CN106970059B

CN106970059B - Preparation and application of two-photon fluorescent probe

Info

Publication number: CN106970059B
Application number: CN201710313248.7A
Authority: CN
Inventors: 刘艳颜; 孟宪福; 李伟; 步文博
Original assignee: Shanghai University of Medicine and Health Sciences
Current assignee: Shanghai University of Medicine and Health Sciences
Priority date: 2017-05-05
Filing date: 2017-05-05
Publication date: 2020-06-16
Anticipated expiration: 2037-05-05
Also published as: CN106970059A

Abstract

The invention belongs to the field of nano biomedical diagnosis and treatment, and mainly relates to preparation of an organic/inorganic composite nano material system with good biocompatibility and application of the organic/inorganic composite nano material system in biological two-photon imaging and two-photon sensitization dynamic treatment. The invention adopts the supermolecule self-assembly drive of the metal porphin complex to construct the functionalized metal-organic framework material ZnTPyP-MOF, and the physical and chemical properties of the original assembly molecule ZnTPyP are kept and simultaneously new optical and structural properties are endowed. The MOF is used as an inner core to grow shell titanium oxide, and then a layer of polylysine is coated, so that the high-performance two-photon diagnosis and treatment type nano biological probe is obtained. The nanoprobe has good biological safety and dispersibility, and ZnTPyP-MOF has good two-photon absorption and fluorescence emission performances, so that sensitization kinetic treatment under two-photon activation is realized; the invention can be widely used for in-situ fluorescence diagnosis and treatment of various diseases, particularly cancers, and has very important significance in clinical medicine.

Description

Preparation and application of two-photon fluorescent probe

Technical Field

The invention belongs to the technical field of nano biomedical imaging, and mainly relates to preparation of a high-efficiency two-photon fluorescent probe with good biocompatibility and application of the high-efficiency two-photon fluorescent probe in two-photon sensitization kinetic treatment.

Background

Porphin and metalloporphin are structural skeletons of various porphyrin tetrapyrrole compounds such as naturally occurring chlorophyll and heme, and are vital substances existing in life. Porphine-like molecules generally have good biocompatibility and at the same time play an important role in the photoelectric conversion process. As a family member of macrocyclic organic molecules, porphin molecules can be used as basic molecules, and through pi-pi accumulation, hydrogen bonds, van der Waals force and the like, the self-assembly among supermolecules is realized, and nanocrystals with various morphologies can be obtained. From basic molecules to assembled nanocrystals, the photoelectric properties of the material change, and new properties are developed, so that the specific applications of porphin molecules are widened.

Zinc

5,10,15, 20-tetra (4-pyridyl) -21H, 23H and porphine (ZnTPyP) are used as good photo-activated molecular matrixes, can be used for assembling and constructing micron/nano metal-organic framework materials (ZnTPyP-MOF) with different shapes and sizes, and are used for carrying out various photoelectric reactions. In fact, photo-generated electrons are critical to the progress of the reaction, whether it be a photocatalytic reaction or a photodynamic therapeutic process. However, few studies focus on the effective generation and utilization of electrons of the assembly material, and the abundant photoelectric properties of ZnTPyP-MOF are not fully exploited.

The multiphoton absorption process refers to the process in which a material absorbs multiple photons simultaneously under the action of intense laser light to activate electrons. Compared with the traditional single photon process, the long-wavelength multi-photon laser has higher tissue penetration depth, and the two-photon absorption is mainly generated at the focus of the super-strong laser generated by the pulse laser, so the two-photon process has good space selectivity, has small damage to a sample, can obtain a higher-resolution imaging effect, and realizes the optical diagnosis and treatment of in-situ diseases. With the development of research, the potential of organic dyes (FITC, DAPI, diiodoeosine) and inorganic nanomaterials (gold nanorods, carbon quantum dots, ZnS) in multi-photon absorption is gradually revealed. Organic two-photon absorption dyes generally have higher quantum yield, but the water delivery property, cytotoxicity, easy fluorescence quenching in an in vivo extreme environment and the like of dye molecules limit the application of the dyes in organisms. The inorganic nonlinear two-photon absorption nano material generally has a stable chemical structure and is easy to modify on the surface, and is relatively suitable for biological research. However, the optical properties of inorganic materials are generally relatively single, which is very limited for practical research. ZnTPyP-MOF coated co-catalyst TiO₂Obtaining ZnTPyP-MOF @ TiO₂(ZMT) that efficiently combines TiO while retaining ZnTPyP-MOF two-photon absorption and high quantum fluorescence yield₂The inorganic material is easy to modify and has excellent catalytic performance, and finally the two-photon absorption characteristic of ZnTPyP-MOF is utilized to realize high-resolution two-photon fluorescence imaging and two-photon sensitization of TiO to cells₂And (4) dynamic treatment.

In summary, it is important to prepare a suitable organic/inorganic complex to construct a ZMT that can fully exert the advantages of each organism and has good biocompatibility, which is suitable for two-photon driven fluorescence imaging and TiO imaging₂The sensitization kinetics has great significance.

Disclosure of Invention

The invention aims to synthesize an organic/inorganic heterogeneous nanocrystal which has good biocompatibility and high-efficiency two-photon fluorescence and two-photon sensitization dynamic therapeutic performance.

In order to solve the technical problems, the invention is realized by the following technical scheme:

a novel high-efficiency organic/inorganic heterogeneous nano-crystal with two-photon fluorescence and two-photon sensitization kinetic therapeutic performance is based on ZnTPyP-MOF assembled particles, and the two-photon absorption and dye activation performance are provided by the organic/inorganic heterogeneous nano-crystal. TiO 2₂The coating can ensure the hydrophilicity and the biological safety of the whole particle, and in addition, the combined action of the ZnTPyP-MOF inner core leads the inner core excited electrons to be led into the outer TiO layer₂Conduction band, to enhance electronic activity, synergistically contributes to the generation of active oxygen.

Firstly, adopting an acid-base neutralization method to assemble and prepare ZnTPyP-MOF nanocrystals in surfactant micelles; then, through titanium source hydrolysis reaction, uniformly coating a layer of amorphous TiO with good water solubility and high catalytic performance on the surface of ZnTPyP-MOF₂(ii) a Finally, the mixture is adsorbed on TiO through the action of static electricity₂The outer layer is coated with Polylysine (PLL) with good biocompatibility, and finally the organic/inorganic heterogeneous nanocrystal with good biological safety is obtained.

The process for preparing the metal-organic framework nano material by adopting the molecular self-assembly method comprises the following specific steps: ZnTPyP is dissolved in 2 millimole/liter (mM) hydrochloric acid solution, and ZnTPyP molecules are protonated to obtain ZnTPyP-H₄ ⁴⁺Adding a mixed solution of NaOH and CTAB, ZnTPyP-H₄ ⁴⁺Deprotonating the ZnTPyP by meeting alkali, enabling the water-conveying ZnTPyP to enter a CTAB micelle, and self-assembling to obtain the ZnTPyP-MOF nanocrystal through the Zn-N bonding effect. And (4) cleaning with ethanol for 2-3 times, and removing the nanocrystalline surfactant.

The ZnTPyP meets H⁺Protonation, adjusting ZnTPyP and H⁺Molar ratio sufficient to protonate ZnTPyP to ZnTPyP-H₄ ⁴⁺To obtain completely dissolved ZnTPyP-H₄ ⁴⁺And (3) precursor.

The ZnTPyP-H₄ ⁴⁺Adding NaOH solution of CTAB, stirring the system for about 5 minutes, ZnTPyP-H₄ ⁴⁺With OH^-After sufficient neutralization, the assembly process is complete and the product is collected by centrifugation immediately.

The pH of the reaction assembly solution, the concentration of CTAB and the assembly time can obviously influence the appearance and the size of a product and need to be strictly controlled.

And repeatedly cleaning the assembled product ZnTPyP-MOF by using ethanol, removing the surfactant, centrifugally cleaning, and dispersing in the ethanol for later use.

A titanium source hydrolysis process with the assistance of CTAB is adopted, and a layer of amorphous TiO grows on the outer surface of ZnTPyP-MOF nano particles₂The method comprises the following steps: ZnTPyP-MOF is dissolved and dispersed in NaOH aqueous solution with 0.2mM CTAB; then, diluting diisopropoxybialkyl diacetone Titanium (TDAA) and methanol according to the volume ratio of 1:100, adding a small amount of the diisopropoxybialkyl diacetone Titanium (TDAA) and methanol into the ZnTPyP-MOF solution batch by batch, and slowly stirring the mixture to ensure that the TDAA slowly hydrolyzes and grows TiO on the surface of the ZnTPyP-MOF₂And cleaning with ethanol for 2-3 times, and removing the nanocrystalline surfactant.

The CTAB assisted growth synthesized ZnTPyP-MOF @ TiO₂(abbreviated as ZMT) particles have good water solubility and dispersibility.

TiO in the ZMT particles₂Successful coating is achieved in a solution with CTAB concentration of about 0.2mmol/L and pH of about 10, and CTAB with too high a concentration will hinder TiO₂Too low a concentration of CTAB leads to soft aggregation of the ZnTPyP-MOF core, affecting the morphology of the final product.

And repeatedly cleaning the assembled product ZMT by using ethanol, removing the surfactant, centrifugally cleaning, and dispersing in water for later use.

A layer of PLL with good biocompatibility is modified on the outer surface of the ZMT nano-particles in an electrostatic adsorption mode. The method comprises the following steps: the aqueous solution of ZMT was added to the PBS solution of PLL, stirred slowly for 20 minutes, and the PLL-modified nanoparticles were collected by water washing.

The PLL solution system is formed by mixing PLL and NaCl in a PBS buffer solution, and the temperature is kept at 4 ℃.

And after the ZMT particles are added into a PLL system, slowly stirring for 20 minutes, and washing for 2-3 times to obtain ZMT @ PLL with uniformly-coated PLL and good dispersibility.

The invention has the beneficial effects that:

(1) the organic porphin molecule assembled inner core ZnTPyP-MOF can absorb near infrared two-photon laser and emit fluorescence, has low phototoxicity, and mainly generates a small amount of singlet oxygen under the action of excited electrons and oxygen.

(2) The surface of the invention is coated with TiO₂Then, electrons generated under the two-photon excitation of ZnTPyP-MOF can be efficiently injected into surface TiO₂The compound is used for enhancing electronic activity, so that ambient oxygen is reduced to be superoxide anion, cytotoxicity is enhanced rapidly, and double-photosensitization dynamic treatment of deep tissue in-situ tumors can be realized by the synergy of the compound and the superoxide anion.

(3) The invention is widely used for the fluorescence diagnosis and photodynamic therapy of various diseases, particularly cancers, and has very important significance in clinical application.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) and a Scanning Electron Microscope (SEM) photograph of nanoparticles prepared in example 1;

FIG. 2 is a graph comparing the hydration kinetic sizes (DLS) of ZnTPyP-MOF and ZMT nanoparticles prepared in example 1 before and after PLL modification;

FIG. 3 is an absorption spectrum of ZnTPyP monomer molecules, ZnTPyP-MOF and ZMT nanoparticles prepared in example 1.

FIG. 4 is a graph of the results of active oxygen detection of the ZnTPyP-MOF and ZMT nanoparticles prepared in example 1 under 500nm visible light illumination.

FIG. 5 is a graph showing the results of the "scavenger test" of active oxygen species for the ZnTPyP-MOF and ZMT nanoparticles prepared in example 1.

FIG. 6 is a graph of the results of active oxygen detection of the ZnTPyP-MOF and ZMT nanoparticles prepared in example 1 under 808nm laser irradiation.

FIG. 7 shows single-photon and two-photon laser fluorescence spectra of ZnTPyP, ZnTPyP-MOF, ZMT prepared in example 1.

FIG. 8 is a fluorescence plot of the ZnTPyP-MOF and ZMT nanoparticles prepared in example 1 under 830nm two-photon excitation.

FIG. 9 is a linear plot of the square of the different dual-light intensities versus the intensity of the emitted fluorescence for the ZMT nanoparticles prepared in example 1.

FIG. 10 is the two-photon fluorescence of ZMT nanoparticles prepared in example 1 at different levels within the cell.

FIG. 11 is a graph of changes in reactive oxygen species of cells under 830nm two-photon irradiation in cells monitored by the simultaneous light stimulation method using the ZnTPyP-MOF and ZMT nanoparticles prepared in example 1.

FIG. 12 shows ZnTPyP-MOF and ZMT nanoparticles and TiO only from example 1₂Influence on cell viability at different ion concentrations (MTT method).

FIG. 13 shows ZnTPyP-MOF and ZMT nanoparticles and TiO only prepared in example 1₂Cell viability after irradiation with 830nm two-photon femtosecond laser (MTT method) at the same ion concentration.

Detailed Description

The invention will now be further illustrated by reference to the following examples:

FIG. 1a is a flow chart showing the detailed preparation of PLL-coated ZMT

Firstly, preparation of ZnTPyP-MOF hydrophobic nanoparticles:

1.ZnTPyP-H₄ ⁴⁺preparation of (2): ZnTPyP is dissolved in 0.2mol/L hydrochloric acid solution to obtain 0.01mol/L ZnTPyP solution.

Preparation of CTAB micellar solution: CTAB was dissolved in a 10mmol/L NaOH solution to give a 2mmol/L CTAB solution.

3. Measuring 0.5mL of ZnTPyP solution, quickly adding 9.5mL of CTAB solution, slowly stirring for 5 minutes, adding alcohol, rotating at 10000 rpm, quickly centrifuging and collecting a product.

FIGS. 1b-c are SEM images of ZnTPyP-MOF nanocrystals prepared in this example, in which: the ZnTPyP-MOF is a hexagonal nanodisk.

FIG. 1d is EDS chart of element distribution of ZnTPyP-MOF nanocrystals prepared in this example, in which: the elements carbon and zinc are present significantly and the elements titanium and oxygen are barely detectable.

FIG. 1h is a TEM image of the distribution of elements of ZnTPyP-MOF nanocrystals prepared in this example, wherein: the size of the ZnTPyP-MOF is about 300 nm.

Bis, ZnTPyP-MOF @ TiO₂Preparation of hydrophilic nanoparticles:

preparation of TDAA methanol solution: 10. mu.L of LTDAA was measured and dissolved in 500. mu.L of methanol solution, and stored at room temperature in the dark.

Preparation of CTAB alkaline solution: firstly, NaOH solution with pH of 11.0 is prepared, CTAB is dissolved in the alkaline solution, and CTAB reaction solution with concentration of 0.2mmol/L is prepared.

3.5 micromoles of ZnTPyP-MOF (calculated as ZnTPyP) was dissolved in 8mL of CTAB alkaline solution, 25. mu. of LTDAA methanol solution was added and reacted for 30 minutes, 25. mu. of LTDAA methanol solution was added and the reaction was continued for 2 hours, and the product was collected by centrifugation.

FIGS. 1c-f are SEM images of ZMT nanoparticles prepared in this example, wherein: the nanoparticles are well dispersed and do not nucleate homogeneously.

FIG. 1g is an EDS plot of the element distribution of ZMT nanoparticles prepared in this example, in which: carbon, zinc, titanium and oxygen are present significantly.

FIGS. 1i-j are TEM images of ZMT nanoparticles prepared in this example, in which: the outer layer of the nano particles is evenly coated with TiO₂And no homogeneous nucleation.

Thirdly, PLL modification of the nanoparticles:

preparation of PLL System: NaCl was dissolved in a PBS solution at pH 7.4, and PLL was dissolved in the PBS to prepare a PLL solution having a concentration of 1 mg/mL.

ZnTPyP-MOF or ZMT was dissolved in 200. mu.L of water or ethanol, respectively, 1mL of PLL system was added quickly, stirred slowly for 20 minutes, washed by centrifugation and the final product was dispersed in water.

Fig. 2a, b are DLS spectra of ZnTPyP-MOF nanoparticles prepared in this example before and after PLL modification, in which: the ZnTPyP-MOF nano-particles are well dispersed after PLL modification, and the particle sizes tend to be consistent.

FIG. 2c, d is a DLS spectrum of ZMT nanoparticles prepared in this example before and after PLL modification, in which: the ZMT nanoparticles are well dispersed after PLL modification, and the particle size range is more concentrated.

Fourthly, detecting active oxygen in the solution:

the prepared ZnTPyP-MOF and ZMT can generate active oxygen under the excitation of visible light, and the existence of titanium oxide can obviously enhance the activation electrons of the ZnTPyP-MOF and reduce oxygen to generate superoxide anions, thereby providing conditions for the application of the ZMT in the biological photodynamic therapy.

FIG. 3 is an absorption spectrum of ZnTPyP-MOF and ZMT prepared in this example and ZnTPyP molecules as an assembly matrix, wherein: the absorption of the assembled nanocrystalline in a visible light region is obviously enhanced.

FIG. 4 shows ZnTPyP-MOF, ZMT and TiO prepared in this example₂And generating a detection spectrum of active oxygen under the irradiation of 500nm visible light. 1, 3-diphenyl isobenzofuran (DPBF) is an active oxygen (superoxide anion and singlet oxygen) capture probe, and is used for judging the generation of active oxygen by monitoring the reduction of the absorption peak intensity at 410nm of DPBF, wherein: under the same illumination condition, the yield of ZMT active oxygen is obviously higher than that of ZnTPyP-MOF and TiO₂Results of the individual action.

FIG. 5 shows the results of the experiments for detecting specific components of active oxygen in this example. The p-benzoquinone is taken as a superoxide anion trapping agent, and the influence of superoxide anions on a DPBF absorption peak can be effectively eliminated by adding a DPBF detection solution of active oxygen, wherein in the figure: under the same illumination condition, the addition of p-benzoquinone leads the active oxygen yield of the ZMT group to be similar to that of ZnTPyP-MOF and TiO₂There was no significant difference when acting alone. The reaction products of the ZnTPyP-MOF and the ZMT combined with oxygen are respectively singlet oxygen and superoxide anion under the same illumination, which fully indicates that TiO₂As a co-catalyst, the photocatalytic activity of ZnTPyP-MOF can be obviously enhanced.

FIG. 6 shows ZnTPyP-MOF, ZMT and TiO molecules of this example₂And generating a detection spectrogram of active oxygen under the irradiation of 830nm near-infrared single-photon laser. 1, 3-diphenyl isobenzofuran (DPBF) is used as an active oxygen (superoxide anion and singlet oxygen) capture probe, and the decrease of the absorption peak intensity at 410nm of the DPBF is monitored to judge the production of active oxygenRaw, in the figure: under the same illumination condition, the yield of ZMT active oxygen is equal to that of ZnTPyP-MOF and TiO₂There was no significant difference in the effect alone.

And fifthly, detecting two-photon fluorescence imaging:

the prepared ZnTPyP-MOF can absorb a plurality of photons and emit strong fluorescence of about 650nm under the irradiation of 830nm two-photon femtosecond laser, which provides conditions for the application of the ZnTPyP assembly in the aspect of biological two-photon fluorescence imaging.

FIG. 7 is a fluorescence spectrum of the ZnTPyP monomer molecule of the present embodiment, ZnTPyP-MOF, under 415nm ultraviolet light and 830nm two-photon laser irradiation, in which: under the irradiation of different exciting lights, the emission fluorescence of the ZnTPyP monomer molecules and the ZnTPyP-MOF are basically consistent, which shows that the ZnTPyP-MOF fully retains the fluorescence excitation and emission properties of the ZnTPyP monomer molecules.

FIG. 8 is a fluorescence emission diagram of the ZnTPyP monomer molecule, ZnTPyP-MOF, under 830nm two-photon laser irradiation in the embodiment, wherein: under the excitation of the same two-photon, the emission fluorescent signals of the ZnTPyP monomer molecule and the ZnTPyP-MOF are basically consistent.

FIG. 9 is a graph of the emission fluorescence intensity of ZnTPyP-MOF of this example under different two-photon excitation, in which: the square of the two-photon laser intensity and the emission fluorescence signal of ZnTPyP-MOF are in a linear relation, and the two-photon fluorescence characteristic of the ZnTPyP-MOF is proved.

FIG. 10 is a graph showing fluorescence emission of materials at different cell levels under two-photon excitation after co-culturing ZnTPyP-MOF @ PLL with human cervical cancer cells (HeLa) in this example, wherein: the Z-axis height is changed, the fluorescence information of the material on different focal planes is collected, and the result proves that ZnTPyP-MOF is phagocytosed by cells to reach the cytoplasm instead of being attached to the cell surface.

Sixthly, detecting active oxygen in the cells:

the prepared ZnTPyP-MOF and ZMT can obviously absorb near-infrared two-photons, emit fluorescence and combine TiO₂Can generate a large amount of active oxygen to kill tumor cells.

FIG. 11 shows ZnTPyP-MOF, ZMT and TiO molecules of this example₂And detecting the active oxygen generated in the cell under the irradiation of a 830nm two-photon femtosecond laser. Use of activitiesThe change of the intensity of the fluorescent dye of the sex oxygen kit monitors the change of the yield of active oxygen in cells under 830nm two-photon stimulation, and in the figure: in a synchronous light stimulation experiment, the active oxygen yield of the cells after ZMT pretreatment is obviously higher than that of ZnTPyP-MOF and TiO₂Results in the culture alone.

ZnTPyP-MOF and TiO are verified on a cellular level through an active oxygen detection probe₂Under the combined action, the generation efficiency of active oxygen is greatly improved, the cytotoxicity is greatly enhanced, and the fact that ZnTPyP-MOF can absorb 830nm two-photon is verified, so that the ZMT photosensitization kinetic therapy under the two-photon excitation is realized.

Seventhly, cell survival rate experiment:

prepared ZnTPyP-MOF, ZMT and TiO₂Cell viability was measured by standard MTT assay under various treatment conditions, in coculture with cells at various concentrations. And (3) detecting the survival condition of the cells pretreated by different materials by combining 830nm two-photon laser stimulation.

FIG. 12 shows ZnTPyP-MOF, ZMT and TiO molecules of this example₂The cytotoxicity test results after co-culture with cells. In the figure: at a suitable ion concentration, ZnTPyP-MOF, ZMT and TiO₂Has no obvious cytotoxicity after being co-cultured with cells.

FIG. 13 shows ZnTPyP-MOF, ZMT and TiO molecules of this example₂Cell viability after irradiation with 830nm two-photon femtosecond laser. In the figure: in the same light stimulation experiment, the cell lethality of the ZMT pretreated cells and the ZMT cells is obviously higher than that of ZnTPyP-MOF and TiO₂Results under the sole action.

In conclusion, compared with the existing two-photon fluorescence and sensitization kinetic diagnosis and treatment probe, the metalloporphin supramolecular assembly provided by the invention can fully play roles of inner core ZnTPyP-MOF and shell TiO while ensuring biocompatibility₂The respective advantages of (a). The ZnTPyP-MOF can emit stronger fluorescence under the excitation of near-infrared two-photon, and has the following remarkable advantages: (1) the near-infrared light with long wavelength is less influenced by scattering than the short wavelength light, and is easy to penetrate through the specimen; (2) the exciting light can reach the focal plane, fluorescent molecules outside the focal plane are not excited, and the image resolution is high; (3) near infrared at long waveLight is less toxic to cells than light of shorter wavelengths. ZnTPyP-MOF @ TiO₂After the medium ZnTPyP-MOF absorbs two photons, the dye molecules can be activated, and the activated electrons are injected into TiO₂The electron activity is obviously enhanced, and the reduced electrons are superoxide anions which are used for killing cancer cells, so that the method has important significance for accurate positioning and optical treatment of clinical in-situ diseases and even tumors.

The above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A two-photon sensitization kinetic organic/inorganic heterogeneous nanocrystal, characterized in that: the high-efficiency photodynamic active oxygen generation performance comes from ZnTPyP-MOF and TiO₂The combined action of (A) and (B);

the preparation method of the ZnTPyP-MOF comprises the following steps: ZnTPyP is dissolved in 0.2mol/L hydrochloric acid solution to obtain 0.01mol/L hydrochloric acid solution of ZnTPyP, ZnTPyP molecules are protonated to obtain ZnTPyP-H₄ ⁴⁺Adding a mixed solution of NaOH and CTAB, ZnTPyP-H₄ ⁴⁺Deprotonating the ZnTPyP by meeting alkali, allowing the water-conveying ZnTPyP to enter a CTAB micelle, and self-assembling to obtain ZnTPyP-MOF nanocrystals through Zn-N bonding;

the ZnTPyP-MOF and TiO₂The combined action steps comprise the following steps: ZnTPyP-MOF is dissolved and dispersed in a mixed solution of NaOH and CTAB; then, diluting diisopropoxybialkyl diacetone Titanium (TDAA) and methanol according to the volume ratio of 1:100, adding a small amount of the diisopropoxybialkyl diacetone Titanium (TDAA) and methanol into the ZnTPyP-MOF solution batch by batch, and slowly stirring the mixture to ensure that the TDAA slowly hydrolyzes and grows TiO on the surface of the ZnTPyP-MOF₂；

The preparation method of the mixed solution of NaOH and CTAB comprises the following steps: CTAB was dissolved in a 10mmol/L NaOH solution to give a 2mmol/L CTAB solution.

2. A method for preparing the two-photon sensitization kinetic organic/inorganic heterogeneous nanocrystal, which is characterized by comprising the following steps: reacting ZnTPyP-MOF withMixing and stirring an alkaline hexadecyl trimethyl ammonium bromide solution, adding a methanol diluted solution of diisopropoxyl bis (acetylacetone) titanium in batches in times, continuously stirring at 280-330 rpm for 4-6 minutes, immediately centrifuging after the assembly is finished, stopping the assembly process to obtain amorphous TiO₂Uniformly coated ZnTPyP-MOF @ TiO₂ZMT for short, and finally uniformly coating a layer of polylysine with good biocompatibility and high water solubility on the surface of the ZnTPyP-MOF nanocrystal in an electrostatic adsorption mode.

3. The method for preparing two-photon sensitization kinetic organic/inorganic heterogeneous nano-crystalline according to claim 2, characterized in that: and (3) diluting the diisopropoxybialkyl diacetone titanium and methanol in a volume ratio of 1:100, adding an alkaline hydrolysis reaction solution, slowly hydrolyzing the TDAA, and uniformly adhering and growing on the surface of ZnTPyP-MOF.