CN113603811B

CN113603811B - PH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer and synthesis method and application thereof

Info

Publication number: CN113603811B
Application number: CN202110914701.6A
Authority: CN
Inventors: 唐汝培; 程旭
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2021-08-10
Filing date: 2021-08-10
Publication date: 2022-06-03
Anticipated expiration: 2041-08-10
Also published as: CN113603811A

Abstract

The invention discloses a pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer, and relates to the technical field of functionalized nano carriers, wherein the polymer has the following structure:

the present invention also provides a method for preparing a polymer, comprising step S1: preparing micromolecular fluoride shown in a formula III; s2: preparing amino fluoride shown in a formula IV; s3: preparing a pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer shown in a formula VI; meanwhile, the polymer prepared by the method is combined with drug molecules and applied to a drug delivery system. The invention has the advantages that: hyaluronic acid is used as a macromolecular skeleton, orthoester modified fluoride is used as a functional monomer, and is grafted to a hyaluronic acid side chain through an amide condensation reaction, so that the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer is finally obtained. The polymer can be assembled with a photosensitizer to obtain the nano drug-loaded micelle, has active targeting property, pH sensitivity and oxygen sensitization performance, and can improve the curative effect of photodynamic therapy.

Description

PH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer and synthesis method and application thereof

Technical Field

The invention relates to the technical field of functionalized nano-carriers, in particular to a pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer and a synthesis method and application thereof.

Background

A large number of research reports prove that tumor tissue metabolism is abnormal, blood vessels are deleted, and the tumor microenvironment with hypoxia and weak acid is caused by solid tumors. Tumor hypoxia further affects the expression of multiple resistant enzymes in cells, such as multidrug resistance proteins MRPs, glutathione reductase GST, and the like, thereby limiting the efficacy of chemotherapy with drugs to a large extent. In addition, tumor hypoxia will also directly affect the photodynamic therapy outcome. It is well known that photodynamic therapy transfers laser energy to oxygen molecules, which are activated to convert into toxic singlet oxygen. Thus, hypoxia would be detrimental to the performance of photodynamic therapy.

In the clinic, the most straightforward approach to tumor hypoxia is to administer hyperbaric oxygen therapy. However, the hyperbaric oxygen chamber has some disadvantages, such as expensive equipment, complicated operation, and inability to specifically deliver oxygen to the tumor site. To ameliorate this deficiency, several tumor in situ oxygen-producing or oxygen-transporting molecules have been extensively developed in recent years. The former uses catalase or manganese dioxide to catalyze the tumor site H₂O₂Decomposing to produce oxygen; the latter is primarily the delivery of oxygen to the tumor site using oxygen binding molecules such as perfluorocompounds, hemoglobin, and the like. Among them, perfluoro compounds are considered as a blood substitute due to their high oxygen carrying and releasing capacity, and are commonly used in some surgical first aid. However, conventional fluoride is difficult to degrade in vivo and lacks targeting specificity, thus having a certain limitation in alleviating tumor hypoxia.

Hyaluronic acid, a biomacromolecule polysaccharide, has good biocompatibility in vivo, and can specifically recognize CD44 receptor overexpressed on cancer cells. In addition, hyaluronic acid has a large number of hydroxyl and carboxyl components, which can be further modified to introduce functional components or drugs. In past studies, a number of hyaluronic acid-based nano-drug delivery systems have been developed, such as hyaluronic acid nanogels, micelles, hybrid particles, etc., and in most cases these nano-systems are capable of significantly increasing intracellular drug levels.

The patent application with the publication number of CN107375199A discloses a nanogel delivery system of polymerized chloroquine and a preparation method thereof, which discloses that the nanogel of the polymerized chloroquine is formed by a polysaccharide skeleton modified by hydroxychloroquine and hydrophobic side chains, however, the currently reported hyaluronic acid derivative lacks stimulation responsiveness or is weak in sensitivity, which results in limited drug release rate of the assembled hyaluronic acid nanomedicine system, compared with normal body tissues, the microenvironment of tumor tissues is abnormal, such as overheating and acid gradient (pH 7.2-5.0), and these special environmental factors also provide necessary preconditions for the development of stimulation-responsive biomacromolecules.

Disclosure of Invention

The invention aims to solve the technical problem that a hyaluronic acid derivative in the prior art is lack of stimulation responsiveness or weak in sensitivity, so that the rate of releasing a medicament of an assembled hyaluronic acid nano-medicament system is limited, and provides a pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer, a synthesis method and application thereof.

The invention solves the technical problems through the following technical means:

a pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer, wherein the structure of the polymer is shown as the formula VI:

has the advantages that: the polymer can be assembled with a photosensitizer together to obtain the nano drug-loaded micelle, has active targeting property, pH sensitivity and oxygen sensitization performance, and can improve the curative effect of photodynamic therapy, and the cumulative release of the polymer in 12 hours reaches more than 45 percent and the cumulative release of the polymer in 48 hours reaches 85 percent under the condition of pH 5.0.

The invention also provides a synthesis method of the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer, and the synthesis route is as follows:

the synthesis of the polymer takes hyaluronic acid as a macromolecular skeleton and acid-sensitive fluoride as a monomer, and the amphiphilic hyaluronic acid fluorinated polymer is synthesized through a grafting reaction catalyzed by amide condensation.

The polymer of the above design has the advantages that: hyaluronic acid is capable of targeting CD44 receptor to enhance cellular uptake; acid-sensitive ortho ester bonds are introduced to trigger the release of the medicine; the stability of the micelle can be improved by regulating and controlling the grafting ratio.

Preferably, the synthesis method of the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer comprises the following steps:

s1, preparation of micromolecular fluoride shown as a formula III:

mixing perfluorodecanol shown as a formula I, 2, 2-trifluoro-N- (2-methoxy- [1,3] -dioxolane-4-methyl) acetamide shown as a formula II and a catalyst, heating to 120 ℃ under reduced pressure of a vacuum pump, and performing post-treatment after the reaction is finished to obtain micromolecular fluoride shown as a formula III;

s2, preparation of amino fluoride shown in formula IV:

dissolving the micromolecule fluorine shown in the formula III prepared in the step S1 in tetrahydrofuran, adding NaOH, stirring at room temperature, reacting, and performing post-treatment to obtain amino fluoride shown in the formula IV;

s3, preparation of the hyaluronic acid fluorinated polymer with pH sensitivity and oxygen sensitization shown in the formula VI:

and adding the amino fluoride shown as the formula IV, hyaluronic acid shown as the formula V, EDC, NHS and triethylamine into a reactor, taking formamide as a solvent, slowly stirring under the protection of nitrogen, reacting, and purifying to obtain the polymer shown as the formula VI.

Preferably, in the step S1, the mol ratio of the perfluorodecanol represented by the formula i, the 2,2, 2-trifluoro-N- (2-methoxy- [1,3] -dioxolan-4-methyl) acetamide represented by the formula ii, and the pyridinium p-toluenesulfonate is 1: 3: 0.002.

preferably, the post-processing operation in step S1 is as follows: after the reaction is finished, standing the reaction solution to room temperature, dissolving the product by using dichloromethane, respectively extracting twice by using 0.5% sodium carbonate solution and saturated saline solution, collecting a lower dichloromethane phase, drying by using anhydrous magnesium sulfate, and performing rotary evaporation and concentration to obtain the micromolecule fluoride shown in the formula III.

Preferably, the catalyst in step S1 is pyridinium p-toluenesulfonate.

Preferably, the post-processing operation in step S2 is as follows: after the reaction, the solvent was removed under reduced pressure, the reaction mixture was dissolved in methylene chloride, extracted with 0.5% sodium carbonate solution and saturated brine, respectively, and then dried over anhydrous magnesium sulfate, filtered under suction, and rotary-evaporated to obtain an amino fluoride represented by formula IV.

Preferably, the amino fluoride shown in formula IV, the hyaluronic acid carboxyl shown in formula V, EDC, NHS and triethylamine are added in step S3 in a molar amount of 3: 1: 3: 3: 1.

preferably, the post-processing operation in step S3 is as follows: after the reaction is finished, dropwise adding the reaction solution into an absolute ethyl alcohol solution according to the volume ratio of 1:10, precipitating a white polymer, fishing out the product, and freeze-drying to obtain the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer shown in the formula VI.

The invention also discloses the application of the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer in a drug delivery system, wherein the drug delivery system comprises the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer, a photosensitizer and an acceptable auxiliary material on a pharmaceutical preparation.

Has the advantages that: the polymer and the photosensitizer are assembled together to obtain the nano drug-loaded micelle, the nano drug-loaded micelle can be used for a drug delivery system, has active targeting property, pH sensitivity and oxygen sensitization performance, and can improve the curative effect of photodynamic therapy.

Preferably, the photosensitizer is selected from chlorin Ce6, but is not limited to Ce 6.

Preferably, the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer and a photosensitizer are synthesized into a drug-carrying micelle, and the preparation method of the drug-carrying micelle comprises the following steps: mixing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer with a photosensitizer, adding DMSO (dimethylsulfoxide), heating for dissolving, dropwise adding deionized water, dialyzing by using a dialysis bag after forming micelle emulsion, centrifuging to remove the non-entrapped photosensitizer, and dispersing precipitate in the deionized water to obtain the drug-loaded micelle.

The invention has the advantages that:

the polymer can be assembled with a photosensitizer together to obtain the nano drug-loaded micelle, has active targeting property, pH sensitivity and oxygen sensitization performance, and can improve the curative effect of photodynamic therapy.

The polymer of the above design has the advantages that: hyaluronic acid is capable of targeting CD44 receptor to enhance cellular uptake; acid-sensitive ortho ester bonds are introduced to trigger the release of the medicine; fluoride grafting imparts oxygen carrying and releasing capabilities to the nanosystem.

Drawings

FIG. 1 is a schematic representation of an amino fluoride of formula IV of example 2 of the present invention¹H NMR chart;

FIG. 2 is a mass spectrum of an amino fluoride represented by formula IV in example 2 of the present invention;

FIG. 3 shows a fluorinated polymer of hyaluronic acid of formula VI in example 2 of the present invention¹H NMR chart;

FIG. 4 is a distribution diagram of the particle size of hyaluronic acid blank micelles in example 3 of the present invention;

FIG. 5 is a particle size distribution diagram of hyaluronic acid drug-loaded micelle in example 3 of the present invention;

FIG. 6 is a morphology of hyaluronic acid blank micelles under a transmission electron microscope in example 4 of the present invention;

FIG. 7 is a morphology of the hyaluronic acid drug-loaded micelle in example 4 of the present invention under a transmission electron microscope;

FIG. 8 shows the oxygen-carrying and oxygen-releasing of hyaluronic acid blank micelles in example 5 of the present invention;

FIG. 9 is a graph showing in vitro release of Ce6 from hyaluronic acid drug-loaded micelles in example 6 of the present invention;

FIG. 10 is a graph showing the results of qualitative uptake of hyaluronic acid-carrying micelle cells in example 7 of the present invention;

FIG. 11 is a graph showing the triggering of ROS generation in cells by hyaluronic acid drug-loaded micelles in example 8 of the present invention;

FIG. 12 is a graph showing the ROS triggering statistics of hyaluronic acid drug-loaded micelles in cells according to example 8 of the present invention;

FIG. 13 is a graph of cytotoxicity of hyaluronic acid blank micelles in example 9 of the present invention;

FIG. 14 is a graph showing photodynamic therapy effect of the hyaluronic acid drug-loaded micelle in normoxic cells in example 9 of the present invention;

fig. 15 is a graph of photodynamic therapeutic effect of the hyaluronic acid drug-loaded micelle in hypoxic cells in example 9 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.

Example 1

A pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer has a structure shown in a formula VI:

example 2

The preparation method of the hyaluronic acid fluorinated polymer with pH sensitivity and oxygen sensitivity comprises the following steps:

the synthesis route of the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer is as follows:

s1, preparation of a compound represented by formula III:

a50 mL round bottom reaction flask was charged with perfluorodecanol of formula I (15g,30mmol), 2,2, 2-trifluoro-N- (2-methoxy- [1,3] -dioxolan-4-methyl) acetamide of formula II (19g,89.9mmol), pyridinium p-toluenesulfonate (Py-PTSA) (15.5mg, 0.062mmol) in this order, and the system was heated to 120 ℃ under vacuum using an oil pump for 6 hours in an oil bath. After the reaction is finished, the temperature is returned to the room temperature, 10mL of dichloromethane is used for dissolving reactants, 0.5% of sodium carbonate solution and saturated saline solution are used for extracting twice respectively, a lower organic phase is collected and is evaporated in a rotary mode, and the micromolecular fluoride shown in the formula III is obtained;

s2, and preparing a compound shown as a formula IV:

dissolving the micromolecular fluoride of the formula III prepared in the step S1 in 50mL of tetrahydrofuran, adding 0.1 mol of NaOH solution, quickly stirring for 6h, and then removing the organic solvent by rotary evaporation. Dissolving with dichloromethane, extracting with saturated saline solution for three times, combining organic phases, drying with anhydrous magnesium sulfate, filtering, and rotary evaporating to obtain the amino compound shown in the formula IV.

The structure of the amino fluoride of the formula IV is detected¹H NMR, as shown in figure 1, can find in the spectrogram corresponding to hydrogen proton signals;

the structure of the amino fluoride shown in the formula IV is detected through mass spectrometry, as shown in figure 2, the actual detection value is consistent with the theoretical calculation value, and the synthetic amino fluoride is correct.

a150 mL beaker was charged with the amino fluoride represented by formula IV (3.73g, 6.2mmol), hyaluronic acid represented by formula V (1.0g,2.0mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (1.0g,6.4mmol), and N-hydroxysuccinimide (NHS) (0.72g, 6.3mmol) in this order, followed by addition of 20mL of formamide as a solvent and addition of 0.27mL (0.2g,1.97mmol) of triethylamine, and the reaction was stirred at room temperature for 24 hours. After the reaction is finished, dropwise adding the reaction liquid into 10 times of volume of absolute ethyl alcohol to separate out floccules. And fishing out the floccule by a glass rod, and freeze-drying to obtain the hyaluronic acid fluorinated polymer.

The structural characterization of the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer is shown in fig. 3.

As can be seen from fig. 3: both the orthoester characteristic peak and the hyaluronic acid methyl peak can be observed in a nuclear magnetic spectrum, which indicates that the small molecule fluoride is successfully grafted on the hyaluronic acid, namely the preparation of the hyaluronic acid fluorinated polymer is successful.

Example 3

Preparation and size determination of hyaluronic acid empty and drug-loaded micelle:

firstly, preparing hyaluronic acid blank micelles:

accurately weighing 40mg of hyaluronic acid fluorinated polymer into a 25mL beaker, adding 2mL of DMSO, heating for dissolving assistance, and slowly dropwise adding deionized water with pH of 8.0 until white emulsion is generated; and (3) putting the solution into a dialysis bag with the molecular weight cutoff of 8-14 KD for dialysis, and removing the organic solvent by taking deionized water as dialysate. Finally, the emulsion in the dialysis bag, i.e. hyaluronic acid blank micelle solution, was collected. The blank micellar solution is diluted by 3 times by deionized water, and the particle size and distribution of the blank micellar solution are measured by a nanometer particle sizer.

As shown in FIG. 4, the size of the hyaluronic acid blank micelle is 156.2nm, and the dispersion coefficient is only 0.178, which shows that the micelle has proper size and good dispersibility.

Secondly, preparing hyaluronic acid drug-loaded micelle:

accurately weighing 40mg of hyaluronic acid fluorinated polymer and 5mg of photosensitizer Ce6 in a 25mL beaker, adding 2mL of DMSO, heating to dissolve, slowly dropwise adding deionized water, and detecting the formation of micelle emulsion by using a laser pen; and dialyzing for 24h by using the dialysis bag, finally centrifuging to remove the non-entrapped photosensitizer, and dispersing the precipitate in deionized water to obtain the hyaluronic acid drug-loaded micelle solution. The drug-loaded micelle solution was diluted 3 times and its size and distribution were measured by a nano-particle sizer.

As shown in fig. 5, the hyaluronic acid drug-loaded micelle size was 175.4nm, slightly larger than the blank micelle, indicating that the photosensitizer was successfully encapsulated inside the micelle.

Example 4

And (3) observing the shapes of hyaluronic acid blank and drug-loaded micelle:

diluting the blank and drug-loaded micelle solution prepared in example 3 to a certain degree, then taking a drop of the diluted micelle solution by using a pipette, dripping the drop of the diluted micelle solution on a copper mesh, absorbing the surrounding water by using filter paper, and drying the solution under a heat lamp; and finally, observing the appearances of the two micelles by using a transmission electron microscope.

As shown in fig. 6, the hyaluronic acid blank micelle has a core-shell structure, is spherical, has a size of about 100nm, and is uniformly dispersed;

as shown in fig. 7, the hyaluronic acid-loaded micelle also appears spherical, but its inner region is further deepened, which further illustrates that the photosensitizer is encapsulated in the hydrophobic core of the micelle.

Example 5

Hyaluronic acid blank micelle oxygen carrying and releasing

Putting 2mL of hyaluronic acid blank micelle solution and perfluorocarbon solution into a two-mouth bottle, and introducing oxygen for 1h to obtain hyaluronic acid oxygen-carrying micelle solution; the above solutions were added to an oxygen-free aqueous solution and the change in dissolved oxygen concentration in the water was monitored at different time points with a dissolved oxygen meter.

As shown in fig. 8, the hyaluronic acid oxygen-carrying micelles and perfluorocarbons can significantly increase the dissolved oxygen concentration in water compared to the control group, and the oxygen release exhibits time dependence; when the dissolved oxygen in water reaches saturation, the oxygen release rate is obviously slowed down.

Example 6

In vitro release of Ce6 in hyaluronic acid drug-loaded micelles

Adding 1mL of hyaluronic acid drug-loaded micelle (320 mu g/mL) into a dialysis bag with the molecular weight cutoff of 8kD-14kD, fastening two ends of the dialysis bag by cotton threads, and then putting the dialysis bag into a 50mL centrifuge tube; 5mL of a buffer solution having a pH of 5.0 or 7.4 was injected into the centrifuge tube, and the tube was shaken on a shaker. At set time points, e.g. 0.5, 1, 2, 4.. 24h, etc., the release medium is removed and new buffer is added. Finally, the drug content in the release medium was calibrated by an enzyme reader, and the cumulative release of the photosensitizer Ce6 was calculated, with the release results shown in FIG. 9.

As can be seen from fig. 9: the pure Ce6 drug solution has a fast release rate, and the release rate is over 80% in 10 h; the hyaluronic acid drug-loaded micelle can release the drug in a controlled manner, and the release is gradually accelerated under the condition of pH 5.0, the accumulative release rate in 12 hours reaches more than 45%, and the accumulative release rate in 48 hours reaches 85%; at pH 7.4, the micelles are relatively stable with an accumulated release stabilized at 15%, and these results indicate that drug-loaded micelles have good pH-responsive capability.

Example 7

Qualitative uptake of hyaluronic acid drug-loaded micelle cells:

human liver cancer cells (HepG2) were planted in cell culture dishes and cultured for 24h to allow the cells to adhere to the wall. Then 1.8mL of fresh medium, 0.2mL of pure drug and hyaluronic acid drug-loaded micelle were added to give a final drug concentration of 4. mu.g/mL. After 2 or 4 hours of culture, the old culture medium is aspirated, the cells are washed by PBS, fixed by paraformaldehyde fixing solution, and the cell nucleus is stained by a staining reagent. Finally, the cells were washed again and observed using a confocal laser microscope. In addition, after the cells are attached to the wall, sufficient free hyaluronic acid is added to incubate the cells for 0.5h, then hyaluronic acid drug-loaded micelles are added to culture, and the subsequent operation is the same as above.

As shown in fig. 10: the pure medicine is mainly distributed in cytoplasm in the cells and is in a dispersed state; the drug-loaded micelles are distributed in a punctate manner in cytoplasm, and the fluorescence intensity in cells is gradually enhanced along with the extension of the incubation time; after the free hyaluronic acid is pre-incubated, the efficiency of the cell to take up the drug-loaded micelle is obviously reduced; these results indicate that hyaluronic acid can enhance cellular internalization, resulting in more drug enrichment in the cell.

Example 8

Hyaluronic acid drug-loaded micelles trigger ROS generation in cells:

human liver cancer cells (HepG2) were cultured in cell culture dishes under normoxic and hypoxic (anaerobic bags were used) environments, respectively, and cultured for 24 hours to allow the cells to adhere to the wall. Then, pure drugs and drug-loaded micelle solution are respectively added for culturing for 4 hours, and then PBS is used for washing cells, and fresh culture medium is added. And (3) performing laser irradiation at 630nm for 10min, adding a ROS probe DCFH-DA, and incubating for 30 min. Then, the cells were washed twice with PBS, fixed in 4% paraformaldehyde solution for 5min, washed again twice, and the generation of green ROS was observed with a fluorescent microscope.

As shown in fig. 11: in the normoxic cells, the pure drugs and the drug-loaded micelles can induce obvious ROS generation; in hypoxic cells, pure drug-induced ROS are significantly attenuated, but drug-loaded micelles maintain a higher induction capacity due to oxygen supplementation;

as shown in fig. 12: ROS fluorescence statistics confirm that drug-loaded micelles result in the highest ROS production in both normoxic and hypoxic cells, suggesting that hyaluronic acid can promote more drug enrichment.

Example 9

Evaluation of cytotoxicity:

planting human liver cancer cells (HepG2) and human fibroblast cells (293T) in a 96-well plate, adhering to the wall, and adding hyaluronic acid blank micelles with the concentration of 0.1-1.6 mg/mL. After 24 hours of culture, the old medium was aspirated, and a new medium (5mg/mL) containing MTT was added to continue the culture for 4 hours. And absorbing the culture medium again, adding 150 mu L of dimethyl sulfoxide, shaking to dissolve the crystal violet, measuring the absorbance under the ultraviolet of 570nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

As shown in fig. 13, cell viability was over 95% at all sample concentrations, both in cancer cells and normal cells, indicating that blank micelles are very biocompatible.

Evaluation of photodynamic performance:

the human liver cancer cells (HepG2) are planted in a 96-well plate, normal oxygen and oxygen-deficient environments are respectively given, and pure drugs and drug-loaded micelles are added after the cells are attached to the wall, wherein the drug concentration is set to be 0.5-16 mu g/mL. After 4h of culture, 630nm laser irradiation is given for 10min, and then culture is continued for 24 h. The subsequent operations are the same as above, and finally the cell survival rate is determined.

As shown in fig. 14, the cell viability exhibited a gradient-dependent decrease in normoxic cells with both pure drug and drug-loaded micelle treatment; the drug-loaded micelle can effectively kill cells;

as shown in fig. 15, in hypoxic cells, the ability of pure drugs to kill cells was significantly inhibited; the drug-loaded micelle can keep higher cell killing performance; the result shows that the hyaluronic acid drug-loaded micelle can improve the cell hypoxia state and improve the photodynamic therapy result.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A pH-sensitive and oxygen-sensitized fluorinated polymer of hyaluronic acid, characterized in that: the structure of the polymer is shown as a formula VI:

2. a process for the preparation of the pH sensitive and oxygen sensitised fluorinated polymer of hyaluronic acid according to claim 1, characterized in that: the synthetic route is as follows:

3. the method for preparing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer according to claim 2, characterized in that: the method comprises the following steps:

s1, preparation of micromolecular fluoride shown as a formula III:

s2, and preparation of amino fluoride shown in formula IV:

4. The method for preparing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer according to claim 3, characterized in that: in the step S1, the adding molar ratio of the perfluorodecanol shown in the formula I, the 2,2, 2-trifluoro-N- (2-methoxy- [1,3] -dioxolane-4-methyl) acetamide shown in the formula II and the pyridinium p-toluenesulfonate is 1: 3: 0.002.

5. the method for preparing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer according to claim 3, characterized in that: the post-processing operation in step S1 is as follows: after the reaction is finished, standing the reaction solution to room temperature, dissolving the product by using dichloromethane, respectively extracting twice by using 0.5% sodium carbonate solution and saturated saline solution, collecting a lower dichloromethane phase, drying by using anhydrous magnesium sulfate, and performing rotary evaporation and concentration to obtain the micromolecule fluoride shown in the formula III.

6. The method for preparing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer according to claim 3, characterized in that: the catalyst in the step S1 is pyridinium p-toluenesulfonate.

7. The method for preparing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer according to claim 3, characterized in that: the post-processing operation in step S2 is as follows: after the reaction, the solvent was removed under reduced pressure, the reaction mixture was dissolved in methylene chloride, extracted with 0.5% sodium carbonate solution and saturated brine, respectively, and then dried over anhydrous magnesium sulfate, filtered under suction, and rotary-evaporated to obtain an amino fluoride represented by formula IV.

8. The method for preparing the pH-sensitive and oxygen-sensitized hyaluronic acid fluorinated polymer according to claim 3, characterized in that: in the step S3, the adding amounts of the amino fluoride shown in the formula IV, the hyaluronic acid carboxyl shown in the formula V, EDC, NHS and triethylamine are 3: 1: 3: 3: 1.

9. use of the pH sensitive and oxygen sensitised hyaluronic acid fluorinated polymer according to claim 1 in a drug delivery system, wherein: the drug delivery system comprises a pH-sensitive and oxygen-sensitive hyaluronic acid fluorinated polymer, a photosensitizer and an auxiliary material acceptable in pharmaceutical preparations.

10. The use of a pH sensitive and oxygen sensitised hyaluronic acid fluorinated polymer according to claim 9 in a drug delivery system, wherein: the photosensitizer is selected from chlorin Ce 6.