CN109054049A - A kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket and its preparation method and application - Google Patents
A kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket and its preparation method and application Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- C12N2533/30—Synthetic polymers
Abstract
The present invention relates to a kind of PEG class temperature-sensitive hydrogel three-dimensional cell brackets and its preparation method and application.The preparation method of the three-dimensional cell bracket, it include: to mix the esters of acrylic acid water-soluble macromolecule polymerized monomer containing oligomeric ethylene glycol (PEG) side chain, initiator, surfactant, crosslinking agent and/or comonomer, reaction obtains PEG class temperature-sensitive nano gel;It is uniformly mixed at normal temperature with cell, by thermal induction embedding in situ cell, forms PEG class temperature-sensitive hydrogel three-dimensional cell bracket.Described preparation method is simple, and mild condition is small to cell damage.Compared with traditional PNIPAM class temperature-sensitive hydrogel bracket, the PEG class temperature-sensitive hydrogel bracket good biocompatibility, it is degradable, property is adjustable, extend syneresis degree very little at any time, the growth and proliferation that can maintain cell for a long time are a kind of comparatively ideal tissue engineering materials, can be applied to the fields such as cell injuring model, tissue repair, oncotherapy.
Description
Technical field
The present invention relates to field of biomedical polymer materials more particularly to a kind of PEG class temperature-sensitive hydrogel three-dimensional cells
Bracket and its preparation method and application.
Background technique
Hydrogel is a kind of to pass through the three-dimensional space network being physically or chemically cross-linked to form by colloidal particle or macromolecular chain
Structure has the physiochemical properties similar with living tissue, such as very high water content, lower surface tension and good
Biocompatibility etc. is widely used in fields of biomedicine such as cytoskeleton, tissue repair, medicine controlled releasings.
Temperature-sensitive hydrogel is that one kind can generate quick response to environment temperature and generate the function hydrogel of reversible transition.Its
Flowable liquid in compared with low temperature environment, can with the uniform mixing such as various seed cells, drug and bioactive molecule,
And gelation occurs rapidly in physiological temp environment (37 DEG C) and forms three dimensional hydrogel, thus by the substances embedding in situ such as cell,
Other any chemical reactions are not needed, thus simpler, safety in vivo, and there is invertibity, is a kind of ideal thin
Born of the same parents' timbering material.
A kind of temperature-sensitive hydrogel of most study is poly(N-isopropylacrylamide) (PNIPAM) hydrogel, body at present
Product phase transition temperature is at 32 DEG C, close to body temperature.But it is easy to happen under physiological temperature conditions and mutually separates, syneresis
Phenomenon is more serious, and it is with certain toxicity and refractory organics, limits its application in hydrogel scaffold.
Therefore, seek one kind and have both Thermo-sensitive, biocompatibility and biodegradability, and syneresis degree is lesser
Temperature-sensitive hydrogel timbering material is just particularly important.
Studies have found that poly- by two kinds of esters of acrylic acid water-soluble macromolecules containing oligomeric ethylene glycol (PEG) side chain
Close monomer methoxypolyethylene glycol methacrylate and 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy) ethyl ester copolymerization, it obtains
To polymer also there is good Thermo-sensitive, while having both biocompatibility and degradability, be expected to become a kind of substitution
PNIPAM temperature sensing material (Journal of the American Chemical Society, 2006,128 (40):
13046-13047).
Have the report of synthesis and application of the part about this PEG class temperature sensing material in document at present, such as:
Polymer, 2010, 51(17): 3926-3933;Polymer, 2014, 55(26): 6717-6724;
Macromolecular Chemistry and Physics, 2015, 216(23): 2230-2240;Cellular and
Molecular Bioengineering, 2016, 10(1):1-14.However, there has been no about this PEG class temperature of utilization at present
Document report of the quick hydrogel as three-dimensional cell bracket.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket and its preparation
Methods and applications.The PEG class temperature-sensitive hydrogel three-dimensional cell bracket have Thermo-sensitive, biocompatibility, biodegradability,
The advantages that syringeability and cell releasable property, extends syneresis degree very little at any time, cell can be maintained for a long time
Growth and proliferation, are a kind of comparatively ideal tissue engineering materials, can be applied to cell injuring model, tissue repair, oncotherapy
Equal fields.
In order to reach goal of the invention above-mentioned, the present invention provides a kind of system of PEG class temperature-sensitive hydrogel three-dimensional cell bracket
Preparation Method, comprising the following steps:
(1) will containing the esters of acrylic acid water-soluble macromolecule polymerized monomer of oligomeric ethylene glycol (PEG) side chain, surfactant,
Crosslinking agent and/or comonomer are mixed to form reaction solution, after deoxygenation, initiator are added, radical polymerization occurs in a heated condition
It closes reaction and obtains PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano gel for obtaining step (1) is configured to centainly after dialysis purification using PBS buffer solution
The nanogel solution of mass fraction is uniformly mixed with certain density seed cell suspension, is increased temperature, is passed through heat
Embedding in situ cell is induced, PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed.
In the above preparation method, it is preferred that contain the acrylic acid of oligomeric ethylene glycol (PEG) side chain in the step (1)
Esters water-soluble macromolecule polymerized monomer is methoxypolyethylene glycol methacrylate and 2- methyl -2- acrylic acid -2-(2- methoxy
Base oxethyl) ethyl ester, the quality of methoxypolyethylene glycol methacrylate is 2- methyl -2- acrylic acid -2-(2- methoxyl group ethoxy
Base) ethyl ester quality 0~50%.
Preferably, the crosslinking agent in the step (1) is N, and N'- methylene diacrylamide BIS, emulsifier is dodecyl
Sodium sulphate SDS, initiator are any one in ammonium persulfate APS or potassium peroxydisulfate KPS, and quality is respectively to contain oligomerization second two
1 ~ 15%, 0 ~ 3%, the 2 ~ 6% of the esters of acrylic acid water-soluble macromolecule polymerized monomer quality of alcohol (PEG) side chain.
Preferably, the comonomer in the step (1) is acrylic acid, methacrylic acid, Sodium Allyl Sulfonate, allyl
The water-soluble polymerisable monomer containing carboxyl, sulfonic group or amino such as amine, quality are third containing oligomeric ethylene glycol (PEG) side chain
The 0~4% of olefin(e) acid esters water-soluble macromolecule polymerized monomer quality.
Preferably, the mass fraction of polymerized monomer is 2~6% in reaction solution in the step (1);Polymeric reaction temperature is
50~90 DEG C;Reaction time is 3~8h.
Preferably, the middle reaction of the step (1), which obtains PEG class temperature-sensitive nano gel, has good temperature sensitivity,
Volume-phase transition temperature at 26 ~ 37 DEG C, 20 DEG C its partial size in 100 ~ 1000nm.
Preferably, the mass fraction of nanogel solution is 1 ~ 10% in the step (2), and the concentration of cell suspending liquid is 1
×105~5×106A/mL, the temperature that thermal induction forms temperature-sensitive hydrogel three-dimensional cell bracket is 26 ~ 37 DEG C.
The present invention also provides a kind of PEG class temperature-sensitive hydrogel three-dimensional cell brackets, are by above-mentioned preparation method system
For what is obtained;The PEG class temperature-sensitive hydrogel three-dimensional cell bracket extends syneresis degree very in physiological temp at any time
It is small, the growth and proliferation of seed cell can be maintained for a long time.
A kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket is applied in cell injuring model, tissue repair, is swollen
The fields such as tumor treatment.
Compared with prior art, the invention has the following advantages:
(1) preparation method is simple for PEG class temperature-sensitive hydrogel three-dimensional cell bracket provided by the invention, and mild condition is right
Cell damage is small;
(2) PEG class temperature-sensitive hydrogel three-dimensional cell bracket provided by the invention has good Thermo-sensitive, biocompatibility, life
Biodegradable and property controllability.It is single by control macromolecular polymeric monomer, surfactant, crosslinking agent and/or copolymerization
The inventory of body etc., it is adjustable to synthesize the property such as obtained partial size, Thermo-sensitive, the electrically charged amount of PEG class temperature-sensitive nano gel
Matter, and then the property for the PEG class temperature-sensitive hydrogel three-dimensional cell bracket being prepared is adjusted, the cell of internal embedding can be regulated and controled
Growth behavior;
(3) PEG class temperature-sensitive hydrogel three-dimensional cell bracket provided by the invention extends syneresis degree very little at any time, can
Cell can be released by cooling down solation, be a kind of more satisfactory by the growth and proliferation for maintaining cell for a long time
Tissue engineering material, can be widely applied to the fields such as cell injuring model, tissue repair, oncotherapy.
Detailed description of the invention
Fig. 1 is the partial size variation with temperature situation for the PEG class temperature-sensitive nano gel that embodiment 1 synthesizes.
Fig. 2 is the forming process of PEG class temperature-sensitive hydrogel in embodiment 1.
Fig. 3 is PEG class temperature-sensitive hydrogel extended steadiness at any time prepared by embodiment 1.
Fig. 4 is the MTT growth curve of cell in the PEG class temperature-sensitive hydrogel cytoskeleton of the preparation of embodiment 1.
Fig. 5 is glimmering after the cell in PEG class temperature-sensitive hydrogel cytoskeleton prepared by embodiment 1 is dyed using AO/EB
Light microscope figure.
Specific embodiment
In order to which technical characteristic of the invention, purpose and beneficial effect are more clearly understood, with reference to embodiments
It is explained further the present invention, but embodiment does not limit in any form the present invention.
Embodiment 1
Present embodiments provide a kind of preparation method and application of PEG class temperature-sensitive hydrogel three-dimensional cell bracket comprising following
Step:
(1) 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy is weighed) ethyl ester 2.2371g, oligomeric ethylene glycol methyl ether methyl-prop
Olefin(e) acid ester 0.5462g, N, N'- methylene-bisacrylamide 0.0431g and lauryl sodium sulfate 0.0284g are in 250mL's
In three neck round bottom, the dissolution of 100mL distilled water is added, is continually fed into nitrogen deoxygenation.70 DEG C are increased the temperature to after 1h, it will
0.0810g initiator potassium persulfate is dissolved in 5mL distilled water, is added in above-mentioned solution, keeps nitrogen atmosphere, the reaction was continued 5h,
Obtain PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano for obtaining step (1) gel permeation 7 days, change 3 water daily, to remove remaining reaction
Object, electrolyte and by-product.After purification, freeze-drying is for 24 hours.Certain density solution is configured to using PBS buffer solution, and it is certain
The MCF-7 breast cancer cell of concentration mixes, and makes the final mass score 3% of nanogel solution, and the ultimate density of cell is 1
×105A/ml.Mixed liquor is added in 48 porocyte culture plates, 0.5mL is added in every hole, is transferred to 37 DEG C of cell culture
PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed in situ by thermal induction in case.Timing observe and measurement cell in the bracket
Growing state.
The partial size and Thermo-sensitive of the PEG class temperature-sensitive nano gel that synthesis obtains in step (1) are characterized.
Partially synthetic obtained PEG class temperature-sensitive nano gel is taken to be configured to certain density weak solution with distilled water, thoroughly
After film filtering, its partial size at different temperatures is measured using nano particle size instrument.As shown in Figure 1, its partial size is about at 20 DEG C
250nm, and increased with temperature, partial size gradually becomes smaller, and shows good Thermo-sensitive, Volume-phase transition temperature is on 30 DEG C of left sides
It is right.
The forming process and stability of PEG class temperature-sensitive hydrogel in step (2) are studied.
It (is not contained by the PEG class temperature-sensitive nano gel solution that step (2) the method configuration final mass score is 3%
Cell), increase the temperature to 37 DEG C.As shown in Fig. 2, being flowable liquid at 20 DEG C, 37 DEG C are warming up to, and after keeping 10min
Form stable hydrogel.As shown in figure 3, with the extension of time, this PEG class temperature-sensitive hydrogel stability is good, volume
It is basically unchanged, has no apparent syneresis phenomenon.
The growing state of cell in the present embodiment in the bracket is studied.
Using proliferative conditions of the mtt assay research cell in PEG class temperature-sensitive hydrogel cytoskeleton.Cultivate specific time
Afterwards, 100 μ L MTT solution are added to hole every in gaging hole to 48 orifice plates respectively, continue after cultivating 4h, take out gel piece, is added
10mL dimethyl sulfoxide, stirring are dissolved out the first a ceremonial jade-ladle, used in libation wherein generated.It is measured at 490nm using ultraviolet specrophotometer
Absorbance value.As shown in figure 4, absorbance value gradually increases with the increase of cultivated days, illustrate that the number of cell gradually increases
It is more.
Using activity and form situation of the AO/EB decoration method research cell in PEG class temperature-sensitive hydrogel cytoskeleton.
After cultivating specific time, the 10 bis- coloring agents of μ L AO/EB are added to hole every in gaging hole to 48 orifice plates respectively, continue after cultivating 1h,
Taking-up gel piece, which is placed under inverted fluorescence microscope, to be observed.As shown in figure 5, green, explanation is presented in cell in incubation time
Cell maintains good activity, and with the extension of cultivated days, cell tends to form many cells group.
The PEG class temperature-sensitive hydrogel three-dimensional cell bracket of the present embodiment has good Thermo-sensitive, extends dehydration at any time
Shrinkage degree very little can maintain the growth and proliferation of cell for a long time, and preparation method is simple, mild condition, to thin
Born of the same parents are a kind of ideal cell scaffold materials without obvious damage.
Embodiment 2
Present embodiments provide a kind of preparation method and application of PEG class temperature-sensitive hydrogel three-dimensional cell bracket comprising following
Step:
(1) 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy is weighed) ethyl ester 2.3664g, oligomeric ethylene glycol methyl ether methyl-prop
Olefin(e) acid ester 0.3368g, N, N'- methylene-bisacrylamide 0.0431g and lauryl sodium sulfate 0.0284g are in 250mL's
In three neck round bottom, the dissolution of 100mL distilled water is added, is continually fed into nitrogen deoxygenation.70 DEG C are increased the temperature to after 1h, it will
0.0810g initiator potassium persulfate is dissolved in 5mL distilled water, is added in above-mentioned solution, keeps nitrogen atmosphere, the reaction was continued 5h,
Obtain PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano for obtaining step (1) gel permeation 7 days, change 3 water daily, to remove remaining reaction
Object, electrolyte and by-product.After purification, freeze-drying is for 24 hours.Certain density solution is configured to using PBS buffer solution, and it is certain
The MCF-7 breast cancer cell of concentration mixes, and makes the final mass score 3% of nanogel solution, and the ultimate density of cell is 1
×105A/ml.Mixed liquor is added in 48 porocyte culture plates, 0.5mL is added in every hole, is transferred to 37 DEG C of cell culture
PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed in situ by thermal induction in case.
Embodiment 3
Present embodiments provide a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket comprising following steps:
(1) 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy is weighed) ethyl ester 2.5004g, oligomeric ethylene glycol methyl ether methyl-prop
Olefin(e) acid ester 0.1260g, N, N'- methylene-bisacrylamide 0.0431g and lauryl sodium sulfate 0.0284g are in 250mL's
In three neck round bottom, the dissolution of 100mL distilled water is added, is continually fed into nitrogen deoxygenation.70 DEG C are increased the temperature to after 1h, it will
0.0810g initiator potassium persulfate is dissolved in 5mL distilled water, is added in above-mentioned solution, keeps nitrogen atmosphere, the reaction was continued 5h,
Obtain PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano for obtaining step (1) gel permeation 7 days, change 3 water daily, to remove remaining reaction
Object, electrolyte and by-product.After purification, freeze-drying is for 24 hours.Certain density solution is configured to using PBS buffer solution, and it is certain
The Hela cervical cancer cell of concentration mixes, and makes the final mass score 3% of nanogel solution, the ultimate density of cell for 1 ×
105A/ml.Mixed liquor is added in 48 porocyte culture plates, 0.5mL is added in every hole, it is transferred to 37 DEG C of cell incubator,
PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed in situ by thermal induction.
Embodiment 4
Present embodiments provide a kind of preparation method and application of PEG class temperature-sensitive hydrogel three-dimensional cell bracket comprising following
Step:
(1) 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy is weighed) ethyl ester 2.2371g, oligomeric ethylene glycol methyl ether methyl-prop
Olefin(e) acid ester 0.5462g, N, N'- methylene-bisacrylamide 0.0431g and lauryl sodium sulfate 0.0142g are in 250mL's
In three neck round bottom, the dissolution of 100mL distilled water is added, is continually fed into nitrogen deoxygenation.70 DEG C are increased the temperature to after 1h, it will
0.0810g initiator potassium persulfate is dissolved in 5mL distilled water, is added in above-mentioned solution, keeps nitrogen atmosphere, the reaction was continued 5h,
Obtain PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano for obtaining step (1) gel permeation 7 days, change 3 water daily, to remove remaining reaction
Object, electrolyte and by-product.After purification, freeze-drying is for 24 hours.Certain density solution is configured to using PBS buffer solution, and it is certain
The HepG2 liver cancer cells of concentration mix, and make the final mass score 3% of nanogel solution, the ultimate density of cell for 1 ×
105A/ml.Mixed liquor is added in 48 porocyte culture plates, 0.5mL is added in every hole, it is transferred to 37 DEG C of cell incubator,
PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed in situ by thermal induction.
Embodiment 5
Present embodiments provide a kind of preparation method and application of PEG class temperature-sensitive hydrogel three-dimensional cell bracket comprising following
Step:
(1) 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy is weighed) ethyl ester 2.1451g, oligomeric ethylene glycol methyl ether methyl-prop
Olefin(e) acid ester 0.5250g, acrylic acid 0.0403g, N, N'- methylene-bisacrylamide 0.0431g and lauryl sodium sulfate
0.0284g is added the dissolution of 100mL distilled water, is continually fed into nitrogen deoxygenation in the three neck round bottom of 250mL.It is risen after 1h
High-temperature is dissolved in 5mL distilled water to 70 DEG C, by 0.0810g initiator potassium persulfate, is added in above-mentioned solution, keeps nitrogen
Atmosphere, the reaction was continued 5h obtain PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano for obtaining step (1) gel permeation 7 days, change 3 water daily, to remove remaining reaction
Object, electrolyte and by-product.After purification, freeze-drying is for 24 hours.Certain density solution is configured to using PBS buffer solution, and it is certain
The MCF-7 breast cancer cell of concentration mixes, and makes the final mass score 3% of nanogel solution, and the ultimate density of cell is 1
×105A/ml.Mixed liquor is added in 48 porocyte culture plates, 0.5mL is added in every hole, is transferred to 37 DEG C of cell culture
PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed in situ by thermal induction in case.
Claims (9)
1. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket, which comprises the following steps:
(1) will containing the esters of acrylic acid water-soluble macromolecule polymerized monomer of oligomeric ethylene glycol (PEG) side chain, surfactant,
Crosslinking agent and/or comonomer are mixed to form reaction solution, after deoxygenation, initiator are added, radical polymerization occurs in a heated condition
It closes reaction and obtains PEG class temperature-sensitive nano gel;
(2) the PEG class temperature-sensitive nano gel for obtaining step (1) is configured to centainly after dialysis purification using PBS buffer solution
The nanogel solution of mass fraction is uniformly mixed with certain density seed cell suspension, is increased temperature, is passed through heat
Embedding in situ cell is induced, PEG class temperature-sensitive hydrogel three-dimensional cell bracket is formed.
2. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1, which is characterized in that
Esters of acrylic acid water-soluble macromolecule polymerized monomer in the step (1) containing oligomeric ethylene glycol (PEG) side chain is poly- second two
Alcohol methyl ether methacrylate and 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy) ethyl ester, methoxypolyethylene glycol methyl-prop
The quality of olefin(e) acid ester be 2- methyl -2- acrylic acid -2-(2- methoxy ethoxy) ethyl ester quality 0~50%.
3. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1, which is characterized in that
Crosslinking agent in the step (1) is N, and N'- methylene-bisacrylamide (BIS), emulsifier is lauryl sodium sulfate
(SDS), initiator is any one in ammonium persulfate (APS) or potassium peroxydisulfate (KPS), and quality is respectively to contain oligomerization second two
1 ~ 15%, 0 ~ 3%, the 2 ~ 6% of the esters of acrylic acid water-soluble macromolecule polymerized monomer quality of alcohol (PEG) side chain.
4. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1, which is characterized in that
Comonomer in the step (1), which is that acrylic acid, methacrylic acid, Sodium Allyl Sulfonate, allyl amine etc. are water-soluble, to be contained
The polymerisable monomer of carboxyl, sulfonic group or amino, quality are that the esters of acrylic acid containing oligomeric ethylene glycol (PEG) side chain is water-soluble
The 0~4% of macromolecular polymeric monomer mass.
5. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1, which is characterized in that
The mass fraction of polymerized monomer is 2~6% in reaction solution in the step (1);Polymeric reaction temperature is 50~90 DEG C;When reaction
Between be 3~12h.
6. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1, which is characterized in that
Reaction obtains PEG class temperature-sensitive nano gel with good temperature sensitivity in the step (1), and Volume-phase transition temperature exists
Its partial size is in 100 ~ 1000nm at 26 ~ 37 DEG C, 20 DEG C.
7. a kind of preparation method of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1, which is characterized in that
The mass fraction of nanogel solution is 1 ~ 10% in the step (2), and the concentration of cell suspending liquid is 1 × 105~5×106A/
ML, the temperature that thermal induction forms temperature-sensitive hydrogel three-dimensional cell bracket is 26 ~ 37 DEG C.
8. a kind of PEG class temperature-sensitive hydrogel three-dimensional cell bracket is prepared by the preparation method described in claim 1~7
It obtains;The PEG class temperature-sensitive hydrogel three-dimensional cell bracket extends syneresis degree very little in physiological temp at any time,
The growth and proliferation of seed cell can be maintained for a long time.
9. a kind of application of PEG class temperature-sensitive hydrogel three-dimensional cell bracket as described in claim 1~8 is any, feature exist
In the PEG class temperature-sensitive hydrogel three-dimensional cell bracket is for fields such as cell injuring model, tissue repair, oncotherapies.
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CN115960305A (en) * | 2022-12-19 | 2023-04-14 | 华中科技大学 | Nano gel, nano medicine based on nano gel, preparation method and application thereof |
CN115960305B (en) * | 2022-12-19 | 2024-04-02 | 华中科技大学 | Nanogel, nanogel-based nano medicine, and preparation method and application thereof |
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