CN113995838B

CN113995838B - Glutathione response photoacoustic probe and preparation method and application thereof

Info

Publication number: CN113995838B
Application number: CN202111317626.1A
Authority: CN
Inventors: 马骋; 梁晓龙; 陈超毅; 张路路
Original assignee: Tsinghua University; Peking University Third Hospital
Current assignee: Tsinghua University; Peking University Third Hospital
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2022-07-26
Anticipated expiration: 2041-11-09
Also published as: CN113995838A

Abstract

The invention provides a photoacoustic nano molecular probe for generating Prussian blue through glutathione response and a preparation method and application thereof. The nano molecular probe provided by the invention comprises a surface modification and an inner core, wherein the inner core comprises potassium ferrocyanide molecules and ferric acetylacetonate molecules; the surface modification is distearoyl phosphatidyl ethanolamine-polyethylene glycol molecules and distearoyl phosphatidyl choline molecules. The nano-particle provided by the invention can realize response type photoacoustic signal enhancement and living body glutathione detection and tumor in-situ imaging under the glutathione environment.

Description

Glutathione response photoacoustic probe and preparation method and application thereof

Technical Field

The invention relates to a photoacoustic imaging technology in the technical field of biomedicine, in particular to a photoacoustic nano molecular probe for producing prussian blue through response of glutathione and a preparation method and application thereof.

Background

Glutathione is the most abundant endogenous active small molecule in cells and tissues, playing an important role in vital activities and maintaining redox balance. Many diseases, including liver and skin diseases, cardiovascular diseases, cancer, lead to abnormal glutathione metabolism. Glutathione is produced by solid tumor cells to reduce abnormal active oxygen, and the concentration of glutathione in tumor cells is 2-20 millimole/liter, which is 1000 times that of normal cells. Therefore, visual detection and quantification of glutathione are of great significance for the diagnosis of early stage tumors and other glutathione-related diseases. Currently, the main method for detecting cellular glutathione is to use fluorescent probes based on glutathione-responsive organic fluorophores. However, due to factors such as shallow tissue penetration depth, strong light scattering and biological background, the application of fluorescence imaging in clinical and preclinical living tissue glutathione detection is greatly limited.

The photoacoustic imaging is a biological imaging technology which can be applied to clinical and preclinical researches, has optical contrast and ultrasonic penetration depth, expands structural images and functional images based on different chromophores to acoustic imaging depth, breaks through optical scattering limit, can detect spectral information of deep biological tissues, and has the advantages of non-invasion, no radiation, high imaging speed, low cost and the like. Conventional photoacoustic imaging methods achieve imaging of deep biological tissues by the contrast of light absorption (e.g., hemoglobin, melanin, fat, etc.) generated by the endogenous photoacoustic chromophores in the organism. Therefore, the photoacoustic imaging based on the binding specificity response type probe has wide application prospect in the detection of deep tissue tumor in clinic and the preclinical research.

The prior art discloses that nanoparticle reagents based on molybdenum-based polyoxometallate or boron fluoride complexed dipyrromethene fluorochromes can be used for photoacoustic imaging of glutathione. However, the above techniques still suffer from poor biocompatibility, complicated preparation process, and the like.

In conclusion, the development of a photoacoustic probe with glutathione response, good biocompatibility and a simple preparation process for living glutathione detection and tumor in-situ imaging is an urgent technical problem to be solved in the field.

Disclosure of Invention

In order to solve the problems, the inventor designs a photoacoustic nano molecular probe for generating Prussian blue in response to glutathione, and provides a preparation method and application. The photoacoustic nano molecular probe for generating Prussian blue through response of glutathione provided by the invention has the advantages that the Prussian blue is generated through response of glutathione in an organism, the enhancement of glutathione response type photoacoustic signals is realized, and meanwhile, the in-situ imaging of tumors is realized.

In one aspect, the application provides a photoacoustic nano molecular probe for producing prussian blue through response of glutathione, which is characterized in that the nano molecular probe comprises a surface modification and an inner core, wherein the inner core comprises potassium ferrocyanide molecules and ferric acetylacetonate molecules; the surface modification is distearoyl phosphatidyl ethanolamine-polyethylene glycol molecules and distearoyl phosphatidyl choline molecules.

Further, the mass ratio of the molecules of potassium ferrocyanide, ferric acetylacetonate, distearoyl phosphatidyl ethanolamine-polyethylene glycol and distearoyl phosphatidyl choline is 2.79: 1.51: 1: 2.54; the respective molar ratio was 18.5:12:1: 9.

Further, the nano-particles have an average particle size of 58 nm.

Further, under the action of glutathione, the nano molecular probe can react to generate Prussian blue, and a near-infrared photoacoustic signal is provided.

Further, the lowest response concentration of glutathione of the nano molecular probe is 0.3 millimole per liter.

Further, the prussian blue generated after the response of the nano molecular probe glutathione has an absorption peak at 700 nm.

In another aspect, the present application provides a method for preparing the above nanomolecular probe, which comprises the following steps:

step 1, adding potassium ferrocyanide into distilled water to obtain a transparent liquid A with the concentration of 30 mmol per ml;

step 2, adding ferric acetylacetonate, distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylcholine into absolute ethyl alcohol, and carrying out ultrasonic treatment for 5-15 minutes to obtain a B solution with the concentration of 200 millimoles per milliliter, wherein the molar ratio of the ferric acetylacetonate to the distearoylphosphatidylethanolamine-polyethylene glycol to the distearoylphosphatidylcholine is 90: 1: 9;

step 3, adding the solution B into the solution A under an ultrasonic condition, and carrying out ultrasonic treatment for 30 minutes to obtain a turbid solution C, wherein the volume ratio of the solution A to the solution B is 9: 1;

step 4, transferring the solution C into a plastic tube, and carrying out ultrasonic treatment for 5 minutes by using a probe with 10% output under the ice bath condition;

and 5, transferring the solution C subjected to the ultrasonic treatment of the probe into a dialysis bag, and dialyzing the solution C in ultrapure water for 2 hours to obtain the photoacoustic nano molecular probe which has good water dispersibility and can generate Prussian blue in response to glutathione.

Further, in the synthesis process of the photoacoustic nano molecular probe capable of generating Prussian blue in response to glutathione, the molar ratio of potassium ferrocyanide, ferric acetylacetonate, distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylcholine molecules is 135: 90: 1: 9; the corresponding mass ratio was 20:11:1: 2.54.

On the other hand, the application provides the application of the nano molecular probe or the nano molecular probe prepared by the method in preparing a tumor in-situ imaging reagent.

In another aspect, the present application provides an application of the above-mentioned nano-molecular probe or the nano-molecular probe prepared according to the above-mentioned method in preparing a tumor photothermal therapy preparation.

Has the beneficial effects that:

the beneficial effects of the invention are:

the invention carries out entrapment on potassium ferrocyanide and ferric acetylacetonate through distearoyl phosphatidyl ethanolamine-polyethylene glycol and distearoyl phosphatidyl choline molecules, realizes hydrophilic modification, improves the biocompatibility, effectively prolongs the circulation time in vivo and enhances the tumor enrichment effect.

Under the action of glutathione, ferric acetylacetonate reacts with glutathione, and ferric iron complexed in the ferric acetylacetonate dissociates to release ferric ions. The released ferric ions can react with the ferrous cyanide ions carried in the photoacoustic nano molecular probe to generate Prussian blue nano particles. The produced Prussian blue nano particles have strong light absorption in a near infrared region, can be used as a photoacoustic imaging contrast agent, and kill tumor cells under a photothermal condition to treat tumors.

The invention provides a method for synthesizing a photoacoustic nano-molecular probe capable of generating Prussian blue through response of glutathione, Prussian blue nano-particles generated by the synthesized photoacoustic nano-molecular probe under the action of the glutathione can be used as a photoacoustic imaging contrast agent, and the process depends on the characteristic of high expression of the glutathione in a tumor microenvironment, so that specific recognition of tumors is realized, and a new strategy for tumor diagnosis is provided.

Drawings

FIG. 1 is an electron microscope image of a photoacoustic nano molecular probe for generating Prussian blue in response to glutathione obtained in example 1;

FIG. 2 is a particle size distribution diagram and an element content diagram of the photoacoustic nano-molecular probe for generating Prussian blue in response to the glutathione obtained in example 1;

FIG. 3 is a graph of the absorption spectrum and the ratio of absorbance at 700 nm and 990 nm of the photo-acoustic nano-molecular probe for generating Prussian blue in response to glutathione obtained in example 1 after co-incubation with different concentrations of glutathione;

FIG. 4 is the ratio of absorbance at 700 nm and 990 nm measured after co-incubation with glutathione or a common amino acid solution of the glutathione-responsive Prussian blue-producing photoacoustic nanomolecular probe obtained in example 1;

FIG. 5 is the ratio of photoacoustic images at 700 nm and 990 nm and photoacoustic intensities at 700 nm and 990 nm of the glutathione-responsive Prussian blue-producing photoacoustic nanomolecular probe obtained in example 1 after co-incubation with different concentrations of glutathione;

fig. 6 is a graph showing the ratio of photoacoustic intensities at 700 nm and 990 nm of a tumor region in photoacoustic images at 700 nm and 990 nm of different time periods after injecting the glutathione solution or phosphate buffer solution for prussian blue obtained in example 1 into a 4T1 tumor by intratumoral injection and in photoacoustic images at 700 nm and 990 nm of different time periods after injecting the glutathione solution or phosphate buffer solution for prussian blue obtained in example 1 into a 4T1 tumor by intratumoral injection;

FIG. 7 is a graph showing the ratio of photoacoustic intensities at 700 nm and 990 nm of a tumor region in photoacoustic images at different time periods before and after injection of the glutathione-responsive Prussian blue producing photoacoustic nanomolecular probe solution obtained in example 1 by systemic administration and before and after injection of the glutathione-responsive Prussian blue producing photoacoustic nanomolecular probe solution obtained in example 1 by systemic administration;

FIG. 8 is a photograph of a thermal infrared imager, taken before the laser irradiation of the photo-acoustic nano-molecular probe solution for producing Prussian blue in response to glutathione obtained in example 1, after the laser irradiation of the photo-acoustic nano-molecular probe solution for producing Prussian blue in response to glutathione obtained in example 1 for 5 minutes, and after the laser irradiation of the photo-acoustic nano-molecular probe solution for producing Prussian blue in response to glutathione obtained in example 1 for 5 minutes after co-incubation;

fig. 9 is a thermal infrared imager picture before and after 5 minutes of laser irradiation and temperature change after irradiation of the tumor site with laser after injecting the same volume of the photo-acoustic nano-molecular probe solution, prussian blue nanoparticle solution, or phosphate buffer solution obtained in example 1, which generates prussian blue in response to glutathione, for 10 hours;

fig. 10 is a schematic diagram of the principle of detecting tumor cells by using the glutathione-responsive prussian blue-producing photoacoustic nano-molecular probe of the present application.

Detailed Description

Example 158 preparation of photo-acoustic NanoTaolecular probes that generate Prussian blue in response to Nano glutathione

The preparation method comprises the following specific steps:

step 3, adding the solution B into the solution A under the ultrasonic condition, and carrying out ultrasonic treatment for 30 minutes to obtain a turbid solution C, wherein the volume ratio of the solution A to the solution B is 9: 1;

The quality of distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylcholine is not changed during the preparation process, and the non-entrapped potassium ferrocyanide and ferric acetylacetonate are reduced during the dialysis process, so that the quality is changed. Therefore, the molar ratio of the molecules of potassium ferrocyanide, ferric acetylacetonate, distearoyl phosphatidyl ethanolamine-polyethylene glycol and distearoyl phosphatidyl choline is 135: 90: 1:9 in the preparation and feeding process; the corresponding mass ratio is 20:11:1: 2.54; the mass ratio of the molecules of potassium ferrocyanide, ferric acetylacetonate, distearoyl phosphatidyl ethanolamine-polyethylene glycol and distearoyl phosphatidyl choline in the finished product is 2.79: 1.51: 1: 2.54; the corresponding molar ratio was 18.5:12:1: 9.

As shown in fig. 1, an electron micrograph of the photoacoustic nano-molecular probe generating prussian blue in response to glutathione obtained in this example is shown.

FIG. 2 is the data of particle size characterization of the photo-acoustic nano-molecular probe generating Prussian blue in response to glutathione obtained in example 1. The left graph is a particle size distribution graph of the photoacoustic nano molecular probe which is obtained by testing the dynamic light scattering method and generates Prussian blue in response to the glutathione, and the right graph is the element content of the photoacoustic nano molecular probe which is obtained by testing the inductive coupling plasma emission spectrometer and generates Prussian blue in response to the glutathione. As can be seen from the left figure of FIG. 2, the average particle size of the photoacoustic nano-molecular probe for generating Prussian blue in response to glutathione is 58 +/-20 nanometers, the particle size distribution is concentrated, the particle size distribution accords with the particle size of the nano-molecular probe for passive aggregation of tumor, and the photoacoustic nano-molecular probe for generating Prussian blue in response to glutathione is enriched in the tumor tissue through the permeation and retention Effect (EPR) of the tumor tissue. As can be seen from the right graph of FIG. 2, the concentrations of potassium element and iron element carried by the photoacoustic nano-molecular probe for producing Prussian blue in response to glutathione are 574 +/-19 and 341 +/-5 mg/L respectively, which indicates that potassium ferrocyanide and iron acetylacetonate molecules are successfully carried by the photoacoustic nano-molecular probe for producing Prussian blue in response to glutathione.

Example 2 glutathione response Performance test

Glutathione solutions with different concentrations are added into the glutathione-responsive Prussian blue-producing photoacoustic nano molecular probe solution obtained in the example 1 for incubation, and after the complete reaction, an ultraviolet visible near-infrared spectrophotometer is adopted to measure the absorption spectrum of the probe.

FIG. 3 is glutathione response performance characterization data of the photo-acoustic nano-molecular probe for generating Prussian blue in response to glutathione obtained in example 1. The left graph shows the absorption spectrum of the photoacoustic nano molecular probe which generates the prussian blue in response to glutathione and is incubated by using glutathione with different concentrations, which is measured by an ultraviolet-visible near-infrared spectrophotometer, the absorbance of the photoacoustic nano molecular probe which generates the prussian blue in response to glutathione at 700 nm is gradually increased along with the increase of the concentration of the glutathione, prussian blue nano particles are generated after the probe is incubated with the glutathione, and the right graph shows the ratio of the absorbance of the photoacoustic nano molecular probe which generates the prussian blue in response to glutathione at 700 nm and 990 nm after the probe is incubated by using glutathione with different concentrations. As can be seen from fig. 3, the obtained prussian blue-responsive photoacoustic nanomolecular probe has good response performance to glutathione, and the ratio of absorbance at 700 nm and 990 nm of the prussian blue photoacoustic nanomolecular probe has good linear relationship with the concentration of glutathione.

Example 3 glutathione specific response Performance test

Glutathione obtained in example 1 responds to the photo-acoustic nano molecular probe solution for generating prussian blue, glutathione or common amino acid solution with the same concentration (10 mmol/L) is respectively added for incubation, and after the sufficient reflection, an ultraviolet-visible near-infrared spectrophotometer is adopted to measure the absorption spectrum.

Fig. 4 is a ratio of absorbance at 700 nm and 990 nm of the prussian blue-producing photoacoustic nanomolecular probe responding to glutathione obtained in example 1 after co-incubation with glutathione or a common amino acid solution. As can be seen from FIG. 4, the photoacoustic nano molecular probe for generating Prussian blue through response of the glutathione has good specific response performance on glutathione and has no response to other common amino acids of human bodies.

Example 4 in vitro photoacoustic Performance testing

Glutathione solutions with different concentrations are added into the glutathione-responsive Prussian blue-generating photoacoustic nano-molecular probe solution obtained in example 1 for incubation, the probe solution is transferred into a polytetrafluoroethylene tube with the inner diameter of 0.30 mm and the outer diameter of 0.60 mm after being fully reflected, and photoacoustic intensities at 700 nm and 990 nm are measured by adopting a photoacoustic tomography imaging system.

Fig. 5 is in vitro photoacoustic performance test data of the prussian blue-producing photoacoustic nanomolecular probe responded by glutathione obtained in example 1. The upper graph is a photoacoustic image at 700 nm and 990 nm after glutathione responses to the photo-acoustic nano-molecular probe solution for producing prussian blue by adding glutathione solutions with different concentrations for incubation, and the lower graph is a ratio of the photo-acoustic intensity at 700 nm and 990 nm after the glutathione responses to the photo-acoustic nano-molecular probe solution for producing prussian blue by adding the glutathione solutions with different concentrations for incubation. As can be seen from fig. 5, the obtained photoacoustic nanomolecular probe for prussian blue generation in response to glutathione has good photoacoustic imaging performance, and the ratio of the photoacoustic intensity at 700 nm and 990 nm of the photoacoustic nanomolecular probe for prussian blue generation in response to glutathione has good linear relationship with the concentration of glutathione.

Example 5 in vivo photoacoustic Performance test

Nude mice seeded with two 4T1 tumors were anesthetized with oxygen containing 2% isoflurane. Two 4T1 tumors were injected intratumorally with the glutathione-responsive Prussian blue-producing photoacoustic molecular probe solutions (50. mu.L, 0.3 mg/mL) or phosphate buffer solutions (50. mu.L) obtained in example 1, respectively. Scanning imaging is carried out by adopting a photoacoustic tomography imaging system at different time periods after injection, and photoacoustic images at 700 nanometers and 990 nanometers are obtained.

Fig. 6 is in-vivo photoacoustic performance test data of the prussian blue-producing photoacoustic nanomolecular probe responded to glutathione obtained in example 1. Wherein the upper graph is photoacoustic images at 700 nm and 990 nm at different time periods after injecting the glutathione obtained from example 1 into the 4T1 tumor by intratumoral injection and responding to the prussian blue-producing photoacoustic nano-molecular probe solution or phosphate buffer, and the lower graph is the ratio of photoacoustic intensity at 700 nm and 990 nm of the tumor region in the photoacoustic images at different time periods after injecting the glutathione obtained from example 1 into the 4T1 tumor by intratumoral injection and responding to the prussian blue-producing photoacoustic nano-molecular probe solution or phosphate buffer. As can be seen from fig. 6, the ratio (11.53 ± 0.08) of the photoacoustic intensities at 700 nm and 990 nm of the tumor region of the prussian blue-producing photoacoustic nanomolecular probe solution injected with glutathione obtained in example 1 was 1.49 times of the photoacoustic intensities (7.78 ± 0.10) at 700 nm and 990 nm of the tumor region injected with phosphate buffer solution 6 hours after the intratumoral injection, indicating that prussian blue nanoparticles were formed by the reaction between the prussian blue-producing photoacoustic nanomolecular probe responded with glutathione obtained in example 1 and the glutathione in the tumor environment, and that the prussian blue-producing photoacoustic nanomolecular probe responded with glutathione obtained in example 1 had good glutathione response performance and photoacoustic imaging performance.

Example 6 tumor detection Performance test

Nude mice bearing 4T1 tumor were anesthetized with oxygen containing 2% isoflurane. The glutathione obtained in example 1 was injected systemically in response to a prussian blue-producing photo-acoustic nanomolecular probe solution (150 μ l, 0.3 mg/ml). Scanning imaging is carried out by adopting a photoacoustic tomography imaging system at different time periods before and after injection, and photoacoustic images at 700 nanometers and 990 nanometers are obtained.

Fig. 7 is tumor detection performance test data of the photo-acoustic nano-molecular probe generating prussian blue in response to glutathione obtained in example 1. Wherein the upper graph is photoacoustic images at 700 nm and 990 nm at different time periods before and after injection of the glutathione solution obtained in example 1 to generate prussian blue in response to prussian blue by systemic administration, and the lower graph is the ratio of photoacoustic intensities at 700 nm and 990 nm of a tumor region in the photoacoustic images at different time periods before and after injection of the glutathione solution obtained in example 1 to generate prussian blue by systemic administration. As can be seen from fig. 7, 10 hours after the glutathione obtained in example 1 was systemically administered to respond to the prussian blue-producing photoacoustic nanoprobe solution, the ratio of the photoacoustic intensities at 700 nm and 990 nm of the tumor region reached the maximum value (3.88 ± 0.31), which was 2.12 times higher than that before the glutathione obtained in example 1 was systemically administered to respond to the prussian blue-producing photoacoustic nanoprobe solution (1.78 ± 0.25), indicating that the prussian blue nanoparticles were formed by the reaction between the prussian blue-producing photoacoustic nanoprobe obtained in example 1 and glutathione in the tumor environment, and that the prussian blue-producing photoacoustic nanoprobe obtained in example 1 had good tumor detection performance.

Example 7 in vitro photothermal Properties test

An equal amount of the photoacoustic nanomolecular probe solution that produces prussian blue in response to glutathione obtained in example 1 was transferred to a plastic tube, added with glutathione (10 mmol/l) or deionized water in the same volume respectively for co-incubation, irradiated with near-infrared laser of 658 nm at a power density of 0.5 w/cm, and the temperature of the solution before and after laser irradiation was recorded using a thermal infrared imager.

Fig. 8 is in vitro photothermal performance test data of the prussian blue-producing photoacoustic nanomolecular probe in response to glutathione obtained in example 1. From left to right, the pictures of the thermal infrared imagers before the laser irradiation of the photoacoustic nanomolecular probe solution which is obtained in example 1 and can generate prussian blue in response to glutathione, the pictures of the thermal infrared imagers after the laser irradiation of the photoacoustic nanomolecular probe solution which is obtained in example 1 and can generate prussian blue in response to glutathione for 5 minutes, and the pictures of the thermal infrared imagers after the laser irradiation of the photoacoustic nanomolecular probe solution which is obtained in example 1 and can generate prussian blue in response to glutathione are respectively shown. As can be seen from fig. 8, the temperature of the solution before laser irradiation was 25 degrees celsius, the temperature of the solution after only laser irradiation for 5 minutes was 35 degrees celsius, and the temperature of the solution after laser irradiation for 5 minutes after co-incubation with glutathione was 62 degrees celsius, which indicates that the photoacoustic nano-molecular probe solution for producing prussian blue in response to glutathione obtained in example 1 had good photothermal properties after co-incubation with glutathione.

Example 8 in vivo photothermal Property test

Mice bearing subcutaneous 4T1 xenograft tumors were injected with the same volume of the glutathione-responsive prussian blue-producing photoacoustic nanoprobe solution, prussian blue nanoparticle solution, or phosphate buffer solution obtained in example 1, respectively, by systemic administration. The tumor site was irradiated with 658 nm near-infrared laser at a power density of 0.8W/cm 10 hours after injection, and the temperature change before and after laser irradiation was recorded with a thermal infrared imager.

Fig. 9 is in-vivo photothermal performance test data of the photo-acoustic nano-molecular probe for generating prussian blue in response to glutathione obtained in example 1. The upper graph is the thermal infrared imager pictures before and after laser irradiation for 5 minutes after injecting the same volume of the photoacoustic nano-molecular probe solution, the prussian blue nanoparticle solution or the phosphate buffer solution which is obtained in example 1 and generates prussian blue in response to glutathione, and the lower graph is the temperature change after the tumor part is irradiated by laser. As can be seen from fig. 9, the tumor surface temperature of the mice injected with the same volume of the glutathione-responsive prussian blue-producing photoacoustic nanoprobe solution or prussian blue nanoparticle solution of example 1 rapidly increased from about 35 degrees celsius to about 60 degrees celsius under laser irradiation, and the tumor surface temperature of the mice injected with the same volume of phosphate buffer showed only a slight change under laser irradiation, indicating that the glutathione-responsive prussian blue-producing photoacoustic nanoprobe solution of example 1 had good optoacoustic and thermal properties in vivo after co-incubation with glutathione.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. The photoacoustic nano molecular probe for generating Prussian blue through response of glutathione is characterized by comprising a surface modification and an inner core, wherein the inner core comprises a potassium ferrocyanide molecule and an iron acetylacetonate molecule; the surface modification is distearoylphosphatidylethanolamine-polyethylene glycol molecules and distearoylphosphatidylcholine molecules; wherein the mass ratio of the potassium ferrocyanide, ferric acetylacetonate, distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylcholine molecules is 2.79: 1.51: 1: 2.54.

2. The nanomolecular probe of claim 1, wherein the average particle size of the nanomolecular probe is 58 nanometers.

3. The nanomolecular probe according to claim 1, wherein the nanomolecular probe reacts to produce prussian blue under the influence of glutathione, providing a photoacoustic signal in the near infrared region.

4. The nanomolecular probe of claim 1, wherein the nanomolecular probe has a glutathione minimum response concentration of 0.3 millimoles per liter.

5. The nanomolecular probe of claim 1, wherein the prussian blue produced after a glutathione response of the nanomolecular probe has an absorption peak at 700 nm.

6. The method for preparing the nanomolecular probe according to claim 1, comprising the steps of:

step 2, adding ferric acetylacetonate, distearoyl phosphatidyl ethanolamine-polyethylene glycol and distearoyl phosphatidyl choline into absolute ethyl alcohol, and carrying out ultrasonic treatment for 5-15 minutes to obtain a B solution with the concentration of 200 millimoles per milliliter, wherein the molar ratio of the ferric acetylacetonate, the distearoyl phosphatidyl ethanolamine-polyethylene glycol to the distearoyl phosphatidyl choline is 90: 1: 9;

step 5, transferring the solution C subjected to the ultrasonic treatment of the probe into a dialysis bag, and dialyzing the solution C in ultrapure water for 2 hours to obtain a photoacoustic nano molecular probe which has good water dispersibility and can generate Prussian blue in response to glutathione;

wherein the molar ratio of the potassium ferrocyanide, ferric acetylacetonate, distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylcholine molecules in the preparation process is 135: 90: 1: 9.

7. Use of the nanomolecular probe according to any of claims 1-5 or prepared according to the method of claim 6 for the preparation of a reagent for in situ imaging of tumors.

8. Use of the nanomolecular probe according to any of claims 1 to 5 or prepared according to the method of claim 6 in the preparation of a tumor photothermal therapy formulation.