CN109232995A - The temperature-sensitive hydrogel of adjustable response temperature contains cell material and the preparation method and application thereof - Google Patents
The temperature-sensitive hydrogel of adjustable response temperature contains cell material and the preparation method and application thereof Download PDFInfo
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- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61L27/52—Hydrogels or hydrocolloids
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- C08J2301/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives
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- C08K3/00—Use of inorganic substances as compounding ingredients
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- C08K5/00—Use of organic ingredients
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- C08K5/053—Polyhydroxylic alcohols
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- C08K5/09—Carboxylic acids; Metal salts thereof; Anhydrides thereof
- C08K5/098—Metal salts of carboxylic acids
Abstract
The invention belongs to intellectual material, high molecular material and biomedical engineering field, and in particular to the temperature-sensitive hydrogel of the adjustable response temperature of one kind contains cell material and the preparation method and application thereof.Carry cell temperature-sensitive hydrogel, the raw material components including following weight percent: methylcellulose, 12wt%；PEG-20000,7.4wt%；Non-toxic inorganic sodium salt, 1.4wt%；Xylitol, 0~16wt%；Surplus is water.The non-toxic inorganic sodium salt is Na2HPO3.12H2The temperature-sensitive hydrogel of the adjustable response temperature of one kind O of the invention contains cell material, can adjust temperature sensitive response temperature and no biotoxicity by adjusting Determination of Xylitol.In low temperature, keep transparent liquid that can be changed into gel state when being warming up to phase transition temperature.And material no biotoxicity can contain protection cell, be injected into vivo.
Description
Technical field
The invention belongs to intellectual material, high molecular material and biomedical engineering field, and in particular to a kind of adjustable
The temperature-sensitive hydrogel of response temperature contains cell material and the preparation method and application thereof.
Background technique
Stem cell therapy has entered clinical test as a kind of promising method of tissue repair, a plurality of types of stem cells
Stage.But direct injection transplanting allogeneic or when heterogenous cell, they the effect of be detained by transplanted cells and implantation amount
Lower limit, the retention rate of cell is very low, and there are inflammation and the potential risks of immune response.Therefore hydrogel is as one
For a cell carrier package cell when being injected into internal, protecting it from being damaged is to have very much important (van Berlo, J.H.；
Molkentin,J.D.An Emerging Consensus on Cardiac Regeneration.Nat.Med.2014,20,
1386-93)。
A kind of attractive strategy is the water-setting that stem cell (such as CSCs) is wrapped in hydrogel, and cell is loaded into
Glue is delivered in injury tissue.Temperature-sensitive hydrogel is due to being easy to administration and minimally-invasive treatment, note with good biocompatibility
It penetrates and polyalcohol hydrogel is used to be widely studied the promising carrier as the lasting various drugs of conveying.Particularly, injectable
Thermosensitive aquagel presents to flow freely to be dissolved under room temperature or low temperature, but as the temperature rises, becomes semisolid mechanical water
Gel.Therefore, frangible therapeutic agent (such as protein, polypeptide, cell or other small molecules) may be easy to fall at low temperature
Then collosol state is carried cell aggregation object solution and spontaneously forms water in situ under body temperature after being injected in vivo using conventional syringe
Gel.Cell is effectively delivered to target site, and not by beneficial cellular uptake and bioavilability and milder
Good reaction improves therapeutic immunization response.In particular, thermosensitive aquagel can be injected into target site by Noninvasive management.
There are manufacturing cost height, complex procedures to have notably and there is a small amount of bio-toxicity for current cell carrier.
Summary of the invention
The object of the present invention is to provide a kind of temperature-sensitive hydrogel of adjustable response temperature contain cell material and
Preparation method and application.Hydrogel provided by the invention is liquid at room temperature, is solidifying when being higher than its phase transition temperature
Colloidal state, and the phase transition temperature of gel can be adjusted by the content of xylitol according to diseased region situation；Preparation method letter
It is single cheap nontoxic, it is suitble to industrialization, avoids the side effect of organic solvent, accomplished the safeguard protection to cell.
In order to achieve the above objectives, the present invention provides the following technical scheme that
It is a kind of it is adjustable response temperature temperature-sensitive hydrogel contain cell material, the group including following weight percent
Point:
Methylcellulose, 12wt%；
PEG-20000,7.4wt%；
Non-toxic inorganic sodium salt, 1.4wt%；
Xylitol, 0~16wt%；
Surplus is water.
Wherein, non-toxic inorganic sodium salt can be sodium citrate or NaCl or Na2HPO3.12H2O etc..
The temperature-sensitive hydrogel of adjustable response temperature proposed by the present invention contains the preparation method of cell material, specifically
Steps are as follows:
Take methylcellulose, polyethylene glycol-20000, non-toxic inorganic sodium salt, xylitol in 70~100 DEG C heat go from
Uniform stirring in sub- water, is uniformly dispersed, and is put into refrigerator cold-storage and stays overnight, is allowed to be completely dissolved, and adjustable response temperature can be obtained
The temperature-sensitive hydrogel of degree contains cell material.
Wherein, the concentration of methylcellulose used is 136.36mg/ml；The concentration of PEG-20000 used is 80mg/ml；
The concentration of non-toxic inorganic sodium salt used is 14.2mg/ml；The concentration of xylitol used is 0~200mg/ml.
The temperature-sensitive hydrogel of the adjustable response temperature of gained contains cell material, the weight percent of each component are as follows:
Methylcellulose, 12wt%；
PEG-20000,7.4wt%；
Non-toxic inorganic sodium salt, 1.4wt%；
Xylitol, 0~16wt%；
Surplus is water.
Further, the non-toxic inorganic sodium salt can be sodium citrate or NaCl or Na2HPO3.12H2O。
Further, the brand that the methylcellulose uses is Adamas, viscosity 15mPa.s, with the amount of 12wt%, system
Make hydrogel；The concentration of methylcellulose improves then too sticky, and methyl cellulose concentration reduces then its phase transition temperature
Also it reduces.It since the osmotic pressure of cell in vivo is 300mmol/L, therefore selects sodium dihydrogen phosphate as salt effect additive, adds
Entering amount is 14.2mg/ml, is cell maximum osmotic pressure value.Xylitol is added simultaneously to adjust the phase transition temperature of hydrogel, in fact
Now respond the adjustable of temperature.80mg/ml PEG-2000 is added to increase its gelation rate.
In the present invention, the adjusting of hydrogel phase transition temperature, such as xylose can be realized by adjusting Determination of Xylitol
When the mass percent of alcohol is 4% (concentration 40mg/ml), phase transition temperature is 34 DEG C, when xylitol mass percent
When for 8% (concentration 80mg/ml), phase transition temperature is 32 DEG C.
The temperature-sensitive hydrogel of adjustable response temperature provided by the invention contains cell material, contains cell infusion
Intracorporal method are as follows:
(1) the temperature-sensitive hydrogel solution and cell suspending liquid for the adjustable response temperature for obtaining the present invention are in low temperature
In being shaken on shaking table under environment, so that uniformly mixing, keeps being completely transformed into a gel state at 37 DEG C；
(2) gel state hydrogel-cell mixture is drawn with the syringe of removal syringe needle, is injected after loading onto syringe.
No. 1.2 syringe needle needle cylinder injections are used in the present invention.
Hydrogel provided by the invention is liquid at room temperature, is gel state when being higher than its phase transition temperature.It can be with
The phase transition temperature of gel is adjusted by the content of xylitol according to diseased region situation.Wherein, non-toxic inorganic sodium salt is due to thin
The effect of born of the same parents' osmotic pressure maintains ion concentration 300mmol/L, and the change of xylitol influences less Premeabilisation of cells pressure, therefore can
Change its phase transition temperature freely to adjust concentration so that material prepared by the present invention is more widely applied, PEG-2000 to
Increase its gelation rate, it is low to solve stem cell retention rate when being injected into internal, loses big problem.
Preparation method provided by the invention is simple cheap nontoxic, is suitble to industrialization, avoids the side effect of organic solvent, do
The safeguard protection for cell is arrived.The Injectable temperature sensitive gel of the prior art all uses external solution state, is injected into body
The technology condensed after interior, and after the present invention is mixed in vitro, solidified with cell using gel, it is injected in vivo, as wrapped up fruit
The jelly that can be inhaled method, yet there are no document report both at home and abroad at present.
Compared with the prior art, the advantages of the present invention are as follows: the present invention uses methylcellulose, various nontoxic by changing
The additive amount of additive effectively changes its phase transition temperature.Raw material sources are extensive, low in cost, and operating method is easy, technique
Simply, the prospect with industrial production application, can need to change the phase transition temperature of material according to the state of an illness, realize effective cell
Intelligence release.And biological nontoxic can carry cell.The hybrid material can realize hydrogel solution when temperature reduces, and release
Cell is put, provides a kind of thinking for treatment, is had in fields such as medicine controlled releasing, biological intelligence switch, biosensors extensive
Application.
Detailed description of the invention
Fig. 1 is to freeze after the 8% xylitol hydrogel solution that the embodiment of the present invention 1 provides is changed into gel state at 37 degree
Dry scanning electron microscope (SEM) photograph.
Fig. 2 is the heating rheogram (heating rate 0.3 for the 8% xylitol hydrogel solution that the embodiment of the present invention 1 provides
DEG C/min, strain 0.05% and frequency 1rad/s).
Fig. 3 is unenhanced, stress 0.1 of the 8% xylitol hydrogel solution that provides of the embodiment of the present invention 1 at 37 degree.
Fig. 4 is the 4% xylitol hydrogel MTT experiment data that the embodiment of the present invention 2 provides.
Fig. 5 is that 1 gel of embodiment provided in an embodiment of the present invention package dil staining cell shows fluorescent microscopy images (are presented solid
Determine state).
Fig. 6 is that 1 gel of embodiment provided in an embodiment of the present invention package dil staining cell discharges cell in pbs solution
(travelling state is presented) in shows fluorescent microscopy images.
Specific embodiment
The following examples are further illustrations of the invention, it is not intended to limit the scope of the invention.
Embodiment 1
Weigh 0.68182g methylcellulose, 0.4g PEG-20000 (80mg/ml), 0.179g Na2HPO3.12H2O
(14.2mg/ml), 0.4g xylitol (80mg/ml) is dissolved in 100 degree of deionized waters of 5ml, is put into 4 degree of ice after mixing evenly
Case is completely dissolved overnight.It tests rheology (0.3 DEG C/min of heating rate strains 0.05% and frequency 1rad/s) and obtains its gel temperature
Degree is at 31.8 degree.
Take wherein 1ml solution mixed with embryonic stem cell suspension, on shaking table after uniform stirring, be put into 37 degree of incubators
Form it into gel state within one hour.The hydrogel for becoming gel state is drawn with the syringe for removing syringe needle, injection lacks after installing syringe needle
Haemal tissue.
Embodiment 2
Weigh 0.68182g methylcellulose, 0.4g PEG-20000 (80mg/ml), 0.179g Na2HPO3.12H2O
(14.2mg/ml), 0.2g xylitol (40mg/ml) is dissolved in 100 degree of deionized waters of 5ml, is put into 4 degree of ice after mixing evenly
Case is completely dissolved overnight.It tests rheology (0.3 DEG C/min of heating rate strains 0.05% and frequency 1rad/s) and obtains its gel temperature
Degree is at 33.4 degree.
Take wherein 1ml solution mixed with embryonic stem cell suspension, on shaking table after uniform stirring, be put into 37 degree of incubators
Form it into gel state within one hour.The hydrogel for becoming gel state is drawn with the syringe for removing syringe needle, injection lacks after installing syringe needle
Haemal tissue.
The hole of comparative example 1:37 degree gel
1 gel of example is placed to holding 3 hours in 37 degree of water-baths, so that it goes completely into gel.It takes on a small quantity in liquid nitrogen
Middle temper is cold, is handled 24 hours with freeze dryer, removes moisture removal.Plane, which is opened, obtains section, observes under scanning electron microscope.
2 maxicell toxicity of comparative example；
MTT experiment method:
A. logarithmic phase cell is collected, concentration of cell suspension is adjusted, 100ul is added in every hole, and bed board makes cell tune density to be measured
To the hole 1000-10000, (edge hole is filled with sterile PBS).
B.5%CO2, 37 DEG C of incubations, until cell monolayer is paved with bottom hole (96 hole flat underside), the gel that concentration gradient is added soaks
Extract.
C.5%CO2, 37 DEG C incubation 16-48 hours, observe under inverted microscope.
D. 20ulMTT solution (5mg/ml, i.e. 0.5%MTT) is added in every hole, continues to cultivate 4h.
E. culture is terminated, culture solution in hole is carefully sucked.
F. 150ul dimethyl sulfoxide is added in every hole, sets low-speed oscillation 10min on shaking table, dissolves crystal sufficiently.In enzyme
Join the light absorption value in each hole of measurement at immune detector OD490nm.
G. it is arranged zeroing hole (culture medium, MTT, dimethyl sulfoxide) simultaneously, (cell, the drug of same concentrations are molten for control wells
Solve medium, culture solution, MTT, dimethyl sulfoxide)
Fig. 1 is to freeze after the 8% xylitol hydrogel solution that the embodiment of the present invention 1 provides is changed into gel state at 37 degree
Dry scanning electron microscope (SEM) photograph.As can be seen from the figure: its pore size can contain cell between 20-30 microns.
Fig. 2 is the heating rheogram (heating rate 0.3 for the 8% xylitol hydrogel solution that the embodiment of the present invention 1 provides
DEG C/min, strain 0.05% and frequency 1rad/s).As can be seen from the figure: its phase transition temperature is at 32 DEG C.
Fig. 3 is unenhanced, stress 0.1 of the 8% xylitol hydrogel solution that provides of the embodiment of the present invention 1 at 37 degree.From figure
In it can be seen that its mechanical strength is moderate, not only can protect cell, but also can be in injectable under curdled appearance.
Fig. 4 is the 8% xylitol hydrogel MTT experiment data that the embodiment of the present invention 2 provides.As can be seen from the figure: solidifying
Glue does not have bio-toxicity and even helps to cell growth.
Fig. 5 is 1 gel of embodiment provided in an embodiment of the present invention in 37 DEG C of package dil staining cell shows fluorescent microscopy images
(stationary state is presented).As can be seen from the figure: cell is contained in gel, is protected.
Fig. 6 is that 1 gel of embodiment provided in an embodiment of the present invention package dil staining cell discharges cell in pbs solution
(travelling state is presented) in shows fluorescent microscopy images.As can be seen from the figure: at low temperatures, cell is discharged from gel.
Foregoing description is only the description to present pre-ferred embodiments, is not any restriction to the scope of the invention.Appoint
Any change or modification what those skilled in the art makes according to the technology contents of the disclosure above should all regard
For equivalent effective embodiment, the range of technical solution of the present invention protection is belonged to.
Claims (9)
1. a kind of temperature-sensitive hydrogel of adjustable response temperature contains cell material, it is characterised in that: including following weight
The component of percentage:
Methylcellulose, 12wt%；
PEG-20000,7.4wt%；
Non-toxic inorganic sodium salt, 1.4wt%；
Xylitol, 0~16wt%；
Surplus is water.
2. the temperature-sensitive hydrogel of adjustable response temperature according to claim 1 contains cell material, feature exists
In: the non-toxic inorganic sodium salt is Na2HPO3·12H2O。
3. the preparation side that the temperature-sensitive hydrogel of adjustable response temperature according to claim 1 contains cell material
Method, which is characterized in that specific step is as follows:
Take methylcellulose, polyethylene glycol-20000, non-toxic inorganic sodium salt, xylitol in the deionized water of 70~100 DEG C of heat
Middle uniform stirring, is uniformly dispersed, and is put into refrigerator cold-storage and stays overnight, is allowed to be completely dissolved, and adjustable response temperature can be obtained
Temperature-sensitive hydrogel contains cell material.
4. the preparation side that the temperature-sensitive hydrogel of adjustable response temperature according to claim 3 contains cell material
Method, it is characterised in that: the concentration of the methylcellulose is 136.36mg/ml；The concentration of the PEG-20000 is 80mg/ml；
The non-toxic inorganic sodium salt is Na2HPO3·12H2O, concentration 14.2mg/ml；The concentration of the xylitol is 0~200mg/
ml。
5. the preparation side that the temperature-sensitive hydrogel of adjustable response temperature according to claim 3 contains cell material
Method, it is characterised in that: the viscosity of the methylcellulose is 15mPa.s.
6. the preparation side that the temperature-sensitive hydrogel of adjustable response temperature according to claim 3 contains cell material
Method, it is characterised in that: the phase transition temperature that the temperature-sensitive hydrogel of the adjustable response temperature contains cell material passes through
The mass percent for adjusting the xylitol is completed.
7. the preparation side that the temperature-sensitive hydrogel of adjustable response temperature according to claim 6 contains cell material
Method, it is characterised in that: when the mass percent of the xylitol is 4%, the Thermo-sensitive water of the adjustable response temperature
The phase transition temperature that gel contains cell material is 34 DEG C；
When the mass percent of the xylitol be 8% (concentration 80mg/ml) when, it is described it is adjustable response temperature it is temperature sensitive
Property hydrogel contain cell material phase transition temperature be 32 DEG C.
8. the temperature-sensitive hydrogel of adjustable response temperature according to claim 1 contains cell material, it is applied to packet
Carry the intracorporal method of cell infusion are as follows:
(1) by the temperature-sensitive hydrogel solution of the adjustable response temperature and cell suspending liquid at low ambient temperatures in shaking table
Upper concussion, so that uniformly mixing, keeps being completely transformed into a gel state at 37 DEG C；
(2) gel state hydrogel-cell mixture is drawn with the syringe of removal syringe needle, is injected after loading onto syringe.
9. the temperature-sensitive hydrogel of adjustable response temperature according to claim 8 contains cell material, it is applied to packet
It carries the intracorporal method of cell infusion: using No. 1.2 syringe needle needle cylinder injections.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109912850A (en) * | 2019-03-11 | 2019-06-21 | 同济大学 | Contain the self-healing hydrogel and its preparation method and application of excretion body |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109912850A (en) * | 2019-03-11 | 2019-06-21 | 同济大学 | Contain the self-healing hydrogel and its preparation method and application of excretion body |
| CN109912850B (en) * | 2019-03-11 | 2021-03-02 | 同济大学 | Self-healing hydrogel entrapping exosomes and preparation method and application thereof |
| CN114042188A (en) * | 2021-08-31 | 2022-02-15 | 上海市第六人民医院 | Composite hydrogel for promoting healing of aponeurosis and preparation method and application thereof |
| CN114042188B (en) * | 2021-08-31 | 2023-03-31 | 上海市第六人民医院 | Composite hydrogel for promoting healing of aponeurosis and preparation method and application thereof |
| CN113842493A (en) * | 2021-09-13 | 2021-12-28 | 深圳先进技术研究院 | Preparation method of temperature-sensitive hydrogel and temperature-sensitive hydrogel |
| CN113842493B (en) * | 2021-09-13 | 2022-12-02 | 深圳先进技术研究院 | Preparation method of temperature-sensitive hydrogel and temperature-sensitive hydrogel |
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