Near-infrared light-responsive injectable thermosensitive hydrogel and preparation method and application thereof

Abstract

The present application relates to a kind of injectable temperature-sensitive hydrogel of near-infrared light response and its preparation method and application.The injectable temperature-sensitive hydrogel of near-infrared light response is: by chitosan, puerarin and near-infrared light response material jointly self-assembled to form the hydrogel with nanofiber network structure;Wherein chitosan and puerarin self-assemble to form nanofiber, and near-infrared light response material is embedded in nanofiber inside and is adsorbed on nanofiber surface;The concentration of solid component in the injectable temperature-sensitive hydrogel of near-infrared light response is 1.5-100 mg / mL, preferably 10-30 mg / mL;The concentration of near-infrared light response material in the injectable temperature-sensitive hydrogel of near-infrared light response does not exceed 1 mg / mL;The near-infrared light response material is selected from at least one of gold nanorod, gold nanoparticle, gold nanocluster and gold nanocage.

Description

TECHNICAL FIELD

[0001] The present application relates to a near-infrared light-responsive injectable thermosensitive hydrogel and its preparation method and application, in particular to a near-infrared light-responsive injectable antibacterial hydrogel for controlled release of drugs and its preparation method and application, and belongs to the field of biological materials. BACKGROUND

[0002] Uveal melanoma (UM) is the most common primary intraocular malignancy with high metastasis rate and mortality, which is one of the medical problems that need to be solved in clinic. Traditional enucleation often leads to permanent blindness, facial disfigurement and mental disorders, so new treatment methods are urgently needed. Injectable hydrogels based on natural polymers have attracted extensive attention of researchers in the field of drug delivery and tumor therapy due to their excellent biocompatibility, mechanically adjustable properties and environmental response characteristics, and become an ideal biomedical application treatment platform. Local treatment with injectable drug-loaded hydrogels can increase the amount of drugs reaching the tumor site of the eyeball, reduce systemic side effects and overcome the blood-ocular barrier.

[0003] Although injectable hydrogels have been widely studied in recent years, it is still a great challenge to design a drug release gel that can be applied to the treatment of intraocular tumors due to the unique microenvironment of the eyeball. An ideal drug-loaded hydrogel platform for injection treatment of ocular tumors should have the following characteristics: (1) It has a suitable mechanical strength after injection, which can resist the intraocular pressure of 12-21 ± 2 mmHg (~1.3-3.0 kPa), and low strength will cause the gel to break, while excessive strength will cause discomfort, disfigurement and functional impairment; (2) On-demand drug release. Although there have been many reports of tumor environment-responsive hydrogels, these hydrogels are difficult to achieve sustained, long-term and intelligent controlled release in vitro and in vivo; (3) High biological safety and biocompatibility. The biological toxicity of chemically cross-linked hydrogels and traditional chemotherapeutic drugs may damage the normal tissues of the eyeball; (4) Antibacterial ability. As an invasive treatment, ocular injection can cause endophthalmitis, which is one of the most destructive eye infections, can cause severe necrosis and cell death of various internal structures, and eventually lead to reversible blindness. In addition, recent studies have shown that bacteria play an important role in tumor occurrence, and the infection rate of bacteria in tumors is about 14.3% to 60%. In order to avoid endophthalmitis caused by ocular injection, effectively kill bacteria at the primary site of the tumor, the biological carrier should have good antibacterial performance. The drug-loaded hydrogels reported in the past can meet one or two of the above requirements, but it is difficult to meet all the requirements. SUMMARY

[0004] In order to achieve safe and effective treatment of ocular tumors, the purpose of the present application is to provide a universally applicable injectable antibacterial and antitumor hydrogel loaded with and controlled release of antitumor drugs, which can achieve high-efficiency antibacterial and antitumor effects without causing damage to normal tissues.

[0005] In a first aspect, the present application provides a near-infrared light responsive injectable thermosensitive hydrogel, which is a hydrogel with a nanofiber network structure formed by self-assembly of chitosan, puerarin and a near-infrared light responsive material; wherein the chitosan and puerarin self-assemble to form nanofibers, and the near-infrared light responsive material is embedded in the nanofibers and adsorbed on the surface of the nanofibers; the concentration of solid components in the near-infrared light responsive injectable thermosensitive hydrogel is 1.5-100 mg / mL, preferably 10-30 mg / mL.

[0006] The concentration of the near-infrared light responsive material in the near-infrared light responsive injectable thermosensitive hydrogel is not more than 1 mg / mL, preferably not more than 0.5 mg / mL, and more preferably not more than 0.1 mg / mL (for example, 0.05-0.1 mg / mL).

[0007] In the present disclosure, gold nanorods are used as photo-thermal responsive elements, chitosan puerarin is used as the main component of the injectable thermosensitive hydrogel, and after loading drugs, the drug release can be controlled by adjusting the light switch timing and quantity. The drug-loaded hydrogel has both antibacterial and antitumor properties. There are a large number of physical bonds such as hydrogen bonds, Π-Π bonds and van der Waals forces in the system. Preferably, the mass ratio of chitosan to puerarin is (1-10):(0.5-20), preferably 1:(0.5-4). Among them, the solid component is at least one of chitosan and puerarin that does not participate in the reaction and the nanofibers self-assembled from chitosan and puerarin.

[0008] Preferably, the near-infrared light responsive injectable thermosensitive hydrogel is irradiated with near-infrared light, and the temperature rises from room temperature to 70°C, while the near-infrared light responsive injectable thermosensitive hydrogel changes from a gel state to a sol state. The change in state is mainly related to the effect of temperature on hydrogen bonds. The wavelength of the near-infrared light is 780-1500 nm, the power of the near-infrared light is not more than 5 W / cm 2 , and the single irradiation time of the near-infrared light is not more than 30 minutes.

[0009] Preferably, the gold nanorods have a uniform rod structure, with a length of 10-100 nm and a width of 5-20 nm; the gold nanoparticles have a particle size of 10-100 nm; the gold nanoclusters have a diameter of 0.1-10 nm; and the gold nanocages have a diameter of 10-100 nm.

[0010] Preferably, the chitosan and puerarin in the injectable self-assembled hydrogel self-assemble to form nanofibers; the diameter of the nanofibers is 50-300 nm, preferably 60-200 nm.

[0011] Preferably, the storage modulus G' of the near-infrared light responsive injectable thermosensitive hydrogel is higher than the loss modulus G'', and the range of G' is 10-10000 Pa, and the range of G'' is 0-5000 Pa.

[0012] Preferably, at least one of a drug, a bioactive factor and an organic small molecule is further loaded in the near-infrared light responsive injectable self-assembled hydrogel, and the loading amount is 0.1-10 wt%, and at this time, it can be called a near-infrared light responsive injectable thermosensitive drug-loaded hydrogel;

[0013] The drug is at least one of fluorouracil, cyclophosphamide, cisplatin, doxorubicin hydrochloride, gefitinib, erlotinib, trastuzumab, goserelin, anastrozole, flutamide, paclitaxel, taxane, daunorubicin, mitoxantrone, methotrexate, vincristine, vindesine, etoposide, teniposide, dexamethasone, methylprednisolone, triamcinolone acetonide, glucocorticoids, cyclosporin A, azathioprine, tripterygium glycosides tablets, mycophenolate mofetil, methotrexate, hydroxychloroquine, gefitinib, erlotinib, afatinib, herceptin, lapatinib, sindi, nivolumab, panitumumab, tislelizumab, atezolizumab, durvalumab, and avastin.

[0014] The bioactive factor is at least one of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGFα and TGFβ), fibroblast growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin (IL-1, IL-1, IL-3, etc.), erythropoietin (EPO), and colony-stimulating factor (CSF).

[0015] The organic small molecule is at least one of indocyanine green, IR783, and IR780.

[0016] Preferably, by adjusting near-infrared light irradiation, the temperature change of the near-infrared light responsive material is caused, the gel-sol transformation of the gel is realized, and the release rate of the drug in the injectable self-assembled drug-loaded hydrogel is regulated. That is, the near-infrared light responsive injectable self-assembled drug-loaded hydrogel in the present application can slowly release the drug, and the drug release can be accelerated under near-infrared light irradiation.

[0017] In a second aspect, the present application provides a preparation method of the near-infrared light responsive injectable thermosensitive hydrogel, comprising:

[0018] (1) Put chitosan, puerarin and a reaction vessel, and then add acetic acid solution as an auxiliary agent to obtain a mixture;

[0019] (2) adding a near-infrared light responsive material (for example, gold nanorods) and a drug into the mixture, self-assembling under the physical mixing effect, and then adding deionized water to dissolve, to obtain the near-infrared light responsive injectable temperature-sensitive hydrogel. In the present application, the porous network structure is formed by using chitosan as the main structure, and when the puerarin self-assembles with the chitosan, the puerarin self-assembles with the chitosan into a nanofiber structure, so that the nanofiber gradually forms on the main body of the porous network structure (chitosan), and the near-infrared light responsive material is embedded in the nanofiber and adsorbed on the surface of the nanofiber. Further, the nanofiber gradually increases to form a nanofiber network structure, and the nanofiber network structure obtained has a certain water storage and water absorption, so that the near-infrared light responsive injectable temperature-sensitive hydrogel is obtained.

[0020] Preferably, the degree of deacetylation of the chitosan is 85-100%; the purity of the puerarin is 90-100%; the mass ratio of the chitosan to the puerarin is 1:(0.5-4); and the ratio of the chitosan to the deionized water is 5mg-10mg:1mL.

[0021] Preferably, the physical mixing effect includes grinding, mechanical ball milling, stirring, or mixing centrifugation.

[0022] The grinding time is 2-20 minutes.

[0023] The mechanical ball milling speed is 60-600 revolutions per minute, and the time is 2 minutes-5 hours.

[0024] The stirring speed is 100-1000 revolutions per minute, and the time is 2 minutes-5 hours.

[0025] The mixing centrifugation speed is 50g-12000g of acceleration due to gravity, and the time is 2 minutes-20 minutes.

[0026] Preferably, in step (1), the concentration of the acetic acid solution is 0.1-10M, and preferably the acetic acid solution has a concentration of 87.5mM; the mass ratio of the amount of the acetic acid solution to the chitosan is (0.1-1mL):50mg; in step (2), the self-assembly time is 0-24 hours; and the deionized water is 10-200 times, and preferably 40-60 times, the total mass of the chitosan and the puerarin. In step (2), after the deionized water is added to dissolve, a second acetic acid solution is added; the concentration of the acetic acid solution is 0.1-10M, and preferably the acetic acid solution has a concentration of 87.5mM; and the mass ratio of the amount of the acetic acid solution to the chitosan is (0.5-10mL):50mg, and preferably 2mL of the acetic acid solution:50mg of the chitosan. The addition of the acetic acid solution can improve the viscosity and mechanical properties of the gel, which is convenient for subsequent use.

[0027] In a third aspect, the present application provides a use of the near-infrared light responsive injectable thermosensitive hydrogel in preparation of an antibacterial and anti-ocular infection material.

[0028] In a fourth aspect, the present application provides a use of the near-infrared light responsive injectable drug-loaded thermosensitive hydrogel in preparation of an anti-tumor material.

[0029] Beneficial effects:

[0030] (1) In the present application, the near-infrared light responsive material gold nanorod is self-assembled with chitosan pterostilbene nanowire fibers to construct a near-infrared light responsive injectable thermosensitive hydrogel. The incorporation of gold nanorods has three functions: ① improving the mechanical strength of the gel, the mechanical strength of the chitosan pterostilbene hydrogel is not enough to resist the intraocular pressure, and it is easy to collapse after being injected into the eye and cannot maintain the gel state and achieve effective drug loading, after the incorporation of gold nanorods, the mechanical strength can be effectively improved, and the gel state can be maintained after being injected into the eye and drug release can be achieved; ② realizing the near-infrared responsive drug release of the gel, near-infrared light can be used as a "switch" for drug release, realizing the on-demand release of drugs, the hydrogel of the present application is mainly formed by hydrogen bond-induced self-assembly, and the hydrogen bond and other actions are extremely sensitive to temperature, when the temperature rises, the hydrogen bond action weakens, and the mechanical strength of the gel decreases, which is manifested as the temperature-sensitive gel-sol transformation of the gel. After the addition of gold nanorods, the good photothermal performance of gold nanorods can realize the temperature rise of the gel system through near-infrared light irradiation, and after further loading of drugs, the gel-sol transformation of the gel can be adjusted to further adjust the drug release; ③ photothermal anti-tumor, when near-infrared light is irradiated, the temperature rises, and tumor cells are more sensitive to temperature, which can achieve better thermal anti-tumor treatment effect;

[0031] (2) In the present application, the prepared hydrogel can be directly injected, and still maintains the gel state after injection, which has been proved in vitro experiments to have: excellent temperature-sensitive and photothermal performance; universal near-infrared light responsive controlled drug release performance; good anti-gram-negative bacteria and anti-gram-positive bacteria effect; good cell compatibility; near-infrared light regulated induction of tumor cell apoptosis performance. In vivo experiments show that the injectable hydrogel synthesized in the present application has the effect of light-controlled drug release, effectively kills bacteria in the eyeball, prevents the occurrence of endophthalmitis, effectively treats eyeball melanoma and does not damage normal tissues, and provides a tumor treatment material with antibacterial / photothermal treatment / controlled drug release treatment;

[0032] (3) In the present application, the preparation process of the near-infrared light responsive controlled drug release injectable hydrogel is stable and controllable, simple, low in cost, easy to realize, good in treatment effect, and convenient for popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1Synthesis of injectable thermosensitive hydrogel for near-infrared light responsive controlled release of drugs and schematic diagram of treatment of eye tumors and eye infections. From Figure 1 As can be seen from a, natural polymer chitosan and traditional Chinese medicine puerarin form nanofiber (CP nanofiber) during grinding, gold nanorods (GNRs) and gene-targeting small molecule inhibitors (DC_AC50) are added, deionized water is added to form hydrogel (CP@Au@DC_AC50 hydrogel), under the irradiation of near-infrared light (NIR) and under the condition of temperature rise, the gel realizes gel-sol transformation (Gel-Sol), and at the same time, the drug is released. From Figure 1 As can be seen from b, after the gel is injected into the eyeball of the mouse, the antibacterial effect is realized through the antibacterial property of the gel itself, and the synergistic photothermal / gene-targeting anti-eye tumor effect is realized through the temperature rise and drug release under the response of near-infrared light;

[0034] Figure 2 Synthesis process and structural schematic diagram of injectable thermosensitive hydrogel for near-infrared light responsive controlled release of drugs. As can be seen from the figure, puerarin, chitosan, gold nanorods and antitumor drugs are added to the mortar and ground to form nanofiber, and after the addition of deionized water, self-assembled nanofiber hydrogel is formed;

[0035] Figure 3 Picture of injectable thermosensitive hydrogel for near-infrared light response. As can be seen from the figure, the chitosan puerarin (CP) hydrogel without gold nanorods is a milky white gel state, and after the addition of gold nanorods, it is a pink gel state, and after heating, the gel is converted into sol, and after cooling, it can be converted into gel state again;

[0036] Figure 4 Scanning electron microscope picture of the gel, a is the structure of chitosan puerarin hydrogel, b is the structure of gold nanorods, c is the structure of near-infrared light responsive injectable thermosensitive hydrogel chitosan puerarin gold nanorod gel, and d is the low magnification structure of injectable thermosensitive hydrogel. From Figure 4 As can be seen from a, the chitosan puerarin hydrogel is nanofiber, and the nanofiber is intertwined with each other, and the diameter is 60-200nm, as can be seen from b in 4, the gold nanorod is a uniform rod structure with a diameter of about 10nm and a length of 50nm, as can be seen from c in 4, the near-infrared light responsive injectable thermosensitive hydrogel contains two structures of nanofiber and gold nanorod, and due to the electrostatic effect, the gold nanorods are regularly and orderly arranged near the nanofiber, and as can be seen from Figure 4 As can be seen from d, the gel has a typical gel porous structure;

[0037] Figure 5UV absorption spectra of chitosan puerarin hydrogel (CP), gold nanorods (GNRs) and near-infrared light responsive injectable thermosensitive hydrogel chitosan puerarin gold nanorod gel (CP@Au). It can be seen from the figure that the gold nanorods have strong absorption peaks near 520 nm and 800 nm, and the CP@Au hydrogel doped with gold nanorods also has absorption peaks at the same position, which proves that the gold nanorods and the hydrogel have photothermal effect in theory;

[0038] Figure 6 DSC curves of CP and CP@Au hydrogels loaded with gold nanorods. It can be seen from the figure that there is an obvious inflection point near 50℃ in the two curves, indicating that the gel-sol transition of the gel occurs near 50℃, which proves the thermosensitive property of the gel;

[0039] Figure 7 FTIR spectra of CP and CP@Au hydrogels loaded with gold nanorods. It can be seen from the figure that with the addition of gold nanorods, the wave number moves to a higher direction from 3254 cm -1 to 3271 cm -1 , and the peak value at 1107 cm -1 disappears. The difference in infrared spectrum may indicate that the interaction between GNRs and CP nanofibers affects the association degree of CP composite, which proves the successful loading of gold nanorods;

[0040] Figure 8 Rheological curves of the storage modulus G' and loss modulus G" of CP and CP@Au hydrogels with time. It can be seen from the figure that the rheological modulus of the hydrogel is significantly improved after adding gold nanorods, which proves that the incorporation of GNRs can improve the mechanical properties of the gel;

[0041] Figure 9 Rheological curves of the storage modulus G' and loss modulus G" of CP@Au hydrogel under cyclic shear strain. It can be seen from the figure that at 1% strain, G' > G", which is in the gel state, and at 500% strain, G' < G", which is in the sol state, and the gel-sol state of the gel can be cyclically converted under cyclic strain, which indicates that the near-infrared light responsive injectable thermosensitive hydrogel has injectable and self-repairing properties;

[0042] Figure 10 The change curves of the storage modulus G' and loss modulus G" of the near-infrared light responsive injectable thermosensitive hydrogel during the heating process. It can be seen from the figure that near 52℃, G' and G" intersect, which is basically consistent with the DSC curve result, which proves the thermosensitive property of the gel;

[0043] Figure 11The near-infrared responsive injectable thermosensitive CP@Au hydrogel exhibited a power density of 0.3 W / cm² after 10 min of irradiation with 808 nm near-infrared light. 2 0.5w / cm 2 and 1.0w / cm 2 Temperature changes. As shown in the figure, the CP@Au hydrogel was heated from 27℃ to 35℃, 48℃ and 65℃ respectively, demonstrating the material's excellent near-infrared light response to temperature rise.

[0044] Figure 12 For CP hydrogel, GNRs solution and CP@Au at 0.5 w / cm 2 Infrared thermographs after near-infrared light irradiation at different power densities for different durations. The figures show that, under the same power density and irradiation time, the near-infrared light-responsive injectable thermosensitive CP@Au hydrogel exhibits the best photothermal effect.

[0045] Figure 13 for Figure 12 The corresponding temperature change curves and their conclusions are consistent with... Figure 12 The conclusions can corroborate each other;

[0046] Figure 14 The figure shows the temperature change of the near-infrared light-responsive injectable thermosensitive CP@Au hydrogel after 5 near-infrared light cycles on / off. As can be seen from the figure, the near-infrared light-responsive injectable thermosensitive CP@Au hydrogel exhibits excellent photothermal cycling capability.

[0047] Figure 15 Near-infrared light-responsive, thermosensitive CP@Au hydrogel at 0.5 w / cm² 2 Infrared thermographs of eye temperature changes after 5 minutes of laser irradiation. As can be seen from the figure, compared with the in vitro warming effect, the warming effect of the Au group is not obvious, which may be related to the dilution of the gold powder by the vitreous fluid in the eye after injection. The CP@Au gel group still has excellent photothermal effect.

[0048] Figure 16 Near-infrared light-responsive, thermosensitive CP@Au hydrogel at 0.5 w / cm² 2 The curve of eye temperature change after 5 minutes of laser irradiation, and Figure 15 The results corroborate this;

[0049] Figure 17 This study investigated the release of doxorubicin (Doxorubicin) hydrogel loaded with the antitumor drug doxorubicin hydrochloride (DOx) via near-infrared light-responsive injectable thermosensitive CP@Au hydrogel after 10 minutes of exposure without and after light exposure. Figure 17 As can be seen from a, Dox has an absorption peak near 470nm. After illumination, the intensity of the absorption peak increases, and the release amount increases. Figure 17It can be seen from the figure that without light (Without NIR), Dox is released slowly, while the near-infrared light has obvious photothermal switching effect when the light is cycled (NIR), and the drug release amount increases when the light is cycled, which proves the near-infrared light responsive release effect of the injectable temperature-sensitive CP@Au hydrogel for Dox;

[0050] Figure 18 The injectable temperature-sensitive CP@Au hydrogel loaded with fluorescent molecule IR783 was injected into the eyeballs of mice, and the near-infrared light 808nm 0.5W / cm

[0051] Figure 19 The injectable temperature-sensitive CP@Au hydrogel loaded with fluorescent molecule IR783 was injected into the eyeballs of mice, and the near-infrared light 808nm 0.5W / cm 2 was irradiated for 5min, and the fluorescence intensity at the beginning and after three days (in b, the ordinate is fluorescence intensity (Fluorescence Intensity)*10 9 , and in c, the ordinate is fluorescence intensity*10 7 ) was compared. It can be seen from the figure that when the materials in different groups were injected into the eyeballs of mice, the fluorescence intensity was basically inhibited due to the consistent concentration of IR783, and there was no significant change. After three days, the fluorescence intensity of the control group was very low, mainly because the drug was degraded and absorbed too quickly, while the gel could achieve slow release of the drug, so the fluorescence intensity was strong after three days, and the fluorescence intensity decreased after photothermal treatment, which was mainly related to the promotion of drug release by photothermal treatment, which proved the near-infrared light responsive release effect of the injectable temperature-sensitive CP@Au hydrogel for IR783 in vivo;

[0052] Figure 20 The injectable temperature-sensitive CP@Au hydrogel loaded with small molecule inhibitor DC_AC50 was injected into the eyeballs of mice, and the near-infrared light release of the injectable temperature-sensitive CP@Au hydrogel was observed. It can be seen from the figure that without light (Without NIR), DC_AC50 is released slowly, while the near-infrared light has obvious photothermal switching effect when the light is cycled (NIR), and the drug release amount increases when the light is cycled, which proves the near-infrared light responsive release effect of the injectable temperature-sensitive CP@Au hydrogel for DC_AC50, and the results prove the universal drug loading and near-infrared light controlled release effect of the near-infrared light responsive injectable temperature-sensitive CP@Au hydrogel; Figures 17-20

[0053] Figure 21 ​The proliferation and live-dead of normal cells ARPE19 and eye tumor cells OM431 and OCM1 after the culture of the near-infrared light responsive injectable thermosensitive CP@Au hydrogel were observed. It can be seen from the figure that the cell proliferation and live-dead are the same as the normal group, indicating that the near-infrared light responsive injectable thermosensitive CP@Au hydrogel has excellent cell compatibility. Figure 20 The near-infrared light responsive injectable thermosensitive CP@Au hydrogel has excellent cell compatibility.

[0054] Figure 22 The cell proliferation of normal eye cells ARPE19 and eye tumor cells OCM1 treated by the control group (Control), drug and gene targeting treatment group (DC_AC50), gel photothermal group (CP@Au+NIR) and gel photothermal synergistic gene targeting treatment group (CP@Au@DC_AC50+NIR) was observed. It can be seen from the figure that for normal cells, the three anti-tumor therapies have little effect on the proliferation of normal cells, while for tumor cells, the near-infrared light responsive injectable thermosensitive drug-loaded hydrogel CP@Au@DC_AC50+NIR can inhibit cell proliferation and has excellent anti-tumor effect.

[0055] Figure 23 The cell apoptosis of eye tumor cells OCM1 treated by the control group (Control), drug and gene targeting treatment group (DC_AC50), gel photothermal group (CP@Au+NIR) and gel photothermal synergistic gene targeting treatment group (CP@Au@DC_AC50+NIR) was observed. It can be seen from the figure that the near-infrared light responsive injectable thermosensitive drug-loaded hydrogel CP@Au@DC_AC50+NIR can significantly induce cell apoptosis.

[0056] Figure 24 The appearance of the eyeball of mice treated by the control group (Control, eyeball injection of eye tumor cells to form tumors), drug and gene targeting treatment group (DC_AC50), gel photothermal group (CP@Au+NIR) and gel photothermal synergistic gene targeting treatment group (CP@Au@DC_AC50+NIR) and normal group (Normal) in the in vivo experiment was observed. After the treatment of the control group with sterile PBS, the eyeball was filled with tumor tissue, and obvious vascular proliferation and eyeball movement disorder could be seen. Compared with the control group, the tumor proliferation degree of the DC_AC50 and CP@Au+NIR groups was lower, but the eyeball was still turbid and swollen, indicating that gene targeting treatment and simple photothermal treatment had certain curative effect. It is worth noting that in the CP@Au@DC_AC50+NIR group, the gel photothermal synergistic gene targeting treatment group was similar to the normal control group, and the eyeball tissue was clear without tumor cell proliferation. It is proved that the near-infrared light responsive injectable thermosensitive drug-loaded hydrogel CP@Au@DC_AC50+NIR has excellent in vivo anti-eye tumor effect.

[0057] Figure 25Figure 6 is a graph showing the eyeball section of a mouse treated with the control group (Control, eyeball injection of tumor cells), the drug gene targeting treatment group (DC_AC50), the gel photothermal group (CP@Au+NIR), the gel photothermal synergistic gene targeting treatment group (CP@Au@DC_AC50+NIR), and the normal group (Normal) after 2 weeks of treatment. As can be seen from the figure, in the control group, the tumor cells broke into the vitreous body and proliferated in large quantities in the eyeball. In the DC_AC50 and CP@Au+NIR groups, the tumor cells only proliferated outside the vitreous body. In the CP@Au@DC_AC50+NIR group, the eyeball was clean and no tumor cells were found inside. This proves that the near-infrared light-responsive injectable thermosensitive drug-loaded hydrogel CP@Au@DC_AC50+NIR has excellent anti-eye tumor effect in vivo;

[0058] Figure 26 Figure 7 is a fluorescence image of each group of materials after the tumor cells were dyed with fluorescence. As can be seen from the figure, the fluorescence of the CP@Au@DC_AC50+NIR group is the weakest, indicating that there are the least tumor cells and the best anti-tumor effect;

[0059] Figure 27 Figure 8 is a graph showing the eyeball mass (a) and eyeball diameter (b) of mice treated with each group of materials in vivo. As can be seen from the figure, the eyeball mass and diameter of the mice treated with the near-infrared light-responsive injectable thermosensitive drug-loaded hydrogel CP@Au@DC_AC50+NIR are not significantly different from those of normal mice, which proves the excellent anti-eye tumor effect of the near-infrared light-responsive injectable thermosensitive drug-loaded hydrogel CP@Au@DC_AC50+NIR;

[0060] Figure 28 Figure 9 is a photograph of the bacterial plate after treatment of E. coli and S. aureus with the control group (PBS, Control), the thermosensitive hydrogel (CP@Au), and the near-infrared light-responsive thermosensitive hydrogel (CP@Au+NIR). As can be seen from the figure, the thermosensitive hydrogel has excellent antibacterial ability;

[0061] Figure 29 Figure 10 is a scanning electron microscope image of bacteria treated with each group of materials. As can be seen from the figure, after treatment with the thermosensitive hydrogel, the bacterial membrane is ruptured, the bacteria are dead, and the bacteria are killed;

[0062] Figure 30The photographs of the eyeballs of the control group (PBS, Control), the thermosensitive hydrogel (CP@Au) and the near-infrared light responsive thermosensitive hydrogel (CP@Au+NIR) after the mice eyeballs were injected with Staphylococcus aureus to induce eyeball bacterial infection. It can be seen from the figure that the eyeballs of the control group of mice were obviously infected, and the eyeballs were swollen and turbid, while the eyeballs of the two gel groups were clean and transparent. HE staining further confirmed that the eyeballs of the control group of rats were filled with a large number of neutrophils, while no inflammatory reaction was found in the two hydrogel groups;

[0063] Figure 31 The bar charts of the weight and diameter of the eyeballs of the control group (PBS, Control), the thermosensitive hydrogel (CP@Au) and the near-infrared light responsive thermosensitive hydrogel (CP@Au+NIR) of mice after the mice eyeballs were injected with Staphylococcus aureus to induce eyeball bacterial infection. It can be seen from the figure that the weight and diameter of the eyeballs of the mice in the gel group were smaller, indicating that there was no bacterial infection and excellent antibacterial ability of the material;

[0064] Figure 32 The scanning electron microscope structure diagram of the near-infrared light responsive thermosensitive hydrogel doped with different concentrations of gold nanorods. It can be seen from the figure that with the increase of the concentration of gold nanorods, the fracture in the structure increases, which may be related to the interaction between the gold nanorods and the nanofibers;

[0065] Figure 33 The X-ray diffraction (XRD) pattern of the near-infrared light responsive thermosensitive hydrogel doped with different concentrations of gold nanorods. It can be seen from the figure that the structure of the material reacted by the XRD pattern of the gel is different with different doping amounts of gold nanorods;

[0066] Figure 34 The rheological properties of the near-infrared light responsive thermosensitive hydrogel doped with different concentrations of gold nanorods. It can be seen from the figure that with the increase of the concentration of gold nanorods, the storage modulus and loss modulus of the gel are improved, indicating that the gold nanorods can improve the mechanical properties of the material. DETAILED DESCRIPTION

[0067] The application is further illustrated by the following embodiments, and it should be understood that the following embodiments are only used to illustrate the application, but not to limit the application.

[0068] In the present disclosure, the injectable near-infrared light responsive thermosensitive drug-loaded hydrogel is self-assembled from chitosan, puerarin, gold nanorods and drugs. Under the induction of weak acid conditions and external force, chitosan and puerarin are assembled into nanofibers through nucleation and growth of nanofibers under the action of physical bonds such as a large number of hydrogen bonds and van der Waals forces in the system, and then further form a network structure. The rod-like structure of the gold nanorods added at the same time is adsorbed on the nanofibers to enhance the mechanical properties of the material, and the drug loading is achieved through hydrogen bonding. The addition of water dissolves to obtain a physically cross-linked shear-thinning near-infrared light responsive injectable thermosensitive hydrogel, which has universal drug release and controlled release performance.

[0069] In the present disclosure, chitosan and puerarin are mixed, and on the basis of adding an acid solution, the material components are self-assembled into nanofibers by one-step grinding method, gold nanorods and drugs prepared are added, and grinding and water dissolution are continued to form a hydrogel. When there is no near-infrared light, the drug is slowly released. When near-infrared light is used for irradiation, the gel gradually warms up, and after reaching the gel sol transition point of the gel, the drug is released in large quantities. The purpose of near-infrared light controlled gel warming and drug release is achieved.

[0070] In a preferred embodiment, the gold nanorods have a uniform rod-like structure with a length of about 50 nm and a width of about 10 nm. The gold nanorods have near-infrared absorption peaks near 520 nm and 800 nm.

[0071] In a preferred embodiment, the gold nanorods can improve the mechanical properties of the gel, and the storage modulus and loss modulus of the gel are improved to a certain extent after loading the gold nanorods.

[0072] In a preferred embodiment, the near-infrared light responsive injectable hydrogel can increase the temperature from room temperature to 70℃ under near-infrared light irradiation. In a preferred embodiment, the gold nanorods are added to the CP nanofibers as the near-infrared light responsive photothermal element, and the photothermal element can be replaced by gold nanoparticles, graphene, black phosphorus and other materials with photothermal response.

[0073] In the present disclosure, the near-infrared light responsive hydrogel can be used in the preparation of antibacterial and anti-eye infection materials.

[0074] In the present disclosure, the near-infrared light responsive injectable thermosensitive hydrogel can be used in the preparation of anti-tumor materials.

[0075] The following further illustrates the embodiments in detail. It should also be understood that the following embodiments are only used to further illustrate the present application, and cannot be understood as limiting the scope of the present application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the present application are within the scope of protection of the present application. The specific process parameters and the like described below are only one example in the appropriate range, i.e. those skilled in the art can make appropriate selection within the range according to the description herein, and are not limited to the specific values of the following examples.

[0076] Example 1

[0077] Gold nanorods were prepared according to the literature method (Wang, D. H.; Ge, N. J.; Yang, T. T.; Peng, F.; Qiao, Y. Q.; Li, Q. W.; Liu, X. Y., NIR-Triggered Crystal Phase Transformation of NiTi-Layered Double Hydroxides Films for Localized Chemothermal Tumor Therapy. Advanced Science 2018, 5 (4), 10.) and 1 mL of gold nanorod solution (concentration of about 0.18 mg / mL) was taken and added to 1.5 mL centrifuge tube with deionized water. After centrifugation twice, it was used.

[0078] 50 mg of chitosan and 100 mg of puerarin were weighed and added to a mortar. 300 μL of 87.5 mM (0.5% by volume) acetic acid was added dropwise, and the mixture was ground until the chitosan and puerarin were evenly mixed and the acetic acid was completely volatilized. Then 0.36 mg of gold nanorods was added and ground until uniform. 8 mL of deionized water was added and stirred until the viscosity of the system gradually increased. 2 mL of 87.5 mM acetic acid was added and stirred to form a gel. The near-infrared light-responsive injectable thermosensitive hydrogel CP@Au-1 was obtained. The concentration of the solid components in the near-infrared light-responsive injectable thermosensitive hydrogel was 15 mg / mL, and the concentration of gold nanorods in the near-infrared light-responsive material was 0.036 mg / mL.

[0079] Example 2

[0080] Take 50 mg of chitosan and 100 mg of puerarin into a mortar. Add 300 μL of 87.5 mM (0.5% volume fraction) acetic acid dropwise, and grind the mixture until the chitosan and puerarin are uniformly mixed and the acetic acid is completely volatilized. Then add 0.72 mg of gold nanorods and continue to grind until uniform. Add 8 mL of deionized water and stir until uniform. At this point, the viscosity of the system gradually increases. Add 2 mL of 87.5 mM acetic acid and stir to form a gel directly. The near-infrared light-responsive injectable thermosensitive hydrogel CP@Au-2 is obtained. The concentration of the solid components in the near-infrared light-responsive injectable thermosensitive hydrogel is 15 mg / mL, and the concentration of gold nanorods in the infrared light-responsive material is 0.072 mg / mL.

[0081] Example 3

[0082] Take 50 mg of chitosan and 100 mg of puerarin into a mortar. Add 300 μL of 87.5 mM (0.5% volume fraction) acetic acid dropwise, and grind the mixture until the chitosan and puerarin are uniformly mixed and the acetic acid is completely volatilized. Then add 0.72 mg of gold nanorods and continue to grind until uniform. Add 8 mL of deionized water and stir until uniform. At this point, the viscosity of the system gradually increases. Add 2 mL of 87.5 mM acetic acid and stir to form a gel directly. The near-infrared light-responsive injectable thermosensitive hydrogel CP@Au-2 is obtained. The concentration of the solid components in the near-infrared light-responsive injectable thermosensitive hydrogel is 15 mg / mL, and the concentration of gold nanorods in the infrared light-responsive material is 0.072 mg / mL.

[0083] Example 4

[0084] Take 50 mg of chitosan and 100 mg of puerarin into a mortar. Add 300 μL of 87.5 mM (0.5% volume fraction) acetic acid dropwise, and grind the mixture until the chitosan and puerarin are uniformly mixed and the acetic acid is completely volatilized. Then add 0.72 mg of gold nanorods and continue to grind until uniform. Add 1 mL of 1000 μM small molecule inhibitor DC_AC50, stir until uniform, and then add 7 mL of deionized water and stir until uniform. At this point, the viscosity of the system gradually increases. Add 2 mL of 87.5 mM acetic acid and stir to form a gel directly. The near-infrared light-responsive drug-loaded injectable thermosensitive hydrogel CP@Au@DC_AC50 is obtained. The concentration of the solid components in the near-infrared light-responsive injectable thermosensitive hydrogel is 15 mg / mL, the concentration of gold nanorods in the infrared light-responsive material is 0.072 mg / mL, and the loading amount of the small molecule inhibitor is 100 μM.

[0085] Example 5

[0086] Take 50 mg of chitosan and 100 mg of puerarin into a mortar. Add 300 μL of 87.5 mM (0.5% by volume) acetic acid dropwise, and grind the mixture until the chitosan and puerarin are evenly mixed and the acetic acid is completely volatilized. Then add 0.72 mg of gold nanorods, and continue to grind until the mixture is evenly mixed. Add 1 mL of 1000 μM of an antitumor drug Dox, stir until the mixture is evenly mixed, add 7 mL of deionized water, and stir until the mixture is evenly mixed. At this time, the viscosity of the system gradually increases. Add 2 mL of 87.5 mM acetic acid, and stir to form a gel directly to obtain a near-infrared light-responsive drug-loaded injectable thermosensitive hydrogel CP@Au@Dox. The concentration of the solid-state component of chitosan in the near-infrared light-responsive injectable thermosensitive hydrogel is 15 mg / mL, the concentration of gold nanorods in the infrared light-responsive material is 0.072 mg / mL, and the drug loading is 100 μM.

[0087] Example 6

[0088] Take 50 mg of chitosan and 100 mg of puerarin into a mortar. Add 300 μL of 87.5 mM (0.5% by volume) acetic acid dropwise, and grind the mixture until the chitosan and puerarin are evenly mixed and the acetic acid is completely volatilized. Then add 0.72 mg of gold nanorods, and continue to grind until the mixture is evenly mixed. Add 1 mL of 1000 μM of an antitumor drug Dox, stir until the mixture is evenly mixed, add 7 mL of deionized water, and stir until the mixture is evenly mixed. At this time, the viscosity of the system gradually increases. Add 2 mL of 87.5 mM acetic acid, and stir to form a gel directly to obtain a near-infrared light-responsive drug-loaded injectable thermosensitive hydrogel CP@Au@Dox. The concentration of the solid-state component of chitosan in the near-infrared light-responsive injectable thermosensitive hydrogel is 15 mg / mL, the concentration of gold nanorods in the infrared light-responsive material is 0.072 mg / mL, and the drug loading is 100 μM.

[0087] Example 6

[0088] Take 50 mg of chitosan and 100 mg of puerarin into a mortar. Add 300 μL of 87.5 mM (0.5% by volume) acetic acid dropwise, and grind the mixture until the chitosan and puerarin are evenly mixed and the acetic acid is completely volatilized. Then add 0.72 mg of gold nanorods, and continue to grind until the mixture is evenly mixed. Add 1 mL of 1000 μM of an antitumor drug Dox, stir until the mixture is evenly mixed, add 7 mL of deionized water, and stir until the mixture is evenly mixed. At this time, the viscosity of the system gradually increases. Add 2 mL of 87.5 mM acetic acid, and stir to form a gel directly to obtain a near-infrared light-responsive drug-loaded injectable thermosensitive hydrogel CP@Au@Dox. The concentration of the solid-state component of chitosan in the near-infrared light-responsive injectable thermosensitive hydrogel is 15 mg / mL, the concentration of gold nanorods in the infrared light-responsive material is 0.072 mg / mL, and the drug loading is 100 μM.

Claims

1. An injectable, temperature-sensitive drug-loaded hydrogel for near-infrared light-responsive controlled-release drugs, characterized in that, The injectable thermosensitive drug-loaded hydrogel for near-infrared light-responsive controlled-release drugs is an injectable self-assembly drug-loaded hydrogel consisting of a hydrogel with a nanofiber network structure formed by the self-assembly of chitosan, puerarin, and a near-infrared light-responsive material, and a drug loaded on the hydrogel. Chitosan and puerarin self-assemble to form a nanofiber network, and the near-infrared light-responsive material is embedded within the nanofibers and adsorbed on their surface. The near-infrared light-responsive material is selected from gold nanorods. The concentration of the solid component in the injectable thermosensitive drug-loaded hydrogel is 10-30 mg / mL. The concentration of the near-infrared light-responsive material in the injectable thermosensitive drug-loaded hydrogel does not exceed 1 mg / mL. By adjusting the near-infrared light irradiation, the temperature of the near-infrared light-responsive material changes, achieving a gel-sol transition and thus controlling the drug release rate in the injectable self-assembly drug-loaded hydrogel. The injectable self-assembly loaded drug hydrogel has a porous gel structure; the mass ratio of chitosan to puerarin is 1:(0.5~4); the injectable thermosensitive drug-loaded hydrogel of near-infrared light-responsive controlled release drug heats up from room temperature to 70°C under near-infrared light irradiation, and at the same time, the injectable thermosensitive drug-loaded hydrogel of near-infrared light-responsive controlled release drug changes from a gel state to a sol state. The injectable thermosensitive drug-loaded hydrogel containing the near-infrared light-responsive controlled-release drug is selected from at least one of antitumor drugs, hormonal drugs, bioactive factors, and small organic molecules, with a loading capacity of 0.1~1000uM; the antitumor drug is selected from fluorouracil, cyclophosphamide, cisplatin, doxorubicin hydrochloride, gefitinib, erlotinib, trastuzumab, goserelin, anastrozole, flutamide, taxanes, daunorubicin, mitoxantrone, methotrexate, vincristine, vindesine, etoposide, teniposide, and alfa. The drug is selected from at least one of the following: tinib, Herceptin, lapatinib, sintilimab, nivolumab, pentazolizumab, tislelizumab, atezolizumab, duvalbumin, and acimetidine; the hormonal drug is selected from at least one of dexamethasone, methylprednisolone, and triamcinolone; the bioactive factor is selected from at least one of platelet growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor, nerve growth factor, interleukin growth factor, erythropoietin, and colony-stimulating factor. The organic small molecule is selected from at least one of indocyanine green, IR783, and IR780.

2. The injectable thermosensitive drug-loaded hydrogel with near-infrared light-responsive controlled-release drug according to claim 1, characterized in that, The wavelength of the near-infrared light is 780~1500nm, the power of the near-infrared light does not exceed 5W / cm2, and the single irradiation time of the near-infrared light does not exceed 30 minutes.

3. The injectable thermosensitive drug-loaded hydrogel with near-infrared light-responsive controlled-release drug according to claim 1, characterized in that, The gold nanorods have a uniform rod-shaped structure with a length of 10-100 nm and a width of 5-20 nm.

4. The injectable thermosensitive drug-loaded hydrogel with near-infrared light-responsive controlled-release drug according to claim 1, characterized in that, The diameter of the nanofibers is 50~300nm.

5. The injectable thermosensitive drug-loaded hydrogel with near-infrared light-responsive controlled-release drug according to claim 4, characterized in that, The diameter of the nanofibers is 60~200nm.

6. The injectable thermosensitive drug-loaded hydrogel with near-infrared light-responsive controlled-release drug according to claim 1, characterized in that, The storage modulus G' of the injectable thermosensitive drug-loaded hydrogel for near-infrared light-responsive controlled-release drugs is higher than the loss modulus G”, and G' ranges from 10 to 10000 Pa, while G” ranges from 0 to 5000 Pa.

7. A method for preparing an injectable thermosensitive drug-loaded hydrogel of an infrared-responsive controlled-release drug as described in any one of claims 1-6, characterized in that, The process includes: (1) adding chitosan and puerarin to a reactor vessel, and then adding acetic acid solution as an excipient to obtain a mixture; (2) adding gold nanorods and drugs to the obtained mixture, which undergo self-assembly under physical mixing, then adding deionized water to dissolve, adding acetic acid solution a second time and stirring to form a gel, thus obtaining an injectable thermosensitive drug-loaded hydrogel with near-infrared light-responsive controlled-release drug.

8. The preparation method according to claim 7, characterized in that, The degree of deacetylation of the chitosan is 85-100%; the purity of the puerarin is 90-100%; and the ratio of chitosan to deionized water is 5 mg-10 mg: 1 mL.

9. The preparation method according to claim 7, characterized in that, In step (1), the concentration of the acetic acid solution is 0.1~10M; the mass ratio of the amount of acetic acid solution to chitosan is (0.1~1mL):50mg; in step (2), the self-assembly time is 0~24 hours; the deionized water is 10~200 times the total mass of chitosan and puerarin.

10. The preparation method according to claim 7, characterized in that, In step (2), after dissolving in deionized water, acetic acid solution is added a second time; the concentration of the acetic acid solution is 0.1-10M, and the mass ratio of the amount of acetic acid solution to chitosan is (0.5-10 mL): 50mg.

11. The use of an injectable thermosensitive drug-loaded hydrogel of a near-infrared light-responsive controlled-release drug according to any one of claims 1-6 in the preparation of anti-ocular tumor drugs or anti-ocular infection drugs.

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