CN113788954A - Chondroitin sulfate polysaccharide probe based on up-conversion nano material and preparation method and application thereof - Google Patents

Chondroitin sulfate polysaccharide probe based on up-conversion nano material and preparation method and application thereof Download PDF

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CN113788954A
CN113788954A CN202110975925.8A CN202110975925A CN113788954A CN 113788954 A CN113788954 A CN 113788954A CN 202110975925 A CN202110975925 A CN 202110975925A CN 113788954 A CN113788954 A CN 113788954A
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刘秀美
巩洁
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Abstract

The invention provides a chondroitin sulfate polysaccharide probe based on an up-conversion nano material, and a preparation method and application thereof. According to the invention, Adipic Dihydrazide (ADH) is used as a bridge, an up-conversion nano material is grafted on a molecular chain of chondroitin sulfate through coupling of carboxyl and amino, and a chondroitin sulfate probe is constructed and obtained through the tumor targeting effect of the chondroitin sulfate and the luminescence property based on a rare earth up-conversion material. Compared with the existing tumor imaging probe, due to the anti-Stokes luminescence characteristic of the up-conversion nano material, the in-vivo tumor imaging of the immune normal mouse under the excitation of 980nm can be realized, the biocompatibility is better, the background fluorescence interference is smaller, and the in-vivo and in-vitro imaging of the tumor can be realized.

Description

Chondroitin sulfate polysaccharide probe based on up-conversion nano material and preparation method and application thereof
Technical Field
The invention relates to a chondroitin sulfate polysaccharide probe based on an up-conversion nano material, and a preparation method and application thereof, and belongs to the technical field of probe synthesis.
Background
Liver cancer is a common cancer death cause in the world, and hepatocellular carcinoma is the most common liver cancer type, has high mortality rate and higher occurrence rate of remote metastasis. The occurrence and development of hepatocellular carcinoma (HCC) is a multi-step and multi-stage pathological process, the molecular mechanism of the onset of HCC is very complex, and early symptoms are not obvious, so that the development of the method for early diagnosis of hepatocellular carcinoma has important significance in improving the early diagnosis rate and the clinical curative effect of hepatocellular carcinoma and preventing and treating hepatocellular carcinoma. The current clinical cancer diagnosis methods include X-ray diagnosis, positron emission tomography (PET-CT), magnetic resonance, pathological tissue section, and the like. X-ray and magnetic resonance techniques are harmful to human body by radiation, and the accuracy is not high. The pathological tissue section has high accuracy, but the operation is complex and takes long time. Although the PET-CT technology has high image definition and accurate detection result, the detection cost is high, and the PET-CT technology also has certain radiation to human bodies. Recently, researchers separate and count tumor cells from normal cells in order to early screen and judge the development of cancer, but the operation method is complex and the error is large.
Tumor visualization analysis belongs to the international frontier research field. The biological imaging technology is used for qualitatively and quantitatively researching the cell and molecule level of the in-vivo biological process in the living body state, the specific molecular probe is used for tracking the target object and imaging, the cell activity and the gene behavior in the living body organism can be directly monitored, the sensitivity is high, the biological processes such as the growth and the metastasis of the tumor in the body, the distribution condition, the expression of specific genes and the like can be intuitively observed, the operation is simple, and the biological imaging method has a great prospect in the tumor diagnosis, the tumor development process tracking and the tumor treatment. The molecular ultrasonic imaging technology which is developed at present and depends on the targeted contrast agent is successfully applied to the early diagnosis and disease course monitoring research of diseases such as malignant tumors and the like, the diagnosis of the diseases and the early diagnosis and disease course monitoring research of the diseases such as the malignant tumors and the like are greatly improved, and the diagnosis rate, the diagnosis rate and the diagnosis accuracy of the diseases are greatly improved. This technique is often limited by the length of time the contrast agent is relied upon to operate. Therefore, the development of a tumor visualization method based on tumor markers is helpful for forming new understanding of malignant tumor essence, and provides scientific basis for explaining the molecular basis of tumorigenesis and determining the biological characterization of each evolution stage of tumor.
At present, organic fluorescent dye and inorganic nano material are commonly used as molecular probes for biological living body imaging. The photobleaching rate of the fluorescent dye is high, quenching is easy to occur, and further development and application of the fluorescent dye are limited. The quantum dot is used as one of inorganic nano materials, has high photochemical stability and good biocompatibility, but can interfere the luminous intensity of the quantum dot due to strong self-luminescence of the epidermis and blood of an organism, so that the application of the quantum dot in vivo imaging is limited. The upconversion nanomaterials (UCNPs) are a nanocomposite material with excellent optical properties, and due to the small size and good biocompatibility, the UCNPs can be easily combined with proteins or other biomolecules, and have the advantages of small damage to biological tissues, strong tissue penetration capacity and no background fluorescence noise interference, so that the UCNPs are greatly developed and applied in the aspects of fluorescent probe targeted labeling, biological detection, biological living body imaging, tumor photothermal therapy and the like. Because UCNPs have no targeting property, how to connect the UCNPs with tumor targeting substances or carry out active group modification on the surface of the UCNPs is realized, thereby realizing the targeting design of a probe and being used for living body imaging and the like, and being worthy of further research.
Chondroitin Sulfate (CS) is a glycosaminoglycan, widely found in all organisms except plants, and is composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine; research shows that CS has various biological activities and pharmacological effects. The presence of tumor stem cells in tumor tissue is a significant cause of affecting tumor formation and invasive metastasis. Liver cancer stem cells are associated with the formation, progression and recurrence of hepatocellular carcinoma. The CD44 molecule is used as a biomarker of liver cancer stem cells, the expression of the CD44 molecule in liver cancer cells is higher than that of paracancerous tissues and normal liver tissues, and the up-regulation of the CD44 molecule is closely related to the differentiation degree, the cancer progression and the prognosis of liver cancer of liver cells. CS is a specific ligand of CD44, can directionally target CD44 by interaction between chondroitin chains and CD44, has tumor targeting property, and is often used as an anti-tumor drug carrier to realize tumor targeted drug delivery to improve drug effect and reduce side effects.
How to combine the tumor targeting effect of CS and the advantages of UCNPs in the aspect of biological imaging to realize in-vivo and in-vitro imaging of HCC provides a new idea for clinical early diagnosis of HCC. Chinese patent document CN103112882A discloses a targeting near infrared NaYF4The upconversion nanocrystalline is prepared by a one-pot method, firstly a stearate precursor is prepared, and then the NaYF is prepared by treating for 18 hours at 160 ℃ through a hydrothermal method4And (4) up-conversion of the nanocrystals. The invention introduces biological targeting molecules into the synthesis process of the up-conversion nanocrystalline, and provides a new method for synthesizing hydrophilic targeting up-conversion nanocrystalline; the synthesized NaYF4The up-conversion nanocrystalline has the excellent properties of regular appearance, uniform size, good biocompatibility, high targeting property and the like, and a fluorescence spectrum is in a near-infrared region window. However, the preparation of the invention needs high pressure and high temperature in a reaction kettle, the reaction time is long, and the up-conversion luminescence is weak.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a chondroitin sulfate polysaccharide probe based on an up-conversion nano material, and a preparation method and application thereof. The HCC tumor imaging method is simulated to be developed through the visualization of the CD44 molecular function based on the key regulation and control effect of CD44 molecules on HCC evolution and diagnosis and the expression up-regulation in HCC cells; according to the invention, Adipic Dihydrazide (ADH) is used as a bridge, an up-conversion nano material is grafted on a molecular chain of chondroitin sulfate through coupling of carboxyl and amino, and a chondroitin sulfate probe is constructed and obtained through the tumor targeting effect of the chondroitin sulfate and the luminescence property based on a rare earth up-conversion material and is used for in-vivo and in-vitro imaging of tumors.
The technical scheme of the invention is as follows:
a chondroitin sulfate polysaccharide probe based on up-conversion nano materials is characterized in that polypropylene is grafted on a chondroitin sulfate molecular chain through a covalent bondAn acid-modified upconverting nanomaterial; the up-conversion nano material is NaYF4Yb, Tm is nuclear, NaYF4Is spherical core-shell structure nano-particle with the shell and the particle diameter of 38-40 nm.
According to the preferable method, the Chondroitin Sulfate (CS) molecular chain is introduced into an adipic dihydrazide bridge structure through the reaction of carboxyl and amino on hydrazide groups in Adipic Dihydrazide (ADH), and the hydrazide groups in the bridge structure are reacted with carboxyl on the surface of the polyacrylic acid modified up-conversion nano material, so that the polyacrylic acid modified up-conversion nano material is grafted on the chondroitin sulfate molecular chain through covalent bonds.
The preparation method of the chondroitin sulfate polysaccharide probe based on the up-conversion nano material comprises the following steps:
(1) preparation of chondroitin sulfate-grafted adipic acid dihydrazide compound (CS-ADH): dissolving Chondroitin Sulfate (CS) in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts, adjusting the pH of a system to 6-7 after dissolving, and activating carboxyl for 10-20min at 30-45 ℃; then adding adipic Acid Dihydrazide (ADH), adjusting the pH of the system to 6-7, reacting for 20-30h at 30-45 ℃, dialyzing, and freeze-drying to obtain CS-ADH;
(2) preparing an up-conversion nano material:
i. mixing yttrium acetate, ytterbium acetate, thulium acetate, oleic acid and 1-octadecene, stirring for 20-40min at 170 ℃ under the protection of inert gas until the solid is completely dissolved and uniformly mixed, and cooling to room temperature; adding NaOH and NH4Heating the methanol solution of F to 45-55 ℃ under the protection of inert gas for reaction for 30-50min, continuously heating to 100-120 ℃ for reaction for 60-90min to remove the methanol in the system, then continuously heating to 295-300 ℃ for reaction for 80-100min, cooling, washing and volatilizing the solvent to obtain the nano material (NaYF)4:Yb,Tm)；
ii. Mixing yttrium acetate, oleic acid and 1-octadecene, heating to 150 ℃ and 170 ℃ under the protection of inert gas, stirring for 20-40min to completely dissolve and uniformly mix solids, and naturally cooling to 70-90 ℃; adding nano material (NaYF)4Yb, Tm) in a dispersion of cyclohexane at 70-90 deg.CRemoving cyclohexane in the system for 20-40min, and naturally cooling to 45-55 deg.C; adding NaOH and NH4Heating the methanol solution of F to 45-55 ℃ under the protection of inert gas for reaction for 30-50min, continuously heating to 100-120 ℃ for reaction for 60-90min to remove the methanol in the system, then continuously heating to 295-300 ℃ for reaction for 80-100min, cooling, washing and volatilizing the solvent to obtain the up-conversion nano material (NaYF)4:Yb,Tm@NaYF4UCNPs)；
(3) Preparation of polyacrylic acid modified up-conversion nano material: dispersing the up-conversion nano material in cyclohexane, adding an aqueous solution of cetyl trimethyl ammonium bromide, and violently stirring at room temperature until the cyclohexane is completely volatilized to obtain the up-conversion nano material with the end capped by the cetyl trimethyl ammonium bromide; addition of NH3·H2O, polyacrylic acid aqueous solution, dropwise adding isopropanol under stirring after ultrasonic dispersion, stirring at room temperature for 10-20min after dropwise adding to uniformly mix the solution, washing, and freeze-drying to obtain polyacrylic acid modified up-conversion nano material (NaYF)4:Yb,Tm@NaYF4@PAA)；
(4) Preparing a probe: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into a polyacrylic acid modified up-conversion nano-material water dispersion, fully mixing and uniformly dispersing, adding a chondroitin sulfate grafted adipic acid dihydrazide compound (CS-ADH), and stirring at 35-40 ℃ for reacting for 20-30 h; centrifuging to remove unreacted polyacrylic acid modified upconversion nanometer material, taking supernate, dialyzing, and lyophilizing to obtain chondroitin sulfate polysaccharide probe (CS-UCNPs-Tm) based on upconversion nanometer material.
Preferably, in step (1), the ratio of the mass of Chondroitin Sulfate (CS) to the volume of water is 0.002-0.3 g/mL; the mass ratio of the Chondroitin Sulfate (CS), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 1:3-5: 0.2-1.
Preferably, in step (1), the mass ratio of adipic Acid Dihydrazide (ADH) to Chondroitin Sulfate (CS) is 7-10: 1.
preferably, in the step (1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added, and after dissolution, hydrochloric acid with the concentration of 0.05-0.2mol/L is used for adjusting the pH of the system; after adding Adipic Dihydrazide (ADH), the pH of the system is adjusted by using 0.05-0.2mol/L NaOH aqueous solution.
According to the invention, in step (1), the cut-off molecular weight of dialysis is 4000Da, and the small-molecule compounds which are not reacted completely in the system are removed.
Preferably, in step (2) i, the molar ratio of yttrium acetate, ytterbium acetate and thulium acetate is 3.95:1: 0.05.
Preferably, in step (2) i, the volume ratio of oleic acid to 1-octadecene is 1: 1.2-1.8; the volume ratio of the mass of the yttrium acetate to the oleic acid is 0.05-0.1 mol/L.
Preferably, according to the invention, step (2) i contains NaOH and NH4The concentration of NaOH in the methanol solution of F is 0.1-0.4mol/L, NH4The concentration of F is 0.2-0.6 mol/L.
Preferably, according to the invention, in step (2) i, NaOH, NH4The molar ratio of F to yttrium acetate is 1:1.6: 0.316.
Preferably according to the invention, in step (2) ii, the volume ratio of oleic acid to 1-octadecene is 0.2-0.6: 1; the volume ratio of the mass of the yttrium acetate to the oleic acid is 0.1-0.2 mol/L.
Preferably, according to the invention, in step (2) ii, the nanomaterial (NaYF)4Yb, Tm) in cyclohexane dispersion, nano material (NaYF)4Yb, Tm) is 0.2-0.3 mol/L; nanomaterial (NaYF)4Yb, Tm) and yttrium acetate at a molar ratio of 1.5: 1.
Preferably, according to the invention, in step (2) ii, NaOH and NH are contained4The concentration of NaOH in the methanol solution of F is 0.4-0.6mol/L, NH4The concentration of F is 0.6-1 mol/L.
According to a preferred embodiment of the invention, in step (2) ii, NaOH, NH4The molar ratio of F to yttrium acetate was 2.5:4: 1.
Preferably, in step (3), the ratio of the mass of the upconversion nanomaterial to the volume of cyclohexane is 0.02 to 0.06 g/mL.
Preferably, in step (3), the concentration of the aqueous solution of cetyltrimethylammonium bromide (CTAB) is 4-6 mg/mL; the mass ratio of Cetyl Trimethyl Ammonium Bromide (CTAB) to the up-conversion nano material is 1-1.5: 1.
According to a preferred embodiment of the invention, in step (3), NH3·H2The concentration of O is 1-3mol/L, and the concentration of polyacrylic acid aqueous solution is 0.1-0.3 g/mL; NH (NH)3·H2The volume ratio of the O to the polyacrylic acid aqueous solution is 1-2: 1.
Preferably, in step (3), the mass ratio of the upconversion nanomaterial to the polyacrylic acid is 10-15: 1.
Preferably, according to the invention, in step (3), the volume ratio of isopropanol to cyclohexane is between 15 and 25: 1.
According to a preferred embodiment of the invention, in step (4), the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 2-5: 1.
Preferably, in the step (4), the mass concentration of the polyacrylic acid modified upconversion nanomaterial in the aqueous dispersion of the polyacrylic acid modified upconversion nanomaterial is 8-10 mg/mL; the mass ratio of the polyacrylic acid modified up-conversion nano material to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is 1-1.5: 1.
Preferably, in the step (4), the mass ratio of the chondroitin sulfate grafted adipic acid dihydrazide compound (CS-ADH) to the polyacrylic acid modified upconversion nanomaterial is 1: 0.5-1.5.
Preferably, in step (4), the cut-off of the dialysis is 4000Da, and unreacted small-molecule compounds in the product are removed.
The application of the chondroitin sulfate polysaccharide probe based on the up-conversion nano material is applied to in-vitro and in-vivo imaging of tumor cells.
The reaction principle of the invention is as follows:
Figure BDA0003227264120000051
Figure BDA0003227264120000061
the invention has the following technical characteristics and beneficial effects:
(1) the construction method is based on NaYF by taking Adipic Dihydrazide (ADH) as a bridge4Namely, a Chondroitin Sulfate (CS) probe which converts nano materials on Yb and Tm. Synthesizing NaYF with core-shell structure4:Yb,Tm@NaYF4Up-conversion of nano material, PAA derivation to obtain NaYF4:Yb,Tm@NaYF4@ PAA Material CS-ADH with NaYF by carboxy-amino covalent coupling4:Yb,Tm@NaYF4@ PAA.
(2) Compared with the prior up-conversion synthetic material, the method for preparing NaYF4The reaction time of Yb and Tm is shorter, the reaction can be realized by a common device without being carried out in a reaction kettle. Compared with NaYF4Yb, Tm, NaYF with unexpectedly found core-shell structure4:Yb,Tm@NaYF4The up-conversion luminescence intensity of the material is significantly enhanced, about 17 times that of the nuclear material alone, and the emission band is red-shifted to a wavelength suitable for small animal in vivo imaging. After the derivation by PAA, the position of an emission band is not obviously changed, and although the conversion strength on the material is weakened compared with that of a core-shell structure, the emission band is still far stronger than NaYF4Up-conversion luminescence intensity of Yb, Tm materials. The upconversion nanometer material (NaYF) prepared by the invention4:Yb,Tm@NaYF4UCNPs) coupled with CS-ADH, although the luminous intensity is reduced, compared with NaYF4:Yb,Er@NaYF4The material obtained by coupling UCNPs and CS-ADH has obviously enhanced luminous intensity, the peak value ratio is about 35:1, and the most intense peak wavelength is red shifted from about 545nm to about 800nm, so that the material is more suitable for small animal living body imaging.
(3) CS with a tumor targeting effect is connected with the up-conversion luminescent nano material through ADH, and an up-conversion polysaccharide probe CS-UCNPs-Tm with the tumor targeting effect is designed and synthesized; compared with the existing tumor imaging probe, due to the anti-Stokes luminescence characteristic of the up-conversion nano material, the in-vivo tumor imaging of the immune normal mouse under the excitation of 980nm can be realized, the biocompatibility is better, the background fluorescence interference is smaller, and the in-vitro in-vivo imaging of the tumor can be realized.
Drawings
FIG. 1 is a chart of the infrared spectra of (a) CS-ADH and (b) CS prepared in example 1.
FIG. 2 shows the reaction of (a) CS with (b) CS-ADH prepared in example 11H NMR spectrum.
FIG. 3 is (a) NaYF prepared in example 14:Yb,Tm、(b)NaYF4:Yb,Tm@NaYF4、(c)NaYF4:Yb,Tm@NaYF4TEM image of @ PAA material.
FIG. 4 is (a) NaYF prepared in example 14:Yb,Tm@NaYF4@PAA、(b)NaYF4:Yb,Tm@NaYF4、(c)NaYF4XRD patterns of Yb, Tm, and (d) standard card JCPDS 16-0334.
FIG. 5 is (a) NaYF prepared in example 14:Yb,Tm@NaYF4、(b)NaYF4:Yb,Tm@NaYF4Infrared spectrum of @ PAA.
FIG. 6 is (a) NaYF prepared in example 14:Yb,Tm@NaYF4、(b)NaYF4:Yb,Tm@NaYF4@PAA、(c)NaYF4Up-conversion luminescence patterns of Yb, Tm and (d) CS-UCNPs-Tm (excitation: 980 nm).
FIG. 7 is a graph comparing the upconversion luminescence intensity of (a) CS-UCNPs-Tm prepared in example 1 with (b) CS-UCNPs prepared in comparative example (excitation: 980 nm).
FIG. 8 is a graph showing cytotoxicity experiments of CS-UCNPs-Tm prepared in example 1.
FIG. 9 shows NaYF as a nanomaterial prepared in comparative example 14TEM image of Yb, Tm.
FIG. 10 is a solid NaYF core-shell structure prepared in comparative example 24:Yb,Tm@NaYF4TEM images of UCNPs.
Detailed Description
The present invention is further illustrated by, but not limited to, the following examples.
Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials and equipment are commercially available, unless otherwise specified.
Example 1
A preparation method of chondroitin sulfate polysaccharide probe based on up-conversion nano material comprises the following steps:
(1) synthesis of CS-ADH
Precisely weighing 1g of CS, dissolving in 50mL of water, adding 4g of EDC and 0.585g of NHS, adjusting the pH to about 6-7 by using 0.1mol/L HCl, and activating carboxyl in water bath at 37 ℃ for 15 min; adding 8.8g of ADH, adjusting the pH value to 6-7 by using 0.1mol/L NaOH aqueous solution, reacting in water bath at 37 ℃ for 24 hours, dialyzing water for three days (the molecular weight cutoff is 4000Da), freeze-drying to obtain CS-ADH solid, and performing FTIR and1h NMR confirmed the coupling of CS to ADH.
In FIG. 1, a and b are the infrared spectra of CS-ADH and CS, respectively. In the IR spectrum of CS, 1603.19cm was mainly observed-1C ═ O characteristic stretching vibration peak; the C ═ O stretching vibration peak in CS-ADH appears at 1649.71cm-1And at 1551.85cm-1A new absorption peak, which is an N-H bending vibration peak, appears, and ADH is coupled to CS through an amide reaction to form amide, indicating that the reaction of CS and ADH is successful.
Of CS and CS-ADH1The H NMR spectrum is shown in FIG. 2. In CS1In H NMR, the a peak has a chemical shift of 1.94ppm, is H in the acetylmethyl group in CS, and the peak having a chemical shift in the range of 3.50-4.67ppm is H in the sugar ring skeleton of CS; in CS-ADH1In H NMR, the b peak and the c peak are H on two methylene groups in ADH, and chemical shift is changed due to the change of the chemical environment of H on CS, so that the CS and ADH can be judged to successfully react.
Determination of free amino groups in CS-ADH
Preparation of OPA reagent: 40mg of o-phthalaldehyde is precisely weighed and dissolved in 1mL of methanol, 2.5mL of freshly prepared 20% SDS solution, 25mL of 0.1M borax buffer solution and 100 mu L of beta-mercaptoethanol are added, and distilled water is added to fix the volume to 50mL to obtain the OPA reagent.
Weighing 10mg of CS, dissolving the CS in 5mL of distilled water, stirring to fully dissolve the CS to obtain a 2mg/mL CS solution, sequentially adding 40mg of EDC and 5.85mg of NHS, adjusting the pH to 6-7 by using 0.1mol/L hydrochloric acid, stirring for 15min at 37 ℃ to activate carboxyl, adding 88mg of ADH, adjusting the pH of the solution to 6-7 by using 0.1mol/L NaOH aqueous solution, and stirring for 24h at 37 ℃ to obtain a solution A to be detected; weighing 40mg of EDC and 5.85mg of NHS, dissolving in 5mL of distilled water, adjusting the pH to 6-7 with 0.1mol/L hydrochloric acid, stirring for 15min at 37 ℃ to activate carboxyl, adding 88mg of ADH, adjusting the pH of the solution to 6-7 with 0.1mol/L NaOH aqueous solution, and stirring for 24h at 37 ℃ to serve as a control solution B; weighing 10mg of CS, dissolving in 5mL of distilled water, stirring to fully dissolve the CS to obtain a 2mg/mL CS solution, sequentially adding 40mg of EDC and 5.85mg of NHS, adjusting the pH to 6-7 by using 0.1mol/L hydrochloric acid, and stirring in a water bath at 37 ℃ for 24 hours to obtain a control solution C.
Adding 200 μ L of the solution A to be detected or 200 μ L of the control solution B or 200 μ L of the control solution C into 4mL of OPA reagent, carrying out water bath at 37 ℃ for 2min, and measuring the absorbance value at 340 nm. The absorbance values are shown in table 1 below.
TABLE 1 Absorbance value at 340nm of solution in free amino assay
Figure BDA0003227264120000081
Figure BDA0003227264120000082
As shown in Table 1, free amino groups exist in CS-ADH, and can be coupled with the prepared polyacrylic acid modified upconversion nanometer material theoretically through carboxyl amino groups.
(2) Up-conversion of nanomaterials: NaYF4:20％Yb,1％Tm@NaYF4Synthesis of UCNPs
Synthesis of NaYF 420% Yb, 1% Tm (hereinafter abbreviated as NaYF)4Yb, Tm): respectively precisely weighing yttrium acetate (3.16mmol), ytterbium acetate (0.8mmol) and thulium acetate (0.04mmol), placing in a 500mL three-neck flask, adding 40mL oleic acid and 60mL 1-octadecene, introducing N2After 20min, heating to 160 deg.C and stirring, maintaining for 30min to completely dissolve the solidMixing, and naturally cooling to room temperature; adding a solution containing 10mmol of NaOH and 16mmol of NH440mL of F in methanol under N2Under protection (continuous introduction of N)2) Heating to 50 ℃ for reaction for 40min, then continuously heating to 110 ℃ for reaction for 80min to remove all methanol in the system, heating the solution to 295 ℃ for reaction for 90min, cooling to room temperature after the reaction is finished, adding absolute ethyl alcohol for centrifugal precipitation for a plurality of times, and volatilizing the solvent to obtain solid NaYF4:Yb,Tm。
Synthesis of upconversion nanomaterial NaYF4:Yb,Tm@NaYF4UCNPs: precisely weighing 2mmol of yttrium acetate, placing the yttrium acetate in a 250mL three-neck flask, adding 12mL of oleic acid and 30mL of 1-octadecene, and introducing N2Heating to 160 deg.C after 20min, stirring, maintaining for 30min to completely dissolve and mix the solid, and naturally cooling to 80 deg.C; to the solution was added 3mmol of prepared NaYF dispersed in 12mL cyclohexane4Yb and Tm react at 80 ℃ for 30min to remove cyclohexane in the system, and the temperature is naturally reduced to 50 ℃; adding a solution containing 5mmol of NaOH and 8mmol of NH410mL of methanol solution of F in N2Under protection (continuous introduction of N)2) Heating to 50 ℃ for reaction for 40min, then continuously heating to 110 ℃ for reaction for 80min to remove all methanol in the system, heating the solution to 300 ℃ for reaction for 90min, cooling to room temperature after the reaction is finished, adding absolute ethyl alcohol for centrifugal precipitation for a plurality of times, and volatilizing the solvent to obtain the solid NaYF with the core-shell structure4:Yb,Tm@NaYF4 UCNPs。
(3)NaYF4:Yb,Tm@NaYF4Synthesis of @ PAA
PAA derivatization preparation UCNPs @ PAA: precisely weighing 80mg of UCNPs, dispersing in 2mL of cyclohexane, adding 20mL of distilled water containing 0.1g of CTAB, and violently stirring at room temperature to volatilize the cyclohexane to form CTAB-terminated UCNPs; adding 45 mu.L of 2mol/L NH3·H2O, 30 mu L of 0.2g/mL PAA aqueous solution, ultrasonically dispersing for 30min, dropwise adding 40mL of isopropanol under the condition of continuous stirring, stirring for 10min at room temperature to uniformly mix, centrifugally washing for a plurality of times by using distilled water, and freeze-drying to obtain NaYF4:Yb,Tm@NaYF4@ PAA solid.
NaYF4:Yb,Tm@NaYF4 UCNDrying the Ps, dispersing the Ps in cyclohexane, performing ultrasonic treatment for 30min to uniformly disperse the Ps, dripping two drops of the Ps on a 200-mesh copper net, and observing the morphology and the size of the nanoparticles under a TEM (transmission electron microscope) after the cyclohexane on the copper net is volatilized; NaYF4:Yb,Tm@NaYF4Freeze-drying and dispersing the @ PAA in distilled water, performing ultrasonic treatment for 45min to uniformly disperse the PAA, dripping two drops of PAA on a 200-mesh copper net, and placing the copper net under a TEM (transmission electron microscope) to observe the morphology and the size of the nanoparticles after water is volatilized.
FIG. 3a is a synthetic NaYF4Yb, Tm, it can be seen that the synthesized NaYF4The Yb and Tm up-conversion material has uniform appearance, uniform size, about 35nm size and good dispersion; in the figure, b is prepared NaYF4:Yb,Tm@NaYF4The shell layer thickness of the core-shell structure material is about 3-4nm, the core-shell structure material is uniform in overall appearance and size and well dispersed in cyclohexane; in the figure c is NaYF after PAA derivatization4:Yb,Tm@NaYF4The @ PAA material can be seen from the figure, the surface of the material is uniformly coated with a layer of PAA, the dispersibility in water is good, and the derivatization process basically has no influence on the shape and the size.
FIG. 4 is a NaYF prepared4:Yb,Tm(c)、NaYF4:Yb,Tm@NaYF4Core-shell material (b) and NaYF4:Yb,Tm@NaYF4The XRD pattern of @ PAA (a) is compared with that of standard card JCPDS16-0334(d), which shows that the prepared material is in a hexagonal phase with good crystallinity, and the material is uniform in appearance, uniform in size and good in crystallinity by combining with a TEM (transmission electron microscope) pattern and can be used for further synthesis.
Before PAA derivation (NaYF)4:Yb,Tm@NaYF4Core-shell material, shown in fig. a) after (NaYF)4:Yb,Tm@NaYF4The infrared spectrum of @ PAA, b) is shown in FIG. 5, and is related to NaYF coated with oleic acid on the surface4:Yb,Tm@NaYF4Core-shell Structure comparison, PAA-derivatized NaYF4:Yb,Tm@NaYF4@ PAA material at 2925cm-1And 2854cm-1The absorption peak of C-H bond stretching vibration is reduced, and the C ═ O bond stretching vibration is reduced at 1720cm-1A new absorption peak is generated nearby, which indicates that PAA is successfully coated on NaYF4:Yb,Tm@NaYF4The above.
(4) Synthesis of chondroitin sulfate polysaccharide probe, namely CS-UCNPs-Tm, of up-conversion nano material
Weigh 100mg NaYF4:Yb,Tm@NaYF4@ PAA, adding 10mL of water, and ultrasonically dispersing for 45 min; precisely weighing 80mg EDC and 11.7mg NHS, and adding into the dispersed NaYF under stirring at 37 deg.C4:Yb,Tm@NaYF4@ PAA water solution is stirred for 15min, then 100.0mg of precisely-weighed CS-ADH is added, stirring is carried out for 24h at 37 ℃, after the reaction is finished, centrifugation is carried out at 5000rpm for 10min to remove the polyacrylic acid derived up-conversion nano material which is not completely reacted, supernatant is taken to dialyze against water for three days (the molecular weight cutoff is 4000Da) to remove small molecular compounds which are not completely reacted, and freeze-drying is carried out to obtain CS-UCNPs-Tm solid.
FIG. 6 is a NaYF4Yb, Tm (curve c in the figure), NaYF4:Yb,Tm@NaYF4(curve a in the figure), NaYF4:Yb,Tm@NaYF4The upconversion luminescence plots (excitation: 980nm) for @ PAA (curve b in the figure) and CS-UCNPs-Tm (curve d in the figure). Under 980nm excitation, the emission band is mainly concentrated at 800nm and is matched with NaYF4Compared with Yb and Tm, the NaYF with a core-shell structure4:Yb,Tm@NaYF4The up-conversion luminous intensity of the material is obviously enhanced and is about 17 times of that of a single core material, the position of an emission band is not obviously changed after PAA (poly (acrylic acid) A) is subjected to derivatization and water transfer, and although the up-conversion intensity of the material is weakened compared with that of a core-shell structure, the up-conversion luminous intensity of the material is still far stronger than that of NaYF4Up-conversion luminescence intensity of Yb, Tm materials.
FIG. 7 is a comparison of (a) the intensity of converted luminescence on CS-UCNPs-Tm prepared in example 1 and (b) CS-UCNPs prepared in comparative example 3 (excitation: 980 nm). After the material is coupled with CS-ADH, the up-conversion luminous intensity of the material is obviously weakened, probably because the CS has large molecular weight and large steric hindrance, and-COOH in a disaccharide structure can not completely participate in the reaction, so that the luminous intensity is greatly weakened. But compared with the CS-UCNPs material synthesized in the comparative example 3, the luminous intensity is obviously enhanced, the peak ratio is about 35:1, and the strongest peak wavelength is red-shifted from about 545nm to about 800nm, as shown in figure 7, the material is more suitable for small animal living body imaging.
Comparative example 1
Nano material NaYF4The synthesis of Yb, Tm comprises the following steps:
synthesis of NaYF 420% Yb, 1% Tm (hereinafter abbreviated as NaYF)4Yb, Tm): respectively precisely weighing yttrium acetate (3.16mmol), ytterbium acetate (0.8mmol) and thulium acetate (0.04mmol), placing in a 500mL three-neck flask, adding 40mL oleic acid and 60mL 1-octadecene, introducing N2Heating to 160 deg.C after 20min, stirring, maintaining for 30min to completely dissolve the solid, mixing, and naturally cooling to room temperature; adding a solution containing 10mmol of NaOH and 16mmol of NH440mL of F in methanol under N2Under protection (continuous introduction of N)2) Heating to 50 ℃ for reaction for 40min, then continuously heating to 110 ℃ for reaction for 80min to remove all methanol in the system, heating the solution to 290 ℃ for reaction for 90min, cooling to room temperature after the reaction is finished, adding absolute ethyl alcohol for centrifugal precipitation for a plurality of times, and volatilizing the solvent to obtain solid NaYF4Yb, Tm. The obtained NaYF4The electron microscope image of Yb, Tm is shown in FIG. 9, and it can be seen from the figure that the prepared nano material has non-uniform size and non-uniform morphology, and cannot be used in the next experiment.
Comparative example 2
Up-conversion of nanomaterials: NaYF4:Yb,Tm@NaYF4Synthesis of UCNPs, comprising the steps of:
synthesis of NaYF 420% Yb, 1% Tm (hereinafter abbreviated as NaYF)4Yb, Tm): respectively precisely weighing yttrium acetate (3.16mmol), ytterbium acetate (0.8mmol) and thulium acetate (0.04mmol), placing in a 500mL three-neck flask, adding 40mL oleic acid and 60mL 1-octadecene, introducing N2Heating to 160 deg.C after 20min, stirring, maintaining for 30min to completely dissolve the solid, mixing, and naturally cooling to room temperature; adding a solution containing 10mmol of NaOH and 16mmol of NH440mL of F in methanol under N2Under protection (continuous introduction of N)2) Heating to 50 ℃ for reaction for 40min, then continuously heating to 110 ℃ for reaction for 80min to remove all methanol in the system, heating the solution to 295 ℃ for reaction for 90min, cooling to room temperature after the reaction is finished, adding absolute ethyl alcohol for centrifugal precipitation for a plurality of times, and volatilizing the solvent to obtain solid NaYF4:Yb,Tm。
Synthesis of upconversion nanomaterial NaYF4:Yb,Tm@NaYF4UCNPs: collecting 2mmol of vinegarPrecisely weighing yttrium acid, placing the yttrium acid into a 250mL three-neck flask, adding 12mL oleic acid and 30mL 1-octadecene, and introducing N2Heating to 160 deg.C after 20min, stirring, maintaining for 30min to completely dissolve and mix the solid, and naturally cooling to 80 deg.C; to the solution was added 2mmol of prepared NaYF dispersed in 12mL cyclohexane4Yb and Tm react at 80 ℃ for 30min to remove cyclohexane in the system, and the temperature is naturally reduced to 50 ℃; adding a solution containing 5mmol of NaOH and 8mmol of NH410mL of methanol solution of F in N2Under protection (continuous introduction of N)2) Heating to 50 ℃ for reaction for 40min, then continuously heating to 110 ℃ for reaction for 80min to remove all methanol in the system, heating the solution to 300 ℃ for reaction for 90min, cooling to room temperature after the reaction is finished, adding absolute ethyl alcohol for centrifugal precipitation for a plurality of times, and volatilizing the solvent to obtain the solid NaYF with the core-shell structure4:Yb,Tm@NaYF4UCNPs. The electron microscope image of the obtained material is shown in FIG. 10, and it can be seen from the image that the prepared UNCPs are not uniform in size, the morphology of the nanoparticles is greatly different, and the dispersion is poor.
Comparative example 3
A preparation method of chondroitin sulfate polysaccharide probe based on up-conversion nano material comprises the following steps:
(1) synthesis of CS-ADH, as in example 1;
(2) synthesis of up-converting nanomaterials as described in example 1, except that: the raw materials for preparing the up-conversion nano material are replaced by yttrium acetate (3.16mmol), ytterbium acetate (0.8mmol) and thulium acetate (0.04mmol) to be yttrium acetate (3.12mmol), ytterbium acetate (0.8mmol) and erbium acetate (0.08mmol), and the up-conversion nano material is of a non-core-shell structure; the method comprises the following specific steps:
respectively precisely weighing yttrium acetate (3.12mmol), ytterbium acetate (0.8mmol) and erbium acetate (0.08mmol), placing in a 500mL three-neck flask, adding 40mL oleic acid and 60mL 1-octadecene, introducing N2Heating to 160 deg.C after 20min, stirring, maintaining for 30min to completely dissolve the solid, mixing, and naturally cooling to room temperature; adding a solution containing 10mmol of NaOH and 16mmol of NH440mL of F in methanol under N2Under protection (continuous introduction of N)2) Heating to 50 deg.C for 40min, and heating to 110 deg.C to removeHeating the solution to 295 ℃ for reaction for 90min, cooling to room temperature after the reaction is finished, adding absolute ethyl alcohol for centrifugal precipitation for a plurality of times, and volatilizing the solvent to obtain solid NaYF4:Yb,Er。
(3)NaYF4Synthesis of Yb, Er @ PAA as in example 1;
(4) synthesis of chondroitin sulfate polysaccharide probe of up-conversion nanomaterial, namely CS-UCNPs, was performed as in example 1.
Test example: cell culture and cytotoxicity assays
Preparing a Bel-7402 cell complete culture medium: gibco RPMI 1640 medium containing 10 wt% of ilex purpurea foetus calf serum and 1 wt% of Solarbio streptomycin mixed solution 100X, and storing at 4 ℃ for later use.
MTT test: toxicity of the CS-UCNPs-Tm probe prepared in example 1 on Bel-7402 cells was examined in the concentration range of 5. mu.g/mL-500. mu.g/mL by MTT assay. The 96-well plate was provided with a zero-adjustment well, a control well, and 5. mu.g/mL, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, and 500. mu.g/mL CS-UCNPs-Tm sample wells. Collecting Bel-7402 cells growing in logarithmic phase, digesting with pancreatin, adding twice volume of cell complete culture medium to stop digestion, centrifuging at 800rpm for 5min, suspending and diluting with cell complete culture medium, inoculating into 96-well plate at 8000/well except for zero-setting well, placing the inoculated 96-well plate at 37 deg.C with 5% CO2After the cells are cultured in a constant temperature incubator until the cells adhere to the wall, 100 mu L of complete cell culture medium is added into each hole of a zero adjusting hole and a control hole, 100 mu L of CS-UCNPs-Tm with corresponding concentration prepared by taking the complete cell culture medium as a solvent is added into a sample hole, the sample hole is respectively placed in the constant temperature incubator to be incubated for 12h and 24h, the solution in each hole is taken out and discarded, after the sample hole is washed twice by sterile PBS, 90 mu L of complete cell culture medium and 10 mu L of MTT solution are added into each hole, the constant temperature incubator is continuously placed for culturing for 4h, 110 mu L of Formazan solution is taken out and added into each hole, and after the sample hole is subjected to low-speed shaking on a shaking table for 10min, the absorbance value under 490nm is detected by using an enzyme reader.
The cytotoxicity of the synthesized CS-UCNPs-Tm is examined through an MTT experiment (see figure 8), and the graph shows that the cell survival rate is more than 60% after the CS-UCNPs-Tm and Bel-7402 are incubated for 12h and 24h at different concentrations in the range of 10 mu g/mL-500 mu g/mL, and the cell survival rate of the 12h group is higher than 24h in total. The CS-UCNPs-Tm probe prepared by the method has good cell compatibility and can be used for in vitro and in vivo experiments.

Claims (9)

1. A chondroitin sulfate polysaccharide probe based on an up-conversion nano material is characterized in that the probe is an up-conversion nano material modified by grafting polyacrylic acid on a chondroitin sulfate molecular chain through a covalent bond; the up-conversion nano material is NaYF4Yb, Tm is nuclear, NaYF4Is spherical core-shell structure nano-particle with the shell and the particle diameter of 38-40 nm.
2. The chondroitin sulfate polysaccharide probe based on the up-conversion nanomaterial as claimed in claim 1, wherein a Chondroitin Sulfate (CS) molecular chain is introduced into an adipic acid dihydrazide bridge structure through a reaction between carboxyl and amino on a hydrazide group in adipic Acid Dihydrazide (ADH), and the hydrazide group in the bridge structure is reacted with the carboxyl on the surface of the up-conversion nanomaterial modified by polyacrylic acid, so that the up-conversion nanomaterial modified by polyacrylic acid is grafted on the chondroitin sulfate molecular chain through a covalent bond.
3. The method for preparing chondroitin sulfate polysaccharide probe of up-conversion nanomaterial as claimed in any one of claims 1 or 2, comprising the steps of:
(1) preparation of chondroitin sulfate-grafted adipic acid dihydrazide compound (CS-ADH): dissolving Chondroitin Sulfate (CS) in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts, adjusting the pH of a system to 6-7 after dissolving, and activating carboxyl for 10-20min at 30-45 ℃; then adding adipic Acid Dihydrazide (ADH), adjusting the pH of the system to 6-7, reacting for 20-30h at 30-45 ℃, dialyzing, and freeze-drying to obtain CS-ADH;
(2) preparing an up-conversion nano material:
i. mixing yttrium acetate, ytterbium acetate, thulium acetate, oleic acid and 1-octadecene, stirring for 20-40min at 170 ℃ under the protection of inert gas until the solid is completely dissolved and uniformly mixed, and cooling to the temperature ofRoom temperature; adding NaOH and NH4Heating the methanol solution of F to 45-55 ℃ under the protection of inert gas for reaction for 30-50min, continuously heating to 100-120 ℃ for reaction for 60-90min to remove the methanol in the system, then continuously heating to 295-300 ℃ for reaction for 80-100min, cooling, washing and volatilizing the solvent to obtain the nano material (NaYF)4:Yb,Tm)；
ii. Mixing yttrium acetate, oleic acid and 1-octadecene, heating to 150 ℃ and 170 ℃ under the protection of inert gas, stirring for 20-40min to completely dissolve and uniformly mix solids, and naturally cooling to 70-90 ℃; adding nano material (NaYF)4Reacting cyclohexane dispersion liquid of Yb, Tm) at 70-90 ℃ for 20-40min to remove cyclohexane in the system, and naturally cooling to 45-55 ℃; adding NaOH and NH4Heating the methanol solution of F to 45-55 ℃ under the protection of inert gas for reaction for 30-50min, continuously heating to 100-120 ℃ for reaction for 60-90min to remove the methanol in the system, then continuously heating to 295-300 ℃ for reaction for 80-100min, cooling, washing and volatilizing the solvent to obtain the up-conversion nano material (NaYF)4:Yb,Tm@NaYF4UCNPs)；
(3) Preparation of polyacrylic acid modified up-conversion nano material: dispersing the up-conversion nano material in cyclohexane, adding an aqueous solution of cetyl trimethyl ammonium bromide, and violently stirring at room temperature until the cyclohexane is completely volatilized to obtain the up-conversion nano material with the end capped by the cetyl trimethyl ammonium bromide; addition of NH3·H2O, polyacrylic acid aqueous solution, dropwise adding isopropanol under stirring after ultrasonic dispersion, stirring at room temperature for 10-20min after dropwise adding to uniformly mix the solution, washing, and freeze-drying to obtain polyacrylic acid modified up-conversion nano material (NaYF)4:Yb,Tm@NaYF4@PAA)；
(4) Preparing a probe: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into a polyacrylic acid modified up-conversion nano-material water dispersion, fully mixing and uniformly dispersing, adding a chondroitin sulfate grafted adipic acid dihydrazide compound (CS-ADH), and stirring at 35-40 ℃ for reacting for 20-30 h; centrifuging to remove unreacted polyacrylic acid modified upconversion nanometer material, taking supernate, dialyzing, and lyophilizing to obtain chondroitin sulfate polysaccharide probe (CS-UCNPs-Tm) based on upconversion nanometer material.
4. The method for preparing chondroitin sulfate polysaccharide probe based on up-conversion nanomaterial as claimed in claim 3, wherein the step (1) comprises one or more of the following conditions:
i. the mass ratio of the Chondroitin Sulfate (CS) to the water is 0.002-0.3 g/mL; the mass ratio of the Chondroitin Sulfate (CS), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 1:3-5: 0.2-1;
ii. The mass ratio of Adipic Dihydrazide (ADH) to Chondroitin Sulfate (CS) is 7-10: 1;
iii, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), dissolving, and adjusting the pH of the system by using hydrochloric acid with the concentration of 0.05-0.2 mol/L; after adding Adipic Dihydrazide (ADH), using 0.05-0.2mol/L NaOH aqueous solution to adjust the pH of the system;
iv, removing the small molecular compound which is not completely reacted in the system when the cut-off molecular weight of dialysis is 4000 Da.
5. The method for preparing chondroitin sulfate polysaccharide probe based on up-conversion nanomaterial as claimed in claim 3, wherein the step (2) i comprises one or more of the following conditions:
a. the molar ratio of yttrium acetate, ytterbium acetate and thulium acetate is 3.95:1: 0.05;
b. the volume ratio of the oleic acid to the 1-octadecene is 1: 1.2-1.8; the volume ratio of the mass of the yttrium acetate to the oleic acid is 0.05-0.1 mol/L;
c. containing NaOH and NH4The concentration of NaOH in the methanol solution of F is 0.1-0.4mol/L, NH4The concentration of F is 0.2-0.6 mol/L;
d、NaOH、NH4the molar ratio of F to yttrium acetate is 1:1.6: 0.316.
6. The method for preparing chondroitin sulfate polysaccharide probe based on up-conversion nanomaterial as claimed in claim 3, wherein the step (2) ii comprises one or more of the following conditions:
a. the volume ratio of the oleic acid to the 1-octadecene is 0.2-0.6: 1; the volume ratio of the mass of the yttrium acetate to the oleic acid is 0.1-0.2 mol/L;
b. nanomaterial (NaYF)4Yb, Tm) in cyclohexane dispersion, nano material (NaYF)4Yb, Tm) is 0.2-0.3 mol/L; nanomaterial (NaYF)4Yb, Tm) and yttrium acetate in a molar ratio of 1.5: 1;
c. containing NaOH and NH4The concentration of NaOH in the methanol solution of F is 0.4-0.6mol/L, NH4The concentration of F is 0.6-1 mol/L;
d、NaOH、NH4the molar ratio of F to yttrium acetate was 2.5:4: 1.
7. The method for preparing chondroitin sulfate polysaccharide probe based on up-conversion nanomaterial as claimed in claim 3, wherein the step (3) comprises one or more of the following conditions:
i. the mass of the up-conversion nano material and the volume ratio of cyclohexane are 0.02-0.06 g/mL;
ii. The concentration of the aqueous solution of Cetyl Trimethyl Ammonium Bromide (CTAB) is 4-6 mg/mL; the mass ratio of Cetyl Trimethyl Ammonium Bromide (CTAB) to the up-conversion nano material is 1-1.5: 1;
iii、NH3·H2the concentration of O is 1-3mol/L, and the concentration of polyacrylic acid aqueous solution is 0.1-0.3 g/mL; NH (NH)3·H2The volume ratio of the O to the polyacrylic acid aqueous solution is 1-2: 1;
iv, the mass ratio of the up-conversion nano material to the polyacrylic acid is 10-15: 1;
v, the volume ratio of the isopropanol to the cyclohexane is 15-25: 1.
8. The method for preparing chondroitin sulfate polysaccharide probe based on up-conversion nanomaterial as claimed in claim 3, wherein the step (4) comprises one or more of the following conditions:
i. the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 2-5: 1;
ii. In the aqueous dispersion of the polyacrylic acid modified upconversion nanomaterial, the mass concentration of the polyacrylic acid modified upconversion nanomaterial is 8-10 mg/mL; the mass ratio of the polyacrylic acid modified up-conversion nano material to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is 1-1.5: 1;
iii, the mass ratio of the chondroitin sulfate grafted adipic acid dihydrazide compound (CS-ADH) to the polyacrylic acid modified up-conversion nano material is 1: 0.5-1.5;
iv, removing the small molecular compounds which are not completely reacted in the product by dialysis with the cut-off molecular weight of 4000 Da.
9. Use of the chondroitin sulfate polysaccharide probe of the up-converting nanomaterial of any one of claims 1 or 2 for in vitro and in vivo imaging of tumor cells.
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