
It, freeze-drying obtains the ALG-GA-N (CH that white sponge solid i.e. degree of substitution is 12%3)2。
The preparation of step 3, liver cell crosslinking agent ALG-GA-N (CH3) 2 (DS=8%):
Method is with step 2, and wherein ALG-TBA feeds intake are as follows: 1.0g, 2.2mmol；N-GA-N(CH3)2It feeds intake are as follows: 0.5g,
0.731mmol。
The preparation of step 4, liver cell crosslinking agent ALG-GA-N (CH3) 2 (DS=4%):
Method is with step 2, and wherein ALG-TBA feeds intake are as follows: 1.0g, 2.2mmol；N-GA-N(CH3)2It feeds intake are as follows: 0.3g,
0.439mmol。
The preparation of step 5, liver cell crosslinking agent ALG-GA-N (CH3) 2 (DS=2%):
Method is with step 2, and wherein ALG-TBA feeds intake are as follows: 1.0g, 2.2mmol；N-GA-N(CH3)2It feeds intake are as follows: 0.15g,
0.220mmol。
The preparation of embodiment 2, liver cell crosslinking aid S CTS-GA-N (CH3) 2:
1, the synthesis of SCTS
The synthesis schematic diagram of sulfated chitosan is as shown in Fig. 3, is added 45mL's in the there-necked flask of 500mL
HSO3The concentrated sulfuric acid that 90mL concentration is 98% is slowly added drop-wise in there-necked flask by Cl under condition of ice bath, stirs 15min.It mentions
3g chitosan solid will be added slowly in reaction system by high mixing speed, after about 10min, after stable system, be removed
Remove ice-water bath；After reacting at room temperature 2h, stop reaction；Reaction solution is slowly dropped in the ice ether of 1000mL and (orders for -26 DEG C to hide
Precipitating in 3h).Then, precipitating decompression is filtered, is first washed once with ether, then repeatedly washed with acetone, collected solid and be used in combination
Suitable distilled water dissolution adjusts pH to neutrality with 1M NaOH；Sample solution is transferred to bag filter (M laterw=12-14KD)
In, after dialysing to distilled water 3 days, dialyzate is lyophilized to obtain flaxen solid to be the sulfated chitosan synthesized, yield
It is 94%.
2、SCTS-GA-N(CH3)2Preparation
SCTS-GA-N(CH3)2To prepare reaction equation as shown in Fig. 4, weigh 225mg SCTS, be dissolved in 5mL water, so
After weigh 256mg scu-GA-N (CH3)2, 92mg EDC and 55.2mg NHS is dissolved in the DMF of 45mL, then will dissolve
Solution is added in the aqueous solution of SCTS, feed ratio are as follows: MSuc-GA:MEDC:MNHS=1:1.2:1.2.Temperature is controlled at 35 DEG C
Under the conditions of stir for 24 hours, reaction terminates, precipitated with acetone, filter, freeze-drying, obtain product i.e. SCTS-GA-N (CH3)2。
Embodiment 3, cellular uptake experiment:
(1) the HepG2 cell of logarithmic growth phase is added in the DMEM cell culture medium of 1mL after digesting 1min with pancreatin
And pancreatin, piping and druming cell strip down it from culture dish, are transferred in centrifuge tube, culture medium is discarded after centrifugation, is pipetted again
New DMEM culture medium 1mL, piping and druming make cell be uniformly dispersed, and count, and press every hole 2 × 105The density of a cell is inoculated into 12
In the cell culture of hole, cell is blown and beaten uniformly with 1mL liquid-transfering gun, at 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator.
(2) culture medium is removed with liquid-transfering gun, washed once with PBS buffer solution.Then, it is added mixed according to 1:1 volume ratio
The nano-particle solution 1mL of conjunction.It places it in 37 DEG C of cell culture phase and cultivates 4h.
(3) culture medium is removed with liquid-transfering gun, is washed three times with PBS buffer solution, each 5min, 1mL/ disk, finally all
It removes.The pancreatin digestion of 200 μ L is added, the DMEM culture medium for adding 200 μ L neutralizes, and makes its stripping with liquid-transfering gun piping and druming cell
From.
(4) cell mixture is transferred in 1.5mL centrifuge tube with liquid-transfering gun, then is rinsed with the DMEM culture medium of 200 μ L
Orifice plate is also transferred in the centrifuge tube of 1.5mL.
(5) adjustment revolving speed is 800r/min, is centrifuged 5min.Supernatant is discarded, the PBS buffer solution of 900 μ L is rejoined,
And open its fully dispersed with liquid-transfering gun piping and druming cell.
(6) it is detected using flow cytometer, is the culture medium that mixes in equal volume as blank control using PBS and DMEM
Group；Using Hela cell as parallel control group.
The results are shown in attached figure 5, and HepG2 cell whole fluorescence intensity to the right after co-culturing with nanoparticle is high as seen from the figure
Direction translation illustrates that HepG2 cell integrally has intake to nanoparticle and intake is more average；Thus HepG2 is also embodied
Cell to the intake of nanoparticle is mediated by interaction between the two.And Hela cell through co-cultivation after, part
The overlap of peaks of cell mass and blank control group illustrates that cell therein is zero to the intake of nanoparticle；Meanwhile also there is part
The fluorescence intensity peak of the group of cell illustrates that cell has intake to nanoparticle to right translation；But the fluorescence intensity of cellular uptake
But be not it is unimodal, illustrate the inhomogeneities that cell absorbs nanoparticle.So modified GA has preferably liver cell
Selectivity.
Embodiment 3, MTT experiment:
The inoculum density of HepG2 cell is 0.8 × 104The hole cells/, 400 μ L of DMEM culture medium, cell is in 37 DEG C, 5%
CO2It is incubated in incubator for 24 hours, then, the DMEM solution concentration of configured sample is followed successively by 1mg/L, 2mg/L respectively,
4mg/L is added in culture plate, and 12h is cultivated in cell incubator, discards upper layer culture medium, washes away dead cell with PBS, is added
The 400 μ L of DMEM culture medium of MTT containing 0.5mg/mL continues to be incubated for, and upper solution is discarded after 4h, and the DMSO that 150 μ L are added fills
Divide Rong Xie formazan precipitating, then take out 80 μ L and be added in 96 orifice plates, measure the absorbance in each hole at 540nm using microplate reader,
That is OD value.Every kind of 3 hole of material parallel, experiment are repeated 2 times.Data are indicated with mean+SD, make t detection, and P < 0.05 is thought to have significant
Sex differernce.See attached drawing 6, according to the experimental results, the material of preparation has good cell compatibility.
Embodiment 4, liver cell ball crosslinking agent cell balling-up experiment:
Cell experiment is HepG2 cell in the present invention, and HepG2 cell origin is widely used in liver group in human liver cancer tissue
In the in vitro study of weaver's journey.The DMEM culture solution that the culture medium used is 90% adds 10% fetal calf serum, then plus 1% it is double
It is anti-, cultivated in cell incubator (37 DEG C, 5%CO2).The crosslinking agent of the different degree of substitution of preparation is dissolved in various concentration
In DMEM cell culture medium, then cell is planted in six hole PS cell cultures, 400,000 holes cell/, 2mL culture medium is every
Cellular morphology is observed in hole, culture afterwards for 24 hours.See attached drawing 7.Different degree of substitution and concentration samples form cell ball situation such as one institute of table
Show, wherein when degree of substitution is 4%, balling-up size is uniform, and cellular morphology is good, and it is best to form cell ball effect at this time.
Table one, different degree of substitution and concentration samples form cell ball situation
Note :-indicate that cell ball cannot be formed；√ expression can form cell ball
Embodiment 5, AO/EB Coloration experiment:
Configuration concentration is the AO/EB mixed solution of 50 μ g/mL, discards cell culture medium, and configured good AO/EB is added
Dyeing liquor, hatch in cell incubator 15min (37 DEG C, 5%CO2), cell survival is observed using inverted fluorescence microscope.
See attached drawing 8, the cell in cell ball has good cell activity as seen from the figure.
Claims (2)
1. a kind of cell crosslinker material for three-dimensional liver cell ball culture, it is characterised in that the cell crosslinker material
It is with polysaccharide sodium alginate (ALG) or sulfated chitosan (SCTS) for carrier, by the enoxolone (GA-N after hydrophilic modifying
(CH3)2) to being prepared on polysaccharide sodium alginate or sulfated chitosan, expression formula is respectively ALG-GA-N (CH for modification3)2Or
SCTS-GA-N(CH3)2, structure is respectively as follows:
Preparation step is: ligand enoxolone (GA) progress that will be interacted with liver plasma membrane is hydrophilically modified, i.e., logical
It crosses and introduces tertiary amine group on hydroxyl as water soluble group to GA progress hydrophilic modifying, through esterification on the hydroxyl of GA
It is reintroduced back to carboxyl, it is finally logical for linking arm using ethylenediamine in order to be modified on polysaccharide sodium alginate and sulfated chitosan
The amidation process of carboxyl is crossed by the enoxolone (GA-N (CH after hydrophilic modifying3)2) modify onto ALG sugar chain, it is prepared for Radix Glycyrrhizae
The polysaccharide sodium alginate of hypo acid Derivatives Modified is ALG-GA-N (CH3)2, degree of substitution DS=2%-12%；Or pass through modification
Amido on the carboxyl and sulfated chitosan of enoxolone carries out amidation process and directly modifies modified enoxolone to sulfonation
On the sugar chain of chitosan, it is prepared for the sulfated chitosan SCTS-GA-N (CH of Enoxolone derivative modification3)2, degree of substitution DS
=2%-10%.
2. the cell crosslinker material according to claim 1 for three-dimensional liver cell ball culture, it is characterised in that described
The molecular weight of polysaccharide sodium alginate is 10,000-5 ten thousand；The molecular weight of sulfated chitosan is 30,000-20 ten thousand.
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| CN101254308A (en) * | 2008-04-08 | 2008-09-03 | 南开大学 | Biogastrone acid-polyethyleneglycol /chitosan liver target composite drug administration system and preparation thereof |
| CN101549158A (en) * | 2009-05-08 | 2009-10-07 | 南开大学 | Sodium alginate liver-targeted nanometer drug delivery system and preparing method thereof |
| CN101642573A (en) * | 2009-08-25 | 2010-02-10 | 南开大学 | Chitosan-based hepatic-targeted nano-particle drug delivery system and preparation method thereof |
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| CN101254308A (en) * | 2008-04-08 | 2008-09-03 | 南开大学 | Biogastrone acid-polyethyleneglycol /chitosan liver target composite drug administration system and preparation thereof |
| CN101549158A (en) * | 2009-05-08 | 2009-10-07 | 南开大学 | Sodium alginate liver-targeted nanometer drug delivery system and preparing method thereof |
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