阅读说明：本技术 一种生物材料负载的钙钛矿-二氧化钛纳米复合光催化剂及其构建方法和应用 (Perovskite-titanium dioxide nano composite photocatalyst loaded by biological material and construction method and application thereof ) 是由 丁杨 周建平 盛钰 陈鑫 于 2021-09-28 设计创作，主要内容包括：本发明涉及生物医药材料技术领域,具体涉及一种生物材料负载的钙钛矿-二氧化钛纳米复合光催化剂及其构建方法和应用,该纳米复合光催化剂包括钙钛矿纳米粒；包覆钙钛矿纳米粒的二氧化钛壳层。本发明以前沿新材料钙钛矿为基础,结合二氧化钛以提高结构稳定性并有效分离电子空穴对,构建用于长效Ⅰ型光动力治疗的纳米复合光催化系统。制备工艺简单,解决了临床常用光敏剂氧依赖性高以及无机光敏剂生物相容性低的问题。相较于其他光动力治疗系统,生物材料负载的钙钛矿-二氧化钛纳米复合光催化系统经激光触发,在低剂量下即可发挥高效肿瘤治疗作用,且在合适的给药剂量下安全无毒,在光动力治疗中更具优势。(The invention relates to the technical field of biological medicine materials, in particular to a perovskite-titanium dioxide nano composite photocatalyst loaded by a biological material as well as a construction method and application thereof, wherein the nano composite photocatalyst comprises perovskite nano particles; a titanium dioxide shell layer coating the perovskite nano-particles. The invention constructs a nano composite photocatalytic system for long-acting I-type photodynamic therapy by combining titanium dioxide to improve the structural stability and effectively separate electron hole pairs on the basis of perovskite which is a novel material. The preparation process is simple, and the problems of high oxygen dependence of the common clinical photosensitizer and low biocompatibility of the inorganic photosensitizer are solved. Compared with other photodynamic therapy systems, the perovskite-titanium dioxide nano composite photocatalysis system loaded by the biological material can play a role in high-efficiency tumor therapy at a low dosage by laser triggering, is safe and nontoxic at a proper dosage and has more advantages in photodynamic therapy.)

1. A perovskite-titanium dioxide nano composite photocatalyst loaded by biological materials is characterized in that: the perovskite-titanium dioxide nano composite photocatalyst loaded by the biological material comprises perovskite nanoparticles, a titanium dioxide shell layer and a biological delivery material; the perovskite nano particles are coated on the titanium dioxide shell layer to form a perovskite-titanium dioxide nano composite photocatalyst; the perovskite-titanium dioxide nano composite photocatalyst is loaded by a biological material;

the molecular structural formula of the perovskite nano particle is CH₃NH₃SnX₃Wherein X is one or more of I, Br and Cl monovalent halogen anions;

the particle size range of the perovskite nano particles is 2-20 nm; the particle size range of the perovskite-titanium dioxide nano composite photocatalyst is 10-100 nm;

preferably, the biomaterial is selected from surface double bond modified high molecular polymer, or derivatives thereof, or combination of high molecular polymer and/or derivatives thereof; further preferably, the biomaterial is selected from one or more of surface double bond modified hyaluronic acid, chitosan, polylactic acid, dextran, gelatin, guar gum, poloxamer, poly (N-isopropylacrylamide);

preferably, the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst responds in the near infrared region.

2. The preparation method of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 1, which is characterized by comprising the following steps:

s1, synthesizing perovskite nano-particles;

s2, preparing the perovskite-titanium dioxide nano composite photocatalyst;

s3, constructing the perovskite-titanium dioxide nano composite photocatalyst loaded by the biological material.

3. The preparation method of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 2, wherein the specific steps of S1 are as follows:

s1-1: separately preparing SnX₂Good solvent mother liquor and CH₃NH₃Mother liquor of good solvent of X, SnX₂Good solvent mother liquor and CH₃NH₃Uniformly mixing the good solvent mother liquor of the X to obtain a mixed liquor, and adding oleic acid and oleylamine into the mixed liquor to prepare a precursor liquor;

preferably, SnX is present in the mixed solution₂And CH₃NH₃The molar ratio of X is (0.8-1.2): (0.8 to 1.2);

s1-2: the precursor solution is stirred vigorously, cooled to room temperature and added dropwise to a miscible anti-solvent to obtain dispersed CH₃NH₃SnX₃Reaction solution;

s1-3: suction CH₃NH₃SnX₃Adding the reaction solution into the immiscible anti-solvent, slightly shaking and uniformly mixing, standing until the solution is layered, collecting the lower oily solution, adding the immiscible anti-solvent again to repeat the operation to obtain the purified CH₃NH₃SnX₃Nanoparticles, i.e. perovskite nanoparticles.

4. The preparation method of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 3,

the good solvent in S1-1 is one or more selected from dimethyl sulfoxide, dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, gamma-butyrolactone and dimethyl propylene urea;

the volume percentage of oleic acid in the precursor liquid in the S1-1 is 8-14%; the volume percentage of oleylamine in the precursor liquid is 0.4-1.0%.

5. The preparation method of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 3,

the miscible anti-solvent in S1-2 is selected from one or more of toluene, acetone, acetonitrile, chlorobenzene, diethyl ether, dichloromethane, nitromethane, isopropanol and ethyl acetate;

the volume ratio of the precursor solution to the miscible anti-solvent in S1-2 is 1: 10 to 20.

6. The preparation method of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 3,

the immiscible anti-solvent in S1-3 is selected from one or more of n-hexane, diethyl ether, sec-butanol, trifluorotoluene, iodinated benzene, anisole, methyl acetate and ethyl acetate;

the amount of each addition of the immiscible anti-solvent in S1-3 is CH₃NH₃SnX₃Volume ratio of reaction liquid to immiscible anti-solvent 1: 5 to 10.

7. The preparation method of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 2, wherein the S2 comprises the following specific steps:

s2-1: taking CH prepared from S1₃NH₃SnX₃Dispersing in mixed solvent, ultrasonic treating, adding butyl titanate and concentrated hydrochloric acid, stirring at normal temperature, condensing and refluxing,obtaining a reaction solution; the mixed solvent consists of ethanol and n-hexane;

s2-2: purifying the reaction solution obtained in S2-1 with n-hexane, centrifuging to collect white precipitate, vacuum drying to remove organic solvent to obtain CH₃NH₃SnX₃-TiO₂The compound is perovskite-titanium dioxide nano composite photocatalyst.

8. The method for preparing the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst according to claim 6, wherein the method comprises the following steps:

the volume ratio of ethanol to n-hexane in the mixed solvent in S2-1 is 2-4: 1; the ultrasonic time is 10-30 min; CH (CH)₃NH₃SnX₃The mass ratio of the titanium dioxide to the butyl titanate is 1: 20-30; CH (CH)₃NH₃SnX₃The mass ratio of the concentrated hydrochloric acid to the concentrated hydrochloric acid is 10: 1-3;

the stirring time is 2-4 h; the condensing reflux time is 5-20 h; the condensation reflux temperature is 40-50 ℃;

the volume ratio of the reaction liquid to n-hexane in S2-2 is 1: 3 to 5.

9. The method for preparing the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst as claimed in claim 2, wherein the method comprises the following steps: the specific steps of S3 are:

s3-1: carrying out surface sulfhydrylation reaction on the perovskite-titanium dioxide nano composite photocatalyst obtained in the step S2 to obtain a perovskite-titanium dioxide nano composite photocatalyst with a sulfhydryl modified surface;

preferably, the surface sulfhydrylation reaction of S3-1 is to disperse the perovskite-titanium dioxide nano composite photocatalyst in ethanol, add mercaptopropyl trimethoxy silane, and perform the surface sulfhydrylation reaction under the protection of nitrogen, wherein the molar ratio of the mercaptopropyl trimethoxy silane to the perovskite-titanium dioxide nano composite photocatalyst is 50: 1;

further preferably, the volume percentage concentration of the ethanol is 98%, the concentration of the perovskite-titanium dioxide nano composite photocatalyst in the ethanol is 0.4mg/mL, and the surface sulfhydrylation reaction time is 5 hours;

s3-2: and (3) carrying out Click reaction on the biomaterial and the perovskite-titanium dioxide nano composite photocatalyst with the surface modified sulfydryl obtained by S3-1 under the initiation of ultraviolet light to prepare the perovskite-titanium dioxide nano composite photocatalyst loaded by the biomaterial.

10. Use of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst as claimed in claim 1 for the preparation of photodynamic therapy drugs; preferably, the photodynamic therapy medicament is a photodynamic therapy medicament for treating tumors; further preferably, the compound is a long-acting type I photodynamic therapeutic drug for treating tumors.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a perovskite-titanium dioxide nano composite photocatalyst loaded by a biomaterial, and a construction method and application thereof.

Background

Photodynamic therapy (PDT), an emerging non-invasive treatment modality, has received much attention in recent years as a non-invasive and mild medical technique that produces cytotoxic Reactive Oxygen Species (ROS) by a Photosensitizer (PS) under the trigger of a light source of a specific wavelength to exert an antitumor effect. Over the past two decades, researchers have successfully designed various PDT systems that contain primarily a photosensitizer and a suitable light source. The Type I PDT (Type I reaction) reaction substrate is mainly H₂O、O₂And H₂O₂Etc., can generate ROS such as hydroxyl radicals, superoxide anion radicals, etc. In Type II PDT (Type II interaction) the photosensitizer releases energy to Oxygen (Oxygen, O)₂) Introducing O₂Conversion to singlet oxygen (¹O₂). Type II PDT has a strong oxygen dependence due to the large consumption of oxygen. In the hypoxic environment of solid tumor tissue, this oxygen dependence greatly impedes the practical clinical application of type II PDT. Therefore, the type I PDT has stronger applicability, and the related research is more urgent.

The photocatalytic material has high efficiency and stability, and can be used in solar energy conversion and ringThe method is widely applied to the fields of environmental purification, organic synthesis and the like. Under the trigger of a proper light source, such as Near Infrared (NIR), ultraviolet visible light (UV-Vis) and X-ray wavelength light and the like, photons can be absorbed to generate a series of photochemical reactions, and Reactive Oxygen Species (ROS) with cytotoxicity are generated to play an anti-tumor role. The specific action process is that after the photocatalyst absorbs photon energy, electrons (e)^-) Transition from the Valence Band (VB) to the Conduction Band (CB), hole (h) on the valence band⁺) Oxidizable H₂O generates hydroxyl radicals (. OH) and electrons reduce oxygen to superoxide radicals (. smallcircle.O)^2-). Compared with the traditional photosensitizer, the catalytic property of the photocatalytic material enables the photocatalytic material to repeatedly generate ROS under the condition of sufficient substrate and illumination, can play a role in high-efficiency treatment at low dose, has no oxygen dependence, and has more advantages in photodynamic treatment.

The photocatalysis material commonly used in type I PDT is metal such as gold nanoparticle; semiconductor-based materials such as titanium dioxide (TiO)₂) Zinc oxide (ZnO); some inorganic nanoparticles such as quantum dots, carbon quantum dots, and the like. In order to improve the anti-tumor capability of the photocatalytic material, the photocatalytic material with the light absorption range matched with near infrared light needs to be found to improve the photon absorption efficiency of the material, so that the ROS yield is improved. Perovskite (CH) is not available in the prior art₃NH₃SnX₃) The reason for the report as a photocatalytic material in type I PDT is CH₃NH₃SnX₃The problem existing as a photocatalyst is that the photoproduction electron hole pair is rapidly recombined in the photocatalysis process, and CH is simultaneously generated₃NH₃SnX₃Poor self-stability, easy decomposition in water, Sn²⁺Is easily oxidized, so that a good photocatalytic effect cannot be obtained.

TiO₂The semiconductor is the most widely applied semiconductor, has stable chemical structure, good biocompatibility, optical, electrical, catalytic and other properties, and can be used for compounding with a photocatalytic material, thereby effectively separating electron hole pairs and improving the photocatalytic efficiency; on the other hand, TiO₂The stable chemical property can prevent the perovskite from being oxidized and hydrolyzed, and the long-term stability of the whole system is improved, so that the PDT effect is continuously exerted. But TiO 2₂Is poorly soluble in water and TiO₂The lack of tumor specific recognition ligand on the surface leads to toxicity of adsorbing serum protein when the tumor specific recognition ligand circulates in vivo, and the insufficient selectivity in clinical application leads to low photodynamic therapy efficiency.

Disclosure of Invention

In order to solve the technical problems in the background technology, the invention provides a perovskite-titanium dioxide nano composite photocatalyst loaded by a biological material, and a construction method and application thereof.

The technical scheme of the invention is as follows:

the invention provides a preparation method of a biomaterial-loaded perovskite-titanium dioxide nano-composite photocatalyst, wherein the biomaterial-loaded perovskite-titanium dioxide nano-composite photocatalyst comprises perovskite nanoparticles, a titanium dioxide shell layer and a biological delivery material; the perovskite nano particles are coated on the titanium dioxide shell layer to form a perovskite-titanium dioxide nano composite photocatalyst; the perovskite-titanium dioxide nano composite photocatalyst is loaded by a biological material;

the perovskite nano-particle is a halide perovskite nano-particle with a molecular structural formula of CH₃NH₃SnX₃Wherein X is one or more of I, Br and Cl monovalent halogen anions;

the particle size range of the perovskite nano particles is 2-20 nm; the particle size range of the perovskite-titanium dioxide nano composite photocatalyst is 10-100 nm;

preferably, the biomaterial is selected from a high molecular polymer with double bond (alkenyl) modification on the surface, or a derivative of the high molecular polymer with double bond modification on the surface, or a combination of the high molecular polymer with double bond modification on the surface and/or the derivative thereof;

if the surface of the biological material has double bonds (alkenyl), the biological material does not need to be modified; if the biomaterial itself has no double bond (alkenyl group), the surface functionalization is carried out by a conventional method to obtain the alkenyl group, so that the mercaptoalkene click reaction is carried out with the mercapto group in the presence of a photoinitiator in the subsequent preparation process.

Further preferably, the biomaterial is selected from one or more of surface double bond modified hyaluronic acid, chitosan, polylactic acid, dextran, gelatin, guar gum, poloxamer, poly (N-isopropylacrylamide).

Preferably, the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst responds in the near infrared region. The optimal tissue penetration depth for photodynamic therapy is in the range of 700-850 nm, referred to as the "optical window" or "Near Infrared (NIR) window". In a specific embodiment, the response wavelength of the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst is 808 nm.

The second purpose of the invention is to provide a preparation method of the biomaterial-loaded perovskite-titanium dioxide nano-composite photocatalyst, which comprises the following steps:

s1, synthesizing perovskite nanoparticles;

s2, preparing a perovskite-titanium dioxide nano composite photocatalyst;

s3, constructing the perovskite-titanium dioxide nano composite photocatalyst loaded by the biological material.

Further, the specific step of S1 is:

s1-1: separately preparing SnX₂Good solvent mother liquor and CH₃NH₃Mother liquor of good solvent of X, SnX₂Good solvent mother liquor and CH₃NH₃Uniformly mixing the good solvent mother liquor of the X to obtain a mixed liquor, and adding oleic acid and oleylamine into the mixed liquor to prepare a precursor liquor;

preferably, SnX is present in the mixed solution₂And CH₃NH₃The molar ratio of X is (0.8-1.2): (0.8 to 1.2);

preferably, the oleylamine is preheated to be liquid before being added into the mixed solution;

s1-2: the precursor solution is stirred vigorously, cooled to room temperature and added dropwise to a miscible anti-solvent to obtain dispersed CH₃NH₃SnX₃Reaction solution;

s1-3: suction CH₃NH₃SnX₃Adding the reaction solution into a non-miscible anti-solvent, slightly shaking, uniformly mixing, and standing until the reaction solution is mixedSeparating the solution, collecting the lower oily solution, adding a certain amount of non-miscible anti-solvent again to repeat the above operation to obtain purified CH₃NH₃SnX₃Nanoparticles, i.e. perovskite nanoparticles.

Further, the good solvent in S1-1 is selected from one or more of dimethyl sulfoxide, dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, gamma-butyrolactone and dimethyl propylene urea;

the volume percentage of oleic acid in the precursor liquid in the S1-1 is 8-14%;

the volume percentage of oleylamine in the precursor liquid is 0.4-1.0%.

Further, the miscible anti-solvent in S1-2 is selected from one or more of toluene, acetone, acetonitrile, chlorobenzene, diethyl ether, dichloromethane, nitromethane, isopropanol and ethyl acetate;

the volume ratio of the precursor solution to the miscible anti-solvent in S1-2 is 1: 10 to 20.

Further, the immiscible anti-solvent in S1-3 is selected from one or more of n-hexane, diethyl ether, sec-butanol, trifluorotoluene, iodinated benzene, anisole, methyl acetate and ethyl acetate;

the amount of each addition of the immiscible anti-solvent in S1-3 is CH₃NH₃SnX₃The volume ratio of the reaction solution to the immiscible anti-solvent is 1: 5 to 10.

Further, the specific step of S2 is:

s2-1: taking CH prepared from S1₃NH₃SnX₃Dispersing in a mixed solvent, adding butyl titanate and concentrated hydrochloric acid after ultrasonic treatment, stirring at normal temperature, and condensing and refluxing to obtain a reaction solution; the mixed solvent consists of ethanol and n-hexane; the ethanol is absolute ethanol;

s2-2: purifying the reaction solution obtained in S2-1 with n-hexane, centrifuging to collect white precipitate, vacuum drying to remove organic solvent to obtain CH₃NH₃SnX₃-TiO₂A compound, namely a perovskite-titanium dioxide nano composite photocatalyst;

further, the volume ratio of ethanol to n-hexane in the mixed solvent in S2-1 is 2-4: 1; the ultrasonic time is 10-30 min; CH (CH)₃NH₃SnX₃The mass ratio of the titanium dioxide to the butyl titanate is 1: 20-30; CH (CH)₃NH₃SnX₃The mass ratio of the concentrated hydrochloric acid to the concentrated hydrochloric acid is 10: 1-3; the stirring time is 2-4 h; the condensing reflux time is 5-20 h; the condensation reflux temperature is 40-50 ℃;

the volume ratio of the reaction liquid to n-hexane in S2-2 is 1: 3 to 5.

Further, the specific step of S3 is:

s3-1: carrying out surface sulfhydrylation reaction on the perovskite-titanium dioxide nano composite photocatalyst obtained in the step S2 to obtain a perovskite-titanium dioxide nano composite photocatalyst with a sulfhydryl modified surface;

s3-2: carrying out Click reaction on a biomaterial and the perovskite-titanium dioxide nano composite photocatalyst with the surface modified with sulfydryl obtained by S3-1 under the initiation of ultraviolet light to prepare the perovskite-titanium dioxide nano composite photocatalyst loaded by the biomaterial; the compound photocatalyst has the characteristics of high hydrophilicity, good biocompatibility, safety and no toxicity;

preferably, the surface sulfhydrylation reaction of S3-1 is to disperse the perovskite-titanium dioxide nano composite photocatalyst in ethanol, add mercaptopropyl trimethoxy silane, and perform the surface sulfhydrylation reaction under the protection of nitrogen, wherein the molar ratio of the mercaptopropyl trimethoxy silane to the perovskite-titanium dioxide nano composite photocatalyst is 50: 1;

further preferably, the volume percentage concentration of the ethanol in S3-1 is 98%, the concentration of the perovskite-titanium dioxide nano composite photocatalyst in the ethanol is 0.4mg/mL, and the surface sulfhydrylation reaction time is 5 hours;

preferably, after the surface sulfhydrylation reaction is finished, separating a reaction product, and drying to obtain the perovskite-titanium dioxide nano composite photocatalyst with the surface modified with sulfhydryls; the separation is that after the centrifugation at 16000rpm for 5min, the ethanol is washed twice for precipitation, and the drying is vacuum drying for 12 h;

s3-2, the biomaterial is selected from surface double bond (alkenyl) modified high molecular polymer, or surface double bond modified high molecular polymer derivative, or surface double bond modified high molecular polymer and/or its derivative combination; the double-bond modified high molecular polymer or the derivative thereof or the combination of the high molecular polymer and the derivative thereof can be purchased directly or prepared by a conventional method;

if the surface of the biological material has double bonds (alkenyl), the biological material does not need to be modified; if the biomaterial itself has no double bond (alkenyl group), the surface functionalization is carried out by a conventional method to obtain the alkenyl group, so that the mercaptoalkene click reaction is carried out with the mercapto group in the presence of a photoinitiator in the subsequent preparation process.

Further preferably, the biomaterial is selected from the group consisting of a combination of one or more of double bond modified hyaluronic acid, chitosan, polylactic acid, dextran, gelatin, guar gum, poloxamer, poly (N-isopropylacrylamide);

the proportion of the biological material S3-2 and the surface-modified sulfhydryl perovskite-titanium dioxide nano-composite photocatalyst obtained by S3-1 is conventionally determined by the type and administration form of the biological material;

the Click reaction of S3-2 is to add a conventional photoinitiator and then carry out ultraviolet illumination, wherein the adding amount of the initiator and the ultraviolet illumination time are conventionally determined according to the type and administration form of the biological material.

The third purpose of the invention is to provide the application of the perovskite-titanium dioxide nano composite photocatalyst loaded by the biological material in the preparation of photodynamic therapy medicines; preferably, the photodynamic therapy medicament is a photodynamic therapy medicament for treating tumors; further preferably, the compound is a long-acting type I photodynamic therapeutic drug for treating tumors. The photodynamic therapy medicament has biological safety and tumor targeting property, can be subjected to intravenous injection, local injection and other administration modes to ensure that the photodynamic therapy medicament has excellent intratumoral retention, and is used for long-acting I-type photodynamic therapy of tumors.

The perovskite-titanium dioxide nano composite lightThe catalyst generates electron hole pairs under the excitation of 700-850 nm illumination, and can generate a large amount of active oxygen under low dosage: the electrons can reduce oxygen molecules to superoxide radicals (. O)^2-) The holes are capable of oxidizing water molecules to hydroxyl radicals (. OH). Under the trigger of multiple rounds of NIR, ROS is continuously generated, the membrane potential of mitochondria is damaged, mitochondria are promoted to release Cyto-c, Caspase-3 is activated, the apoptosis of tumor cells is induced, and the long-acting I type photodynamic therapy effect is exerted.

The perovskite-titanium dioxide nano composite photocatalyst is loaded by a biological material, is safe and non-toxic while effectively inhibiting the growth of tumors under a proper administration dosage, has no damage to liver, kidney and thyroid tissue, has no obvious genetic toxicity and genotoxicity, has biological safety and tumor targeting property, can be applied to the body, and has excellent intratumoral retention by the administration modes of intravenous injection, local injection and the like.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the perovskite structure metal material is mainly used in the field of solar cells and photocatalysis at present, and the invention starts from the principle of perovskite photocatalysis and I-type PDT commonality, and the perovskite material is used as a photosensitizer for carrying out anti-tumor treatment in vivo for the first time. With halide perovskite nano-particles CH₃NH₃SnX₃Compared with other common perovskite structures such as lead-based perovskites, the tin halide hybrid perovskite is a base material and does not contain toxic heavy metal elements, and the contained tin and halogen elements are essential trace elements in human bodies, so that the tin halide perovskite has the possibility of biological application and has higher safety when being applied to the field of tumor treatment.

2. In order to improve the photocatalytic performance, the perovskite semiconductor and the titanium dioxide semiconductor are combined in the aspect of material design, the recombination of electron hole pairs is effectively inhibited, the stability of the perovskite semiconductor and the titanium dioxide semiconductor is improved, and meanwhile, the light wave band which can be responded by the photocatalyst is extended to the near infrared wave band, so that the perovskite semiconductor and the titanium dioxide semiconductor have the basis of being applied to the body.

3. By means of tumor targeting retention of the biological delivery material, the perovskite-titanium dioxide nano composite photocatalyst continuously generates ROS like an energy station, and plays a long-acting PDT role.

Drawings

Fig. 1 is an electron microscope image of perovskite nano-particles prepared in example 1 and titanium dioxide-perovskite nano-particles prepared in example 9; wherein, fig. 1A is a transmission electron microscope result of perovskite nanoparticles (scale bar is 100nm), fig. 1B is a high-resolution transmission electron microscope result of perovskite nanoparticles (scale bar is 2nm), fig. 1C is a transmission electron microscope result of titanium dioxide-perovskite nanoparticles (scale bar is 50nm), and fig. 1D is a high-resolution transmission electron microscope result of titanium dioxide-perovskite nanoparticles (scale bar is 2 nm);

FIG. 2 is Fourier transform infrared spectra of perovskite nano-particles prepared in example 1 and perovskite-titanium dioxide nano-composite photocatalyst constructed in example 9;

FIG. 3 is an electron spin resonance diagram of a perovskite-titanium dioxide nano-composite photocatalyst constructed under the implementation example 9;

FIG. 4 is a stability test of the perovskite nanoparticles prepared in example 1 and the MT constructed in example 9 stored in an aqueous solution at 4 ℃; wherein FIG. 4A is a graph showing the change in absorbance at the maximum absorption wavelength after a certain time; FIG. 4B shows the change in photocatalytic ability;

FIG. 5 is a cytotoxicity investigation of the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst constructed under the implementation example 13;

FIG. 6 is an in vivo distribution of the biomaterial-supported perovskite-titanium dioxide nanocomposite photocatalyst constructed under the implementation example 13 in tumor-bearing mice;

FIG. 7 shows a pathological section of a tumor tissue of a mouse after the perovskite-titanium dioxide nano-composite photocatalyst prepared in example 9 and the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst constructed in example 13 are applied to tumor treatment;

FIG. 8 shows that the perovskite-titanium dioxide nano composite photocatalyst prepared in example 9 and the biomaterial-supported perovskite-titanium dioxide nano composite photocatalyst constructed in example 13 are used for detecting the biochemical indicators of blood samples of healthy mice; wherein fig. 8A is alkaline phosphatase (ALP) for evaluating liver function, fig. 8B is alanine Aminotransferase (ALT) for evaluating liver function, fig. 8C is Creatinine (CREA) for evaluating kidney function index, fig. 8D is urea nitrogen (BUN) for evaluating kidney function index, fig. 8E is free triiodothyronine (FT3) for evaluating thyroid function, and fig. 8F is free thyroxine (FT4) for evaluating thyroid function;

FIG. 9 shows the gene toxicity of mice after tumor therapy by using the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst constructed in example 13; fig. 9A is a comet experiment picture, and fig. 9B is corresponding statistical data.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are further described below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The "MA" in the invention refers to: CH (CH)₃NH₃；

The invention of "MASnX₃"means that: CH (CH)₃NH₃SnX₃Perovskite nanoparticles;

the term "MT" as used herein means: CH (CH)₃NH₃SnX₃-TiO₂Perovskite-titanium dioxide nano composite photocatalyst;

the term "HMT" as used herein means: a perovskite-titanium dioxide nano composite photocatalyst loaded by biological material hyaluronic acid;

the "OA" of the present invention means: oleic acid;

the "OAm" refers to: oleylamine;

the term "TBOT" in the present invention means: butyl titanate;

the "NIR" in the present invention means: near infrared;

the "ROS" in the invention refers to: active oxygen;

the term "Cyto-c" as used herein means: cytochrome c.

Preparation process of perovskite-titanium dioxide nano composite photocatalyst

1) Perovskite nanoparticle CH₃NH₃SnX₃(MASnX₃) Preparation of

S1-1: separately preparing SnX₂Good solvent mother liquor and CH₃NH₃Mother liquor of good solvent of X, SnX₂Good solvent mother liquor and CH₃NH₃Uniformly mixing the good solvent mother liquor of the X to obtain a mixed liquor, and adding oleic acid and preheated oleylamine into the mixed liquor to prepare a precursor liquor;

the good solvent is dimethyl sulfoxide (DMSO);

separately preparing SnX₂And CH₃NH₃And (2) uniformly mixing the DMSO mother liquor of X with the type of univalent anion of halogen shown in the table 1 to obtain a mixed solution, and adding Oleic Acid (OA) and oleylamine (OAm) preheated to be liquid at 55 ℃ into the mixed solution to prepare a precursor solution. SnX in the mixed solution described in each example₂And CH₃NH₃The X molar ratio and the volume percentages of oleic acid and oleylamine in the precursor liquid are respectively shown in Table 1;

s1-2: the precursor solution is stirred vigorously, cooled to room temperature and added dropwise to a miscible anti-solvent to obtain dispersed CH₃NH₃SnX₃Reaction solution;

the miscible anti-solvent is toluene;

the volume ratio of the precursor liquid to the toluene is 1: 15;

vigorously stirring the precursor solution obtained in S1-1 at 55 deg.C for 15min, cooling to room temperature, and dropwise adding into toluene to obtain dispersed CH₃NH₃SnX₃Reaction solution;

s1-3: sucking CH obtained from S1-2₃NH₃SnX₃Adding the reaction solution into the immiscible anti-solvent, slightly shaking and uniformly mixing, standing until the solution is layered, collecting the lower oily solution, adding a certain amount of immiscible anti-solvent again to repeat the above operation to obtain purified CH₃NH₃SnX₃Nanoparticles, i.e., perovskite nanoparticles;

the immiscible anti-solvent is n-hexane;

the CH₃NH₃SnX₃The volume ratio of the reaction liquid to n-hexane is 1: 10;

absorbing the perovskite reaction liquid obtained from S1-2 into ten times of n-hexane, slightly shaking, uniformly mixing, standing until the solution is layered, collecting the lower oily solution, adding ten times of n-hexane again, and repeating the above operation to obtain purified CH₃NH₃SnX₃And (3) nanoparticles.

TABLE 1 MASnX₃Synthetic formula of

2) Perovskite-titanium dioxide nano composite photocatalyst MASnX₃-TiO₂Preparation of (MT)

S2-1: taking CH prepared from S1₃NH₃SnX₃Dispersing in a mixed solvent of ethanol and n-hexane, adding butyl titanate and concentrated hydrochloric acid after ultrasonic treatment, stirring at normal temperature, and condensing and refluxing;

taking CH prepared in 1)₃NH₃SnX₃The nanoparticles are dispersed in a mixed solvent of ethanol and n-hexane, and the volume ratio of ethanol to n-hexane in the mixed solvent is 3: 1; after sonication for 15min butyl titanate (TBOT), CH in each example₃NH₃SnX₃The mass ratio to TBOT is as shown in table 2; concentrated hydrochloric acid, CH are dripped₃NH₃SnX₃The mass ratio of the concentrated hydrochloric acid to the concentrated hydrochloric acid is 5: 1; after stirring at room temperature for 3h, the mixture was refluxed at 45 ℃ for a certain period of time, and the reflux reaction time is shown in Table 2.

S2-2: adding n-hexane with the volume 3 times that of the reaction liquid into the reaction liquid obtained in S2-1 for purification, centrifugally collecting separated white precipitate, and removing the organic solvent by vacuum drying to obtain CH₃NH₃SnX₃-TiO₂(MT) composite powder, namely the perovskite-titanium dioxide nano composite photocatalyst.

TABLE 2 MASnX₃-TiO₂(MT) synthesis formula

3) Construction of perovskite-titanium dioxide nano composite photocatalyst loaded by biological material

Biological material and 2) prepared MASnX₃-TiO₂The Click reaction is initiated under ultraviolet light, the biological material is Hyaluronic Acid (HA), and the specific operation of the reaction is as follows:

the method comprises the following steps: the surface of the hyaluronic acid is not modified by double bonds, and the double bond modification of the hyaluronic acid is required to be carried out firstly

Weighing 0.1g of HA, dissolving in pure water, adding 800 mu L of Methacrylic Anhydride (MA), adjusting the pH value to be 8-9, and reacting for 48 hours at the temperature of 4 ℃ under stirring. After the reaction is finished, purifying by using glacial ethanol, redissolving the separated white precipitate in pure water, and dialyzing and freeze-drying to obtain double-bond modified hyaluronic acid (m-HA);

step two: synthesis of thiolated MT (MT-SH)

2mg MT was dispersed in 5mL of a mixed solvent of ethanol/water (49/1, v/v), and 30. mu.L of mercaptopropyltrimethoxysilane (MPTMS) (Mol) was added_MPTMS:Mol_MT50:1) under nitrogen protection for 5 h. Centrifuging at 16000rpm for 5min, washing with ethanol twice, precipitating, and vacuum drying for 12 hr to obtain MT-SH;

step three: conducting Click reaction

Respectively preparing 0.2mg/mL MT-SH aqueous solution, 2mg/mL 2, 2-dimethoxy-2-phenylacetophenone (DMPA) ethanol solution serving as a photoinitiator and m-HA aqueous solution, wherein the concentration of the m-HA aqueous solution is 0.2-0.4 mg/mL; sucking 500 mu L MT-SH solution, 20 mu L DMPA solution and 500 mu L m-HA solution, and mixing the biological material and MASnX₃-TiO₂The mass ratios of (a) to (b) are shown in table 3; placing the reactor under 365nm ultraviolet lamp for illumination to generate a mercaptoene Click reaction, wherein the illumination time is shown in table 3; after the reaction is finished, the reaction is dialyzed for 24 hours in a dark place to remove free DMPO and organic solvent, and the perovskite-titanium dioxide nano composite photocatalyst (HA @ MASnI) loaded by the biological material is obtained₃-TiO₂，HMT)。

TABLE 3 HA @ MASnX₃-TiO₂(HMT) Synthesis recipe

Secondly, the property research of the perovskite-titanium dioxide nano composite photocatalyst loaded by the biological material

Subsequent studies were conducted using the perovskite nanoparticles prepared in example 1, the perovskite-titanium dioxide nano-composite photocatalyst prepared in example 9, and the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst constructed in example 13.

1. Perovskite characterization

MASnI prepared in example 1 was analyzed by Transmission Electron Microscope (TEM)₃Morphology of nanoparticles and MT prepared in example 9. As a result, as shown in FIG. 1, it can be seen from A that the perovskite is uniformly distributed in a spherical shape and has a size of about 5 nm. The reason is that the long-chain oleic acid oleylamine ligand exists on the outer layer of the crystal, so that the size of the crystal is well controlled, and the nano crystal forms micelle and is in a spherical shape. The titanium dioxide is coated on the titanium dioxide, and the titanium dioxide is also uniformly distributed in a spherical shape (figure C), and the size of the titanium dioxide is about 50 nm. The MASnI prepared in example 1 was analyzed by High-resolution Transmission Electron microscope (HRTEM)₃The crystal structures of the nanoparticles (Panel B) and the MT prepared in example 9 (Panel D), HRTEM image showed clear lattice fringes, indicating MASnI₃And MT has high crystallinity.

2. Fourier transform infrared spectroscopy

For MASnI prepared in example 1₃、TiO₂The results of Fourier transform Infrared testing of MT prepared in example 9 are shown in FIG. 2, MASnI₃3424cm in spectrum^-1Is the stretching vibration peak of N-H, 1407cm^-1Is C-N stretching vibration peak, 1021cm^-1Is NH₂The results show that MASnI₃Middle CH₃NH₃ ⁺Presence of (a); 500-800 cm^-1Is TiO₂Characteristic peak of (a); 1120cm appearing in the MT peak spectrum^-1And 838cm^-1The absorption peak represents the existence of Ti-O-C bond and is combined with 500-800 cm^-1To TiO 2₂Can demonstrate MASnI in MT prepared in example 9₃With TiO₂Successful combination of (1).

MT radical generating type

The MT prepared in example 9 was triggered by 808nm light source, and electron spin resonance test was performed with lutidine N-oxide electron capture agent DMPO as electron spin capture agent to verify the kind of free radical generated by perovskite photoactive material MT under illumination. The results are shown in FIG. 3, and no peak appears in the MT spectrum without light irradiation, indicating that no free radical is generated; when MT is irradiated by laser, four intensity ratios of 1: 2: 2: 1, which is a characteristic peak of hydroxyl radicals, indicating that MT can generate a large number of hydroxyl radicals under light.

4. Stability testing of perovskite and MT

Preparation of the same MASnI₃Concentration of MASnI prepared in example 1₃And the MT aqueous solution prepared in example 9, stored standing at 4 ℃ for 7 days, samples were taken at predetermined time points to determine the absorbance at the maximum absorption wavelength. And examine MASnI after a certain period of storage₃And MT catalytic methylene blue degradation ability. ROS generated by the photocatalytic material after laser irradiation can react with methylene blue to degrade the material, so that the ability of the photocatalytic material to generate free radicals can be evaluated in vitro through a photocatalytic methylene blue degradation experiment. Preparing 2mg/mL MT water dispersion and methylene blue water solution (0.02mg/mL), stirring in dark for 30min to reach adsorption-desorption balance, and exposing to 808nm laser irradiation (2W/cm)²) Then, 200. mu.L of the reaction solution was aspirated at a predetermined time point, centrifuged, and the supernatant was measured for absorbance at 664nm using a microplate reader to examine the degradation of methylene blue. MASnI₃Maximum absorbance at absorption wavelength after storage of MT aqueous solution at 4 ℃ for a certain period of timeThe change and the change in photocatalytic ability are shown in fig. 4. Results show that within one week of storage, MASnI₃The solution is decomposed when meeting water, and the colorless absorbance of the solution is close to 0, so the solution has no function of catalyzing and degrading methylene blue. The perovskite photosensitive material MT still has the capability of catalyzing and degrading methylene blue, and the photocatalytic capability is not obviously reduced, which shows that the MT structure has good stability.

HMT cytotoxicity assay

mu.L of suspension (about 5000 cells/well) of mouse breast cancer 4T1 cells (source: Shanghai Biochemical and cell biology institute of Chinese academy of sciences) was added to each well of a 96-well plate, after incubation overnight, the culture medium was discarded and washed with PBS 2 times, the biomaterial-supported perovskite-titanium dioxide nano-composite photocatalyst HMT prepared in example 13 was diluted to 0.1, 1, 5, 10, 50, 100. mu.g/mL (in terms of MT concentration) with serum-free medium, 5 duplicate wells were set at each concentration, and 100. mu.L of drug-containing medium was added to each well. After incubation for 8h in each well, the wells were exposed to 808nm laser light (2W/cm)²) Cycles (t) were followed 1, 2, 3, 4, 5, 6, 7, 8 times. In order to avoid overheating of the culture medium after illumination, the illumination cycle is performed at an interval of 5min after each illumination of 5 min. And after incubation is continued for 24h after illumination, adding 20 mu L of MTT solution with the concentration of 5mg/mL into each hole under the condition of keeping out of the sun, continuing culturing for 4h, removing the culture medium, adding 100 mu L of DMSO into each hole, shaking until the formazan crystals are dissolved, measuring the absorbance under the wavelength of 570nm by using a multifunctional microplate reader, and calculating the cell activity according to a formula.

Cell viability/％＝(OD_sample-OD_blank)/(OD_control-OD_blank)×100％

Wherein, OD_sampleIs the absorbance, OD, of the test solution well treated with the test solution_controlIs the absorbance, OD, of control wells treated with blank medium only_blankThe absorbance of the wells was zeroed using complete medium as a blank.

As shown in FIG. 5, the cell survival rate gradually decreased with the increase of the concentration of HMT and the number of times of light irradiation, and the PDT effect of the cells was significant. The perovskite material is used as a photocatalytic material, the dose can be reduced and the effect can be improved by increasing the number of times of illumination in a certain range, and the advantages of the perovskite photocatalytic material applied to PDT are reflected.

6. Distribution in vivo

A mouse breast cancer 4T1 cell (source: Shanghai Biochemical and cell biology research institute of Chinese academy of sciences) is selected to establish an in-situ breast cancer model for a BALB/c mouse. Construction of Cy5 as a fluorescent Probe^Cy5HMT, in order to observe the distribution of HMT prepared in example 13 in mice. Intratumoral injection^Cy5Fluorescence distribution in tumor-bearing mice was observed by a small animal living body imager at 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h after HMT (with 0.9% physiological saline as a solvent and an MT dose of 0.1mg/kg, the administration dose being 50. mu.L). The results are shown in FIG. 6, intratumoral injection^Cy5Obvious fluorescence signals (in a dotted circle in fig. 6) appear at the tumor part immediately after HMT, and the fluorescence signals in the body are gradually weakened along with the time lapse, which shows that HMT can be efficiently accumulated in tumor tissues under the action of the HA nano-gel delivery carrier, and the obvious observation is approved at 72h, so that the detention of photodynamic therapy drugs in the prior art is stronger, and the long-term illumination is facilitated.

7. Pathological section of mouse tumor tissue

The mouse in-situ breast cancer model successfully constructed in '6' has the tumor volume of 100mm³The mice were randomly divided into six groups, each group being: (1) a physiological saline solution group; (2) a set of NIR illuminations; (3) an MT group; (4) MT and NIR irradiation groups; (5) a HMT group; (6) HMT and NIR irradiation groups.

The treatment modes of each group are respectively as follows:

(1) saline group (Saline): administering 50 μ L of normal saline by intratumoral injection every three days for six times;

(2) NIR illumination group (NIR): administering 50 μ L of normal saline by intratumoral injection on the first day, then performing 3 cycles of laser irradiation (each cycle is 5min interval of illumination), continuing to administer 3 cycles of NIR laser irradiation on the second day, and leaving no treatment on the third day; six times of treatment;

(3) MT group (MT): administering 50 μ L of the MT liquid medicine of example 9 by intratumoral injection every three days, taking 0.9% physiological saline as a solvent, and calculating by MT 10 mg/kg; six times of injection;

(4) MT and NIR irradiation group (MT + NIR): the first day, 50 μ L of MT liquid medicine in example 9 was administered by intratumoral injection, and 0.9% physiological saline was used as a solvent, and MT 10mg/kg was calculated; then 3 cycles of laser irradiation are carried out (each cycle is 5min of illumination and 5min of interval), 3 cycles of NIR laser irradiation are continuously given on the second day, and no treatment is carried out on the third day; the medicine is administrated for six times;

(5) HMT group (HMT): administering 50 μ L of the HMT solution of example 13 by intratumoral injection every three days, using 0.9% physiological saline as a solvent, and calculating by MT 10 mg/kg; six times of injection;

(6) HMT and NIR illumination set (HMT + NIR): the first day, 50 μ L of the MT liquid medicine of example 13 was administered by intratumoral injection, with 0.9% physiological saline as a solvent, calculated as MT 10 mg/kg; then 3 cycles of laser irradiation are carried out (each cycle is 5min of illumination and 5min of interval), 3 cycles of NIR laser irradiation are continuously given on the second day, and no treatment is carried out on the third day; the medicine is administrated for six times.

Tumors from each group of mice were prepared as H & E stained sections on day 18 from the start of the first dose, and the degree of cell damage of tumor tissues was examined, and the results are shown in fig. 7. The nuclei were stained purple with hematoxylin and the cytoplasm pink with eosin. The tumor cells in the normal saline group grow vigorously and are arranged closely, the cell nucleus is deeply colored and is irregular in shape, the cytoplasm is less, and the tumor cells have typical pathological characteristics of tumor tissues; the NIR, MT, HMT treatment group showed similar tissue morphology to the saline group with no significant tumor cell death; the MT + NIR group tumor tissues show a certain degree of tumor cell necrosis areas, the number of cell nuclei is reduced, the staining is shallow, and the cytoplasmic areas are increased; in contrast, the HMT + NIR group had the strongest tumor cell killing effect, with essentially total necrosis of tumor cells, significantly reduced number of nuclei and insignificant staining, with a pink cytoplasm in the field of view. The results again show that HMT exerts a long-acting PDT therapeutic effect under NIR triggering with excellent antitumor activity.

Biochemical indicator assay of HMT in blood samples

Healthy BALB/c mice are taken and randomly divided into three groups for carrying out a safety evaluation experiment, wherein the safety evaluation experiment comprises the following steps: (1) saline group (Saline); (2) an MT group (MT); (3) HMT group (HMT). According to GLP regulations, the drug administration period of the long-term toxicity test is 2 weeks, and the drug administration is performed 2-3 times per week. Mice were administered 50 μ L of the MT prepared in example 9 (calculated as MT 10mg/kg in 0.9% physiological saline), the HMT prepared in example 13 (calculated as MT 10mg/kg in 0.9% physiological saline) and 0.9% physiological saline every 3 days by intravenous injection for two weeks. After the administration, each group of mice was bled by removing eyeballs, centrifuged at 2000rpm for 5min, and the supernatant was collected. And performing biochemical index detection on each group of mouse serum samples, wherein the biochemical index detection comprises alkaline phosphatase (ALP) for evaluating liver function, alanine Aminotransferase (ALT), urea nitrogen (BUN) and Creatinine (CREA) for evaluating kidney function indexes, free triiodothyronine (FT3) and free thyroxine (FT4) for evaluating thyroid function, and the long-term toxicity of the perovskite in-situ nano gel system HMT on healthy mouse tissue organs is evaluated through a blood sample biochemical index. The poisoning of tin element contained in HMT mainly causes liver damage, and the absorption of iodine element on tissue protein may cause damage to various tissues and organs, especially kidney damage, and has certain damage to thyroid function after long-term administration. The results are shown in FIG. 8. The results show that the biochemical indexes of each blood sample of the MT group mice and the HMT group mice are changed in a normal range compared with the physiological saline group, which indicates that the perovskite in situ nano gel system HMT does not cause the liver, kidney and thyroid gland organ damage of the mice.

Genotoxicity Studies of HMT

The damage condition of the preparation to mouse DNA is inspected through a single cell gel electrophoresis experiment and a comet electrophoresis experiment, and the genotoxicity of the preparation is evaluated. The hyaluronic acid-supported perovskite-titanium dioxide nano-composite photocatalyst HMT prepared in example 13 (50 μ L was administered by intravenous injection in terms of MT 10mg/kg in 0.9% physiological saline) and normal saline (50 μ L was administered by intravenous injection) were administered to healthy mice for 2 weeks 2-3 times per week. After the administration period, the peripheral blood lymphocytes of each group of mice are separated to carry out gel electrophoresis experiments. After being embedded in agarose gel, peripheral blood lymphocytes of mice are cracked and uncoiled, broken DNA fragments of damaged cells carry negative charges, migrate or extend to an anode under the action of electrophoresis, form a tail and form a comet shape. The cell tailing condition is observed and photographed under an inverted fluorescence microscope as soon as possible, four indexes of tail length, tail DNA content percentage, tail moment and Olive tail moment of each group of cells are analyzed by CASP image analysis software, the damage condition of the perovskite in-situ nano hydrogel system HMT to the mouse DNA is evaluated, and the result is shown in figure 9. The comet experimental picture of the A picture shows that the cell forms of the normal saline group and the HMT group are oval bright spheres without obvious head and tail, and the tail length, the tail DNA content, the tail moment and the Olive tail moment of the HMT group cell are changed in a normal range by combining the statistical data of the B picture, so that the comet experimental picture has no significant difference compared with the normal saline group. The results show that the perovskite in-situ nano hydrogel system HMT has no obvious DNA damage to mouse peripheral blood lymphocytes, and the HMT has no genotoxicity.