CN108912327B - Degradable living cell fluorescence imaging material with water-soluble unconjugated structure, and preparation method and application thereof - Google Patents
Degradable living cell fluorescence imaging material with water-soluble unconjugated structure, and preparation method and application thereof Download PDFInfo
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- 239000000463 material Substances 0.000 title claims abstract description 76
- 238000002360 preparation method Methods 0.000 title claims abstract description 60
- 238000000799 fluorescence microscopy Methods 0.000 title claims description 54
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 105
- 229920000642 polymer Polymers 0.000 claims abstract description 97
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 39
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 39
- OEIJHBUUFURJLI-UHFFFAOYSA-N octane-1,8-diol Chemical compound OCCCCCCCCO OEIJHBUUFURJLI-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000012632 fluorescent imaging Methods 0.000 claims abstract description 15
- 150000004756 silanes Chemical class 0.000 claims abstract description 10
- 238000012719 thermal polymerization Methods 0.000 claims abstract description 8
- 238000003384 imaging method Methods 0.000 claims abstract description 4
- 238000003756 stirring Methods 0.000 claims description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 44
- 239000008367 deionised water Substances 0.000 claims description 43
- 229910021641 deionized water Inorganic materials 0.000 claims description 43
- 238000006243 chemical reaction Methods 0.000 claims description 36
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 34
- 239000000178 monomer Substances 0.000 claims description 33
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 30
- 238000004108 freeze drying Methods 0.000 claims description 27
- 239000003054 catalyst Substances 0.000 claims description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims description 17
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 15
- 239000007795 chemical reaction product Substances 0.000 claims description 14
- 238000002844 melting Methods 0.000 claims description 14
- 230000008018 melting Effects 0.000 claims description 14
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- -1 dimethyl imine Chemical class 0.000 claims description 9
- 239000004593 Epoxy Substances 0.000 claims description 8
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 8
- 229910000077 silane Inorganic materials 0.000 claims description 8
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 6
- 239000011261 inert gas Substances 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 abstract description 8
- 230000003287 optical effect Effects 0.000 abstract description 5
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- 230000015572 biosynthetic process Effects 0.000 abstract description 3
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- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 14
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- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 10
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- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 2
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- PUAQLLVFLMYYJJ-UHFFFAOYSA-N 2-aminopropiophenone Chemical compound CC(N)C(=O)C1=CC=CC=C1 PUAQLLVFLMYYJJ-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
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- 150000001721 carbon Chemical class 0.000 description 1
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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Abstract
The invention discloses a degradable living cell fluorescent imaging material with a water-soluble non-conjugated structure, a preparation method and application thereof, wherein citric acid, 1, 8-octanediol and polyethylene glycol are subjected to thermal polymerization to obtain PCG prepolymer; then grafting certain amino acid or silane derivatives onto PCG prepolymer to synthesize the polymer. The synthesis method used by the invention is simple and environment-friendly, the cost of the synthesis raw materials is low and the polymer is nontoxic, and the prepared polymer has good blood compatibility and cell compatibility, and meanwhile, the material also has good fluorescence characteristic and optical stability, so that the polymer has good application prospect in living cell imaging.
Description
Technical Field
The invention belongs to the technical field of degradable biomedical materials, and particularly relates to a preparation method and application of a degradable living cell fluorescence imaging material with a water-soluble unconjugated structure.
Background
In recent years, life sciences have rapidly developed with the application of various new technologies and new methods. For example, by using a fluorescence microscope, intracellular fine structures, interactions between cells, cell signal transduction, and the action of macromolecular proteins can be known. However, the two problems of the coverage of signals of fluorescent materials by autofluorescence of cells in the visible light region and easy photobleaching of fluorescent organic dye molecules have hindered the development and research of fluorescent imaging technology and fluorescent probes, so that along with the continuous development of scientific technology, some fluorescent nanomaterials with excellent characteristics are continuously emerging. Compared with the traditional fluorescent molecules, the fluorescent nano material has the advantages of light stability, multifunction, easy regulation and control and the like.
The polymer is a novel fluorescent nano material, and has the advantages of excellent optical characteristics, good stability, convenient modification and the like, so that the polymer has wide application prospect in the fields of biological imaging, disease treatment and the like. The polymer with fluorescence characteristics has conjugated structure and unconjugated structure, and the polymer with conjugated structure has the characteristic of fluorescence intensity, but has poor water solubility and biocompatibility, so that the application of the polymer in biological imaging is greatly limited; the polymer of the non-conjugated structure itself has almost no fluorescence emission, but the polymer prepared by high temperature treatment or chemical crosslinking may have visible fluorescence due to surface state luminescence caused by the carbon nucleus generated by hydrothermal carbonization and the polymer chain of which the surface is self-passivated, or due to crosslinking of the polymer chain, vibration and rotation of a potential fluorophore (carbon-oxygen double bond, nitrogen-containing group, etc.) carried by the polymer itself are limited, non-radiative transition is suppressed, and fluorescence emission in the visible region is enhanced.
At present, polyester biological materials have great development potential in the biological material neighborhood due to the characteristics of controllable mechanical and degradation performance and excellent biocompatibility. The poly (1, 8-octanediol ester) -polyethylene glycol (PCG) is a polymer formed by condensing citric acid, 1, 8-octanediol and polyethylene glycol at high temperature, has the advantages of no toxicity of synthetic monomers, good biocompatibility, simple reaction and no impurity introduction, and can be further modified due to three carboxyl groups of the citric acid, but the PCG without modification has no fluorescent characteristic, so that if the PCG can be modified to have fluorescent emission capability, the application of the PCG in biological imaging can be greatly improved.
Disclosure of Invention
The invention aims to provide a preparation method and application of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure, wherein the method is simple in process, and the prepared material has good biocompatibility and living cell fluorescence imaging capability.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
the preparation method of the degradable living cell fluorescence imaging material with the water-soluble unconjugated structure comprises the following steps:
1) Preparation of PCG prepolymer: carrying out thermal polymerization reaction on citric acid and glycol in a molar ratio of 1:1 at 140-160 ℃ under the conditions of nitrogen, inert gas or vacuum to obtain PCG prepolymer;
2) PCGA polymer preparation: the PCG prepolymer with the molar ratio of 1 (3-10) and amino acid react in MES buffer solution by taking EDC and NHS as catalysts to obtain PCGA polymer;
PCGS polymer preparation: the PCG prepolymer and silane derivative with the mol ratio of 1 (3-10) are reacted in dimethyl imine by using DCC and DMAP as catalysts to obtain the PCGS polymer.
3) Preparation of fluorescent imaging material: the synthesized PCGA polymer or PCGS polymer is dissolved in deionized water to obtain a solution of the degradable living cell fluorescence imaging material with a water-soluble unconjugated structure.
As a further development of the invention, the diol in step 1) is a mixture of 1, 8-octanediol and polyethylene glycol, the molar ratio of 1, 8-octanediol to polyethylene glycol being 7:3.
3. The method of claim 1, wherein the amino acid in step 2) is L-arginine or polylysine.
As a further improvement of the invention, the pH value of the MES buffer in the step 2) is 5-6.
5. The method of claim 1, wherein the silane derivative in step 3) is epoxy silane or amino siloxane.
As a further improvement of the invention, the specific steps of step 1) are:
stirring and melting citric acid and glycol in 140-160deg.C oil bath; after the reaction monomers are completely melted, the temperature is immediately reduced to 140 ℃, and the thermal polymerization reaction is carried out for 5 hours under the conditions of nitrogen, inert gas or vacuum, the reaction product is dialyzed and purified in deionized water for 2 days, and the PCG prepolymer is obtained after freeze drying; .
As a further improvement of the invention, the reaction of step 2) is carried out at room temperature, the final product is purified by dialysis in deionized water for 2 days to remove unreacted monomers and catalyst, and the polymer is obtained by freeze drying.
A degradable living cell fluorescence imaging material with a water-soluble unconjugated structure prepared by the method.
The degradable living cell fluorescence imaging material with the water-soluble unconjugated structure is applied to living cell imaging.
Compared with the prior art, the invention has the following beneficial effects:
aiming at the defects of poor hydrophilicity, light stability, stability in organisms and the like of the traditional fluorescent molecules, the invention provides a preparation method of a degradable living cell fluorescent imaging material with a water-soluble non-conjugated structure. In the invention, the PCG prepolymer obtained by thermal polymerization reaction of citric acid, 1, 8-octanediol and polyethylene glycol has good biocompatibility and hydrophilicity, but the polymer does not have any fluorescence emission, so that some polymers with potential fluorophores (carbon-oxygen double bonds, nitrogen-containing groups and the like) are introduced, for example: the grafting of L-arginine, polylysine, epoxy silane and aminosilicone on the PCG prepolymer can effectively lead the PCG prepolymer to have fluorescence emission capability, and the application in biological imaging can be greatly improved. The preparation method of the invention is simple and convenient, and the synthesis method of the thermal polymerization reaction and the catalytic reaction is environment-friendly, simple to operate and low in raw material cost. The experimental results prove that: the degradable living cell fluorescence imaging material with the water-soluble unconjugated structure prepared by the method has good hydrophilicity, light stability and biocompatibility.
Furthermore, the poly (citrate) Prepolymer (PCG) used in the invention is a degradable aliphatic polyester polymer, and has good biocompatibility, good hydrophilicity, simple preparation method and low-cost and easily obtained monomer.
Furthermore, the invention utilizes L-arginine, polylysine, epoxy silane and aminosiloxane to modify the poly (citrate) Prepolymer (PCG), thereby improving the fluorescence characteristic of the poly (citrate) prepolymer, leading the poly (citrate) prepolymer to have fluorescence emission capability and greatly improving the application in biological imaging.
Furthermore, the degradable living cell fluorescence imaging material with the water-soluble unconjugated structure prepared by the invention has strong fluorescence under ultraviolet excitation, and can monitor the movement of the material in cells in real time.
Further, the water-soluble non-conjugated structure degradable living cell fluorescence imaging material (PCGR) prepared in the invention can effectively improve the biological activity of cells.
Drawings
FIG. 1 is a structural formula of each monomer and polymer in the synthesized water-soluble non-conjugated structure degradable living cell fluorescence imaging material.
FIG. 2 is a 1H-NMR spectrum of the resulting PCG prepolymer and PCGR polymer.
FIG. 3 is a FT-IR spectrum of a PCG prepolymer and a PCGR polymer in a degradable living cell fluorescent imaging material of water-soluble non-conjugated structure synthesized according to the invention;
FIG. 4 shows the optical properties of the water-soluble non-conjugated structure degradable living cell fluorescence imaging material prepared by the invention.
FIG. 5 shows the measurement of myoblast cytotoxicity of the degradable living cell fluorescence imaging material with the water-soluble unconjugated structure.
FIG. 6 shows the results of live cell fluorescence imaging of the degradable live cell fluorescence imaging material with a water-soluble non-conjugated structure prepared by the invention.
Detailed Description
The invention is described in further detail below with reference to the attached drawing figures:
the invention aims to prepare a degradable living cell fluorescence imaging material with a water-soluble unconjugated structure and good biocompatibility and fluorescence characteristics, so that the degradable living cell fluorescence imaging material has the capability of living cell fluorescence labeling. The preparation method comprises the following steps:
1) Preparation of PCG prepolymer: and (3) carrying out thermal polymerization reaction on citric acid and glycol in a molar ratio of 1:1 at 140-160 ℃ under nitrogen or vacuum condition to obtain the PCG prepolymer. The diols are 1, 8-octanediol and polyethylene glycol (PEG). The structural formula of the PCG prepolymer is as follows (as in FIG. 1):
2) PCGA polymer preparation: the PCG prepolymer with the molar ratio of 1 (3-10) and amino acid are reacted in MES buffer with the pH of 5-6 at 50mM and EDC and NHS as catalysts to obtain the PCGA polymer. The amino acid is L-arginine and polylysine. The structure is as follows
PCGS polymer preparation: the PCG prepolymer and silane derivative with the mol ratio of 1 (3-10) are reacted in dimethyl imine by using DCC and DMAP as catalysts to obtain the PCGS polymer. The silane derivative is epoxy silane and amino siloxane. The structural formula is as follows:
3) Preparation of fluorescent imaging material: the synthesized PCGA polymer or PCGS polymer is dissolved in deionized water to obtain a solution of the degradable living cell fluorescence imaging material with a water-soluble unconjugated structure.
Among them, the poly (citrate) Prepolymer (PC) has been widely used in the biomedical field due to its controllable biodegradation, good biocompatibility and low cost. However, PC is extremely poorly water-soluble and does not possess any fluorescence properties in itself, resulting in limited application in live cell fluorescence imaging; polyethylene glycol (PEG) is a nontoxic and nonirritating polymer with good hydrophilicity, and if PC is modified by PEG, the solubility of PC can be improved, so that a water-soluble poly (citrate) (PCG prepolymer) is prepared; l-arginine is a semi-essential amino acid required by human body and plays an important role in biological activity of the organism, such as cell division, wound repair and the like, so that the biocompatibility of the polymer can be obviously improved through modification of the PCG prepolymer by the L-arginine, the L-arginine has a nitrogen-containing group and potential fluorescence emission capability, the PCG prepolymer can have certain fluorescence emission capability through modification of the PCG prepolymer by the L-arginine, and in addition, the PCG can also have certain fluorescence emission capability through modification of the PCG by polylysine, epoxysilane and aminosiloxane. Therefore, in the invention, the degradable living cell fluorescence imaging material with a water-soluble unconjugated structure formed by grafting the amino acid or the silane derivative to the Poly Citrate (PCG) has good biocompatibility, has fluorescence characteristics under ultraviolet irradiation, can monitor the movement of the material in cells in real time, and is a material which can be used for living cell imaging.
For a better understanding of the present invention, the present invention will be described in detail with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGR (3) Polymer: 1mmol of Polycitrate (PCG) is weighed and stirred in 30mL of MES buffer solution with 50mM and pH of 5-6 to be completely dissolved, 4mmol of EDC is added, stirring is carried out at room temperature for 30min, then 4mmol of NHS is added, stirring is carried out at room temperature for 12h, finally 3mmol of L-arginine is added, stirring is carried out at room temperature for 12h, the product is dialyzed and purified in deionized water for 2 days to remove unreacted monomers and catalysts EDC and NHS, and the PCGR (3) polymer is obtained after freeze drying and is used.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGR (3) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 2
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGR (5) Polymer: 1mmol of Polycitrate (PCG) is weighed and stirred in 30mL of MES buffer solution with 50mM and pH of 5-6 to be completely dissolved, 4mmol of EDC is added, stirring is carried out at room temperature for 30min, then 4mmol of NHS is added, stirring is carried out at room temperature for 12h, finally 5mmol of L-arginine is added, stirring is carried out at room temperature for 12h, the product is dialyzed and purified in deionized water for 2 days to remove unreacted monomers and catalysts EDC and NHS, and the PCGR (5) polymer is obtained after freeze drying and is used.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGR (5) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 3
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGR (10) Polymer: 1mmol of Polycitrate (PCG) is weighed and stirred in 30mL of MES buffer solution with 50mM and pH of 5-6 to be completely dissolved, 4mmol of EDC is added, stirring is carried out at room temperature for 30min, then 4mmol of NHS is added, stirring is carried out at room temperature for 12h, finally 10mmol of L-arginine is added, stirring is carried out at room temperature for 12h, the product is dialyzed and purified in deionized water for 2 days to remove unreacted monomers and catalysts EDC and NHS, and the PCGR (10) polymer is obtained after freeze drying and is used.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthetic polymer PCGR (10) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 4
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGK (3) Polymer: 1mmol of Polycitrate (PCG) was weighed and completely dissolved in 30mL of MES buffer solution of 50mM and pH 5-6, 4mmol of EDC was added, stirring was carried out at room temperature for 30min, then 4mmol of NHS was added, stirring was carried out at room temperature for 12h, finally 3mmol of polylysine (EPL) was added, stirring was carried out at room temperature for 12h, and the product was dialyzed and purified in deionized water for 2 days to remove unreacted monomers and catalysts EDC and NHS, and the PCGK (3) polymer was obtained by freeze drying and was left for later use.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGK (3) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 5
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGK (5) Polymer: 1mmol of Polycitrate (PCG) was weighed and completely dissolved in 30mL of MES buffer solution of 50mM and pH 5-6, 4mmol of EDC was added, stirring was carried out at room temperature for 30min, then 4mmol of NHS was added, stirring was carried out at room temperature for 12h, finally 5mmol of polylysine (EPL) was added, stirring was carried out at room temperature for 12h, and the product was dialyzed and purified in deionized water for 2 days to remove unreacted monomers and catalysts EDC and NHS, and the PCGK (5) polymer was obtained by freeze drying and was left for later use.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGK (5) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 6
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGK (10) Polymer: 1mmol of Polycitrate (PCG) was weighed and completely dissolved in 30mL of MES buffer solution of 50mM and pH 5-6, 4mmol of EDC was added, stirring was carried out at room temperature for 30min, then 4mmol of NHS was added, stirring was carried out at room temperature for 12h, finally 10mmol of polylysine (EPL) was added, stirring was carried out at room temperature for 12h, and the product was dialyzed and purified in deionized water for 2 days to remove unreacted monomers and catalysts EDC and NHS, and the PCGK (10) polymer was obtained by freeze drying and was left for later use.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGK (10) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 7
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGM (3) Polymer: 1mmol of Polycytidylate (PCG) is weighed and stirred in DMSO to be completely dissolved, 5mmol of DCC is added, stirring is carried out at room temperature for 30min, then 2mmol of DMAP is added, stirring is carried out at room temperature for 1h, finally 3mmol of aminosilicone is added, stirring is carried out at room temperature for 24h, the product is dialyzed and purified in deionized water for 2 days, unreacted monomers, catalysts DCC and DMAP are removed, and PCGM (3) polymer is obtained after freeze drying, and is used after being left.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGM (3) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 8
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGM (5) Polymer: 1mmol of Polycytidylate (PCG) is weighed and stirred in DMSO to be completely dissolved, 5mmol of DCC is added, stirring is carried out at room temperature for 30min, then 2mmol of DMAP is added, stirring is carried out at room temperature for 1h, finally 5mmol of aminosilicone is added, stirring is carried out at room temperature for 24h, the product is dialyzed and purified in deionized water for 2 days, unreacted monomers, catalysts DCC and DMAP are removed, and PCGM (5) polymer is obtained after freeze drying, and is used after being left.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGM (5) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 9
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGM (10) Polymer: 1mmol of Polycytidylate (PCG) is weighed and stirred in DMSO to be completely dissolved, 5mmol of DCC is added, stirring is carried out at room temperature for 30min, then 2mmol of DMAP is added, stirring is carried out at room temperature for 1h, finally 10mmol of aminosilicone is added, stirring is carried out at room temperature for 24h, the product is dialyzed and purified in deionized water for 2 days, unreacted monomers, catalysts DCC and DMAP are removed, and PCGM (10) polymer is obtained after freeze drying, and is used after being left.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGM (10) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 10
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGE (3) Polymer: 1mmol of poly (citrate) (PCG) is weighed and stirred in DMSO to be completely dissolved, 5mmol of DCC is added, stirring is carried out at room temperature for 30min, then 2mmol of DMAP is added, stirring is carried out at room temperature for 1h, finally 3mmol of epoxy silane is added, stirring is carried out at room temperature for 24h, the product is dialyzed and purified in deionized water for 2 days, unreacted monomers, catalysts DCC and DMAP are removed, and the PCGE (3) polymer is obtained after freeze drying and is reserved for use.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGE (3) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 11
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGE (5) Polymer: 1mmol of poly (citrate) (PCG) is weighed and stirred in DMSO to be completely dissolved, 5mmol of DCC is added, stirring is carried out at room temperature for 30min, then 2mmol of DMAP is added, stirring is carried out at room temperature for 1h, finally 5mmol of epoxy silane is added, stirring is carried out at room temperature for 24h, the product is dialyzed and purified in deionized water for 2 days, unreacted monomers, catalysts DCC and DMAP are removed, and the PCGE (5) polymer is obtained after freeze drying and is reserved for use.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthesized polymer PCGE (5) is dissolved in deionized water, and the polymer has good hydrophilicity and certain fluorescence emission capacity, so that the solution of the degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure can be obtained.
Example 12
1) Preparation of PCG prepolymer: adding citric acid, 1, 8-octanediol and polyethylene glycol into a round-bottom flask according to the molar ratio of 1:0.7:0.3, and stirring and melting in an oil bath at 160 ℃ under the condition of nitrogen; after the citric acid, the 1, 8-octanediol and the polyethylene glycol serving as the reaction monomers are all melted, the temperature is immediately reduced to 140 ℃ and the reaction is carried out for 5 hours under the vacuum condition. Dialyzing and purifying the reaction product in deionized water for 2 days, and freeze-drying for later use;
2) Preparation of PCGE (10) Polymer: 1mmol of poly (citrate) (PCG) is weighed and stirred in DMSO to be completely dissolved, 5mmol of DCC is added, stirring is carried out at room temperature for 30min, then 2mmol of DMAP is added, stirring is carried out at room temperature for 1h, finally 10mmol of epoxy silane is added, stirring is carried out at room temperature for 24h, the product is dialyzed and purified in deionized water for 2 days, unreacted monomers, catalysts DCC and DMAP are removed, and the PCGE (10) polymer is obtained after freeze drying and is reserved for use.
3) Preparation of a degradable living cell fluorescence imaging material with a water-soluble non-conjugated structure: the synthetic polymer PCGE (10) is dissolved in deionized water, and the polymer has good non-conjugated structure and certain fluorescence emission capacity, so that a solution of the water-soluble degradation living cell fluorescence imaging material can be obtained.
The degradable living cell fluorescence imaging material (PCGR, PCGK, PCGM and PCGE) with the water-soluble non-conjugated structure has the advantages of good biocompatibility, good hydrophilicity, simple preparation method, low price and easy obtaining of monomers, and meanwhile, due to the introduction of amino acid or silane derivatives, the polymer shows certain fluorescence characteristics and optical stability, so that the application of the polymer in living cell fluorescence imaging is greatly improved. The detailed analysis is performed in conjunction with experimental data.
FIG. 1 shows the structural formulas of various monomers and polymers in the water-soluble non-conjugated structure degradable living cell fluorescent imaging material synthesized by the invention, wherein A in FIG. 1 shows the structural formula of citric acid, B in FIG. 1 shows the structural formula of 1, 8-octanediol, C in FIG. 1 shows the structural formula of polyethylene glycol, D in FIG. 1 shows the structural formula of PCG prepolymer, and E in FIG. 1 shows the structural formula of PCGR polymer.
FIG. 2 shows the PCG prepolymer and PCGR polymer of the synthetic water-soluble non-conjugated structure of the degradable living cell fluorescent imaging material of the present invention 1 H-NMR spectrum, wherein A in FIG. 2 is PCG prepolymer 1 H-NMR spectrum, multiple peaks of methylene protons of citric acid at 2.6-3.0ppm, methylene proton peaks on 1, 8-octanediol at 1.2,1.5,3.9 and 4.1ppm, respectively, methylene proton peaks on polyethylene glycol at 3.5,4.2 and 4.3ppm, respectively, and occurrence of 3.9,4.1,4.2 and 4.3ppm proton peaks therein indicates successful synthesis of PCG; FIG. 2B is a PCGR prepolymer 1 H-NMR spectrum, wherein the appearance of the 7.8ppm peak indicated that L-arginine had been successfully grafted onto PCG prepolymer and formed a PCGR polymer.
FIG. 3 is a FT-IR spectrum of PCG prepolymer and PCGR polymer in a degradable living cell fluorescent imaging material of a water-soluble non-conjugated structure synthesized according to the invention, wherein 3300-3700cm -1 The absorption peak of hydroxyl (-OH) is 2800-3000cm -1 The part is a sub-partMethyl (-CH) 2 (-) absorption peak, 1747cm -1 At-c=o absorption peak and 1620cm -1 the-C (=O) NH-absorption peak at this point illustrates the success of the synthesis of the PCG prepolymer and the PCGR polymer.
FIG. 4 is an optical property of a synthetic water-soluble non-conjugated structured degradable living cell fluorescent imaging material of the present invention. It can be seen from fig. 4 a and fig. 4B that PCGA exhibits the strongest fluorescence intensity at 395nm excitation wavelength, and its emission wavelength is 490nm, and fig. 4C illustrates that the material exhibits a red shift phenomenon at an increasing excitation wavelength, and fig. 4D shows that the prepolymer PCG does not have fluorescence properties under 365nm ultraviolet lamp excitation, but exhibits certain fluorescence properties under ultraviolet excitation after grafting L-arginine.
FIG. 5 is a cytotoxicity and blood compatibility assay of myoblasts of the synthetic water-soluble non-conjugated structured degradable living cell fluorescent imaging material of the present invention. In FIGS. 5A and 5B, the cytotoxicity of the material against myoblasts was measured, from which it can be seen that the cytotoxicity of the PCG prepolymer and PCGA polymer was particularly low, and that the material had a certain promoting effect on myoblast proliferation after grafting L-arginine; FIG. 5C shows the hemolysis ability of the material, and from the results, it can be seen that the material does not have hemolysis phenomenon under the condition that the solubility reaches 1.25mg/ml, thus indicating that the material has good blood compatibility.
FIG. 6 shows the measurement of living cell fluorescence imaging of the degradable living cell fluorescence imaging material with the water-soluble unconjugated structure synthesized by the invention, wherein the blue light represents the PCGA polymer, the red light is the PI-marked cell nucleus, the green light is the FITC-marked cell skeleton, and the result shows that the PCGA is expected to become a living cell fluorescence imaging material, and has a certain development prospect in the biomedical neighborhood.
The degradable living cell fluorescence imaging material (PCGR, PCGK, PCGM or PCGE) with the water-soluble unconjugated structure, which is synthesized by the invention, has simple preparation process, and the polymer synthesized by grafting certain amino acid or silane derivatives onto the PCG prepolymer has strong and stable photoluminescence capability, has good blood compatibility and cell compatibility, can have a certain promotion effect on proliferation of cells, and can effectively image the whole cells. Therefore, they have a certain development prospect in the biomedical field as a material for living cell fluorescence imaging.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (6)
1. The preparation method of the degradable living cell fluorescence imaging material with the water-soluble unconjugated structure is characterized by comprising the following steps of:
1) Preparation of PCG prepolymer: carrying out thermal polymerization reaction on citric acid and glycol in a molar ratio of 1:1 at 140-160 ℃ under the conditions of nitrogen, inert gas or vacuum to obtain PCG prepolymer;
2) PCGS polymer preparation: the PCG prepolymer and silane derivatives with the molar ratio of 1 (3-10) react in dimethyl imine by taking DCC and DMAP as catalysts to obtain a PCGS polymer;
3) Preparation of fluorescent imaging material: the synthesized PCGS polymer is dissolved in deionized water to obtain a solution of the degradable living cell fluorescent imaging material with a water-soluble non-conjugated structure; wherein the silane derivative is epoxy silane or amino siloxane.
2. The method of claim 1, wherein the glycol in step 1) is a mixture of 1, 8-octanediol and polyethylene glycol, and the molar ratio of 1, 8-octanediol to polyethylene glycol is 7:3.
3. The method of claim 1, wherein the specific steps of step 1) are as follows:
stirring and melting citric acid and glycol in 140-160deg.C oil bath; after the reaction monomers are completely melted, the temperature is immediately reduced to 140 ℃, the thermal polymerization reaction is carried out for 5 hours under the conditions of nitrogen, inert gas or vacuum, the reaction product is dialyzed and purified in deionized water for 2 days, and the PCG prepolymer is obtained after freeze drying.
4. The method of claim 1, wherein the reaction of step 2) is performed at room temperature, and the final product is purified by dialysis in deionized water for 2 days to remove unreacted monomers and catalyst, and then lyophilized to obtain the polymer.
5. A degradable living cell fluorescent imaging material of water-soluble non-conjugated structure produced by the method of any one of claims 1-4.
6. Use of the degradable living cell fluorescent imaging material of water-soluble non-conjugated structure as claimed in claim 5 in living cell imaging.
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