Spraying type operation auxiliary imaging agent based on in-situ adaptation as well as preparation method and application of spraying type operation auxiliary imaging agent

Abstract

The invention discloses a spraying type operation auxiliary imaging agent based on in-situ adaptation as well as a preparation method and application of the spraying type operation auxiliary imaging agent. According to the invention, polydopamine is grafted with various tumor targeting ligands to serve as a photo-thermal / fluorescent material for specifically targeting tumors, biological signals of the tumors are converted into thermal signals or fluorescent signals in situ through laser irradiation or excitation, and then the thermal signals are converted into visual image signals through a thermal imager. By adjusting the laser irradiation time, the signal sensitivity independent of the tumor microenvironment can be further adjusted, and the display of the tumor image is enhanced, so that the imaging signal difference caused by the heterogeneity of the microenvironment is overcome. The spray imaging agent can realize specific temperature rise of a target part by targeting and overexpressing tumor cells of a certain receptor, so that damage to normal tissues is avoided. An obvious temperature difference exists between the tumor tissue and the normal tissue, so that lower background noise is displayed, and the tumor and the surrounding normal tissue can be distinguished more clearly.

Description

Technical Field

[0001] The present invention belongs to the field of materials and biomedicine, and particularly relates to a spray-type surgical auxiliary imaging agent based on in-situ adaptation, and a preparation method and application thereof. Background Art

[0002] Surgical resection has always been the main means of treating tumors, especially solid tumors, and the diagnostic information of tumor images is directly related to the effect of surgical resection. At present, the commonly used imaging diagnostic methods for cancer in clinical practice include ultrasound diagnosis, magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography-computed tomography (PET-CT). These methods have a positive effect on the macroscopic presentation of tumor distribution and anatomical position before surgery, but because they carry radiation, are costly, and have cumbersome procedures, they still cannot provide real-time image navigation during surgery. Accordingly, research in the field mainly focuses on improvements based on traditional imaging technologies, such as intraoperative nuclear magnetic resonance imaging (iMRI) and the development of new intraoperative imaging technologies such as fluorescence imaging and photothermal imaging.

[0003] Although fluorescence imaging shows great potential for intraoperative delineation of tumor boundaries, the inevitable false positives of biological autofluorescence and rapid photochemical destruction reduce its clinical practicality; iMRI can be used during surgery through the transformation of the operating room, but it cannot provide real-time tumor imaging information. Other emerging imaging technologies, such as ultrasound angiography and photoacoustic imaging, are easily damaged by contrast agents during imaging, showing a limited surgical time window and cannot provide sufficient time to complete the operation. In comparison, photothermal imaging has attracted attention due to its advantages such as simple operation, stable signal supply, and flexible application in intraoperative guidance. Photothermal imaging refers to a type of technology that visualizes various physiological signals and lesion locations through changes in photothermal parameters. After a period of laser irradiation, photothermal materials absorb light radiation and generate thermal radiation signals due to non-radiative relaxation of the absorbed energy. Thermal imagers can convert thermal radiation signals into visual images of object temperature changes. Compared with fluorescence imaging, photothermal material-assisted thermal imaging can adjust signal sensitivity by adjusting the irradiation time that is unrelated to background fluorescence.

[0004] The prior art discloses that integrin is an important class of cell surface receptors. Studies have shown that integrin αβυ3 is the most widely studied integrin family member. It is lowly expressed or even not expressed in normal tissues, but its expression level increases sharply in many tumor cells. v3 can bind to RGD (Arg-Gly-Asp) sequence proteins, so it can be used as an ideal tumor imaging agent target. Studies have shown that compared with traditional photothermal materials, polydopamine has superior photothermal conversion efficiency, good biocompatibility and easy modification. Therefore, polydopamine is expected to become a photothermal material carrier for tumor nanoaptamers.

[0005] In addition, whether it is fluorescence, ultrasound, photoacoustic, CT or MRI, they all rely on oral or intravenous contrast agents, which increases the metabolic burden of the human liver and poses a risk of adverse reactions. Among the tumor imaging agents that have been developed, there are no tumor imaging agents that can be adapted in situ in vitro. Therefore, it is very necessary to develop a universal strategy to safely and conveniently image tumors in vitro. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation.

[0007] Another object of the present invention is to provide a spray-type surgical auxiliary imaging agent based on in-situ adaptation obtained by the above preparation method.

[0008] Another object of the present invention is to provide the application of the above-mentioned spray-type surgical auxiliary imaging agent based on in-situ adaptation.

[0009] The purpose of the present invention is achieved through the following technical solution: A method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation, comprising the following steps:

[0010] (1) Synthesis of polydopamine / polyethyleneimine polymer dot composite nanospheres (PDA);

[0011] (2) Grafting tumor targeting peptides to obtain a sprayable imaging agent with photothermal / fluorescence dual-modality imaging and capable of targeting tumors, namely the sprayable surgical auxiliary imaging agent based on in situ adaptation.

[0012] In the polydopamine / polyethyleneimine polymer dot composite nanospheres described in step (1), polydopamine is synthesized using dopamine (DA) as a monomer to form polydopamine nanospheres containing covalent bonds and non-covalent bonds. In a weakly alkaline solution at room temperature, dopamine hydrochloride is used as a monomer, dopamine is oxidized to dopaquinone, and then 5,6-dihydroxyindole (DHI) is obtained by nucleophilic reaction. DHI is polymerized into DHI dimer or trimer by covalent oxidation, and polyethyleneimine polymer dots that can covalently polymerize with DHI are added during the above polymerization process to obtain polydopamine / polyethyleneimine polymer dot composite nanospheres composed of two components; specifically preferably, the following steps are included:

[0013] A. Dissolve dopamine in water to obtain solution A;

[0014] B. mixing polyethyleneimine carbonized polymer dots, water, and an organic solvent to obtain a solution B;

[0015] C. Add solution B to solution A, add ammonia solution, mix well, and react; collect the obtained reaction product by centrifugation, wash the obtained precipitate, and dry it to obtain polydopamine nanospheres.

[0016] The dopamine described in step A is preferably dopamine hydrochloride.

[0017] The concentration of solution A described in step A is preferably 45-55 mg / mL; more preferably 50 mg / mL.

[0018] The water described in step A is preferably deionized water.

[0019] The polyethyleneimine carbonized polymer dots described in step B are prepared by a one-step method, which is prepared by a mixed solution of citric acid and polyethyleneimine (molecular weight 600-25000) by microwave-assisted method. Specifically, it includes the following steps:

[0020] 50-300 mg of citric acid and 10-200 mg of polyethyleneimine polymer dots were dissolved in 5 mL of 0.05 M HCl aqueous solution, stirred evenly, and placed in a microwave reactor for reaction at 100-500 W for 1-10 min. After the reaction was completed, the mixture was cooled to room temperature, dialyzed, and freeze-dried to obtain polyethyleneimine carbonized polymer dots with fluorescent properties.

[0021] The organic solvent described in step B is preferably at least one of methanol and ethanol; more preferably anhydrous ethanol.

[0022] The concentration of the polyethyleneimine carbonized polymer dots in step B is preferably 0.2 to 5 mg / mL.

[0023] The volume ratio of water to organic solvent in step B is preferably 5:8-10; more preferably 5:9.

[0024] The concentration of the ammonia solution in step C is preferably 26% to 30% by volume; more preferably 28% by volume.

[0025] The amount of the ammonia solution in step C is preferably calculated as ammonia solution: solution A = volume ratio of 1:6-7; more preferably, ammonia solution: solution A = volume ratio of 1:6.6.

[0026] The reaction time in step C is preferably 20 to 28 hours, more preferably 24 hours.

[0027] The centrifugal conditions in step C are preferably 10000-14000 rpm for 15-30 min; more preferably 12000 rpm for 20 min.

[0028] The washing solvent in step C is preferably deionized water.

[0029] The number of washing times in step C is preferably 2 to 4 times; more preferably 3 times.

[0030] The drying described in step C is preferably vacuum drying.

[0031] The grafted tumor targeting peptide in step (2) is grafted onto the polyamine nanospheres through Michael addition reaction or Schiff base reaction; preferably, the process comprises the following steps:

[0032] D. dispersing polydopamine / polyethyleneimine polymer dot composite nanospheres in an aqueous solution to obtain a PDA solution;

[0033] E. Add various ligand molecules such as RGD targeting peptide, lactose, glycoprotein, folic acid and biotin OC12 to the polydopamine solution for reaction;

[0034] F. Centrifuge the obtained product, take the precipitate, wash, and dry to obtain the nanoaptamer PDA-ligands in the photothermal imaging agent.

[0035] The aqueous solvent described in step D is preferably deionized water.

[0036] The polydopamine solution in step D preferably has a concentration of 45 to 55 mg / mL; more preferably, the concentration is 50 mg / mL.

[0037] The mass ratio of the ligand described in step E is preferably 5:0.8-1.2 based on the mass of polydopamine; more preferably, the mass ratio of polydopamine:ligand molecule is 5:1.

[0038] The centrifugal conditions in step F are preferably 10,000-14,000 rpm for 15-30 min; more preferably 12,000 rpm for 20 min.

[0039] The washing solvent in step F is preferably deionized water.

[0040] The drying in step F is preferably vacuum drying.

[0041] A spray-type surgical auxiliary imaging agent based on in-situ adaptation is prepared by the above preparation method.

[0042] The above-mentioned spray-type surgical auxiliary imaging agent based on in situ adaptation is used in the preparation of anti-tumor drugs and tumor diagnostic agents.

[0043] The present invention provides a method for solving the safety hazard problem of traditional injectable tumor imaging agents by using in situ spraying tumor nanoaptamer imaging agents. The present invention utilizes polydopamine / polyethyleneimine polymer dot composite nanospheres grafted with various ligands as photothermal / fluorescent materials that specifically target tumors, and converts the biological signals of tumors into thermal signals in situ through laser irradiation or fluorescence excitation, and then converts the thermal signals into intuitive image signals through a thermal imager. By adjusting the laser irradiation time, the signal sensitivity independent of the tumor microenvironment can be further adjusted, and the display of tumor images can be enhanced, thereby overcoming the imaging signal differences caused by the heterogeneity of the microenvironment. The photothermal probe can achieve specific heating of the target site by targeting tumor cells that overexpress integrins, thereby avoiding damage to normal tissues. Due to the obvious temperature difference between tumor tissue and normal tissue, a lower background noise is displayed, and a clearer distinction can be made between tumors and surrounding normal tissues.

[0044] Compared with the prior art, the present invention has the following advantages and effects:

[0045] (1) The present invention proposes a spray-type surgical auxiliary imaging agent based on in situ adaptation and its preparation and use methods. It is prepared from ligand-functionalized polydopamine / polyethyleneimine polymer dot composite nanospheres that can target various tumor cells. It can convert and amplify biological signals into intuitive thermal image signals and visualize tumor boundaries in situ. The invention adopts a method of in situ local spraying combined with a portable infrared camera, which has the characteristics of low cost, convenient use, no radiation hazard, real-time imaging guidance during surgery, and avoidance of the adverse risks of oral or intravenous contrast agents. It can be used as a new general tool to help surgeons make real-time decisions during surgery and improve the effectiveness of tumor resection surgery.

[0046] (2) The present invention adopts a small bottle spray form, which is easy to carry and use. Unlike the large medical equipment that traditional imaging technology relies on, our photothermal imaging agent only needs to be sprayed in situ near the tumor surface and heated for a short time by near-infrared rays, and the boundary between tumor cells and normal tissue cells can be displayed using a portable thermal imager.

[0047] (3) To date, photothermal imaging technology has been mainly used in the biomedical field for photothermal therapy of tumors and bacterial infections, while in terms of sensing applications, it is limited to the detection of cells or molecules. There are no patent reports on precise surgical resection of tumors guided by in vitro assisted thermal imaging of photothermal materials. The present invention innovatively applies photothermal nanomaterials to the field of tumor thermal imaging, providing a new path for intraoperative imaging of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1TEM and SEM images with size distribution of PDA and PDA-RGD inserted respectively; a and c are TEM and SEM images with size distribution of PDA inserted respectively, and b and d are TEM and SEM images with size distribution of PDA inserted respectively (scale bar: 200 nm).

[0049] Figure 2 The size distribution of PDA and PDA-RGD was determined by DLS.

[0050] Figure 3 UV-visible-near-infrared spectra of DA, PDA, and PDA-RGD.

[0051] Figure 4 This is the photothermal response of 100 μg / mL PDA-RGD solution irradiated with 808 nm, 3 W / cm2 near-infrared laser for 6 minutes and the cooling curve after the irradiation was turned off.

[0052] Figure 5 The linear fit graph is a graph of the -lnθ (negative natural logarithm of temperature) data obtained during the cooling period and the time data.

[0053] Figure 6 Cell viability graph of 3T3 cells in vitro at different concentrations of PDA-RGD nanospheres.

[0054] Figure 7 The power density is 3W / cm 2 , Photothermal heating curves of PDA-RGD suspensions with different concentrations under 808nm laser irradiation.

[0055] Figure 8 : is the temperature change curve (ΔT) of PDA-RGD dispersion (100 μg / mL) depending on laser power and irradiation time; wherein a is laser power and b is irradiation time.

[0056] Fig. 9 This is the temperature response curve of 100 μg / mL PDA-RGD when the 808 nm laser is repeatedly turned on / off, and the preset time interval is five cycles.

[0057] Fig.10 The linear fitting graph of ΔT value and the number of B16 cells, 4T1 cells, HUVEC cells, MCF-7 cells and 3T3 cells after treatment with 100 μg / mL PDA-RGD in culture medium for 6 minutes.

[0058] Fig.11 The figure shows the binding ability of FAM-labeled PDA-RGD to 3T3 and 4T1 cells determined by flow cytometry.

[0059] Fig.12 3T3 cells, HUVEC cells, 4T1 cells, and B16 cells were cultured in 96-well plates with 100 μg / mL PDA-RGD at 808 nm and 3 W / cm 2 Infrared images before and after 6 min of laser treatment (cell count: 50,000).

[0060] Fig.13 This is a comparison of the quantitative temperature changes of 3T3 cells, HUVEC cells, 4T1 cells and B16 cells treated with PDA-RGD after 808 nm laser irradiation for 6 minutes (n=3).

[0061] Fig.14 Infrared images of the regional distribution of 3T3 and 4T1 cells treated with PDA-RGD in the same culture dish before and after 808 nm laser irradiation for 6 minutes, with a power density of 3 W / cm 2 .

[0062] Fig.15 This is a graph of relative temperature changes in different regions after co-culture of the above 4T1 cells and 3T3 cells measured by infrared thermal imaging (n=3). The significance is determined by the p value, * represents p<0.05; ** represents p<0.01; *** represents p<0.001.

[0063] Fig.16 The normal muscle tissue treated with PDA-RGD was used as the control group, and the leg muscles of BALB / C mice bearing 4T1 tumors were treated with PDA-RGD (100 μg / mL) or saline as the experimental group. Thermal images were obtained under 808 nm laser irradiation; the red circle indicates the tumor site.

[0064] Fig.17 Infrared image of a BALB / C mouse bearing a 4T1 tumor in the abdominal epithelium sprayed with PDA-RGD (100 μg / mL) before 808 nm laser irradiation for 6 minutes; the white circle indicates the tumor site (scale bar, 1 cm).

[0065] Fig.18 The quantitative temperature change diagram of mouse muscle tumor tissue, normal tissue and edge measured by thermal imaging (n=3).

[0066] Fig.19 The quantitative temperature change diagram of tumor tissue, normal tissue and edge of mouse abdominal model measured by thermal imaging (n=3).

[0067] Fig. 20 H&E staining results of tumors removed under thermal imaging guidance and without thermal imaging guidance (scale bar, 100 μm).

[0068] Fig.21The present invention is a flow chart of imaging using the spray-type surgical auxiliary imaging agent of the present invention. DETAILED DESCRIPTION

[0069] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific conditions are not specified, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0070] Example 1: Preparation and characterization of PDA-RGD

[0071] The preparation method of PDA-RGD comprises the following steps:

[0072] Dopamine (DA) is used as a monomer to synthesize polydopamine nanospheres containing covalent bonds and non-covalent bonds. RGD is grafted onto polydopamine nanospheres through Michael addition reaction or Schiff base reaction to obtain a photothermal imaging agent. The specific steps are as follows:

[0073] (1) Preparation of PDA nanospheres

[0074] A. Dissolve 500 mg of dopamine (DA) completely in 10 mL of deionized water.

[0075] B. The above solution was injected into a mixture containing 50 mL of ethanol, 90 mL of water and 1.5 mL of ammonia solution (28 wt% aqueous solution) was added. The color of the solution gradually changed from transparent to brown and finally to black.

[0076] C. Allow the reaction to proceed for 24 hours. Collect the PDA nanospheres by centrifugation (12000 RPM) and rinse three times with water.

[0077] (2) PDA nanospheres are grafted with RGD to obtain PDA-RGD:

[0078] A. Disperse 100 mg of pretreated PDA nanospheres in 2 mL of water to obtain a PDA solution.

[0079] B. Then, 20 mg of Arg-Gly-Asp(RGD) was added to the PDA solution, and the Schiff base reaction was allowed to proceed for 24 hours to obtain PDA-RGD.

[0080] C. Finally, the product was collected by centrifugation (12000RPM) and washed three times with water.

[0081] The morphological characteristics of the prepared nanospheres were obtained by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1). Using scanning electron microscopy and transmission electron microscopy, the images of PDA-RGD nanospheres showed regular spherical shapes with an average diameter of about 250nm (PDA) and 280nm (PDA-RGD). Compared with the surface of bare PDA nanospheres, the surface of PDA-RGD is rougher and wavy, with a large number of raised spots that may be made of RGD. Under dynamic light scattering (DLS) ( Figure 2 ), PDA and PDA-RGD also showed similar results to the electron microscopy images. The average hydrodynamic diameters of PDA and PDA-RGD were 357.8±6.9nm and 400.3±4.3nm, respectively, which were larger than the electron microscopy results due to the hydration of water.

[0082] DA, PDA, and PDA-RGD were characterized by infrared spectroscopy (IR) in the range of 500–4000, and by ultraviolet-visible-near infrared absorption spectroscopy (UV-vis-NIR) absorbance spectroscopy in the range of 200–900. The data were analyzed using GraphPad 7 ( Figure 3 ). Combined with previous reports, such as Figure 3 As shown, in the infrared absorption range of 1610-1250cm-1, there are obvious overlapping peaks, corresponding to the hydrogen bonds in the self-assembly process of PDA. In addition, there are strong absorption peaks near 1604cm-1 and 1491cm-1, which are mainly attributed to the presence of the intermediate product 5,6-dihydroxyindole (DHI), which proves the successful preparation of PDA. PDA-RGD has a strong absorption peak near 1642cm-1, which is attributed to the stretching vibration of -C=O of the secondary amide in RGD. In addition, compared with dopamine, the prepared PDA and PDA-RGD nanospheres show a wide absorption from visible light to near-infrared wavelengths. The absorption in the ultraviolet-infrared region of the spectrum is attributed to the oxidation of dopamine to dopachrome and dopamine, while the subsequent self-polymerization process leads to significant absorption from visible light to near-infrared wavelengths. The results show that PDA-RGD has near-infrared absorption characteristics, indicating its feasibility as a photothermal material.

[0083] Example 2: Testing the biocompatibility and photothermal performance of PDA-RGD at different concentrations

[0084] Thermal conversion efficiency is an important indicator for evaluating the performance of photothermal agents. The heating / cooling curve is shown in Figure 4 ,right Figure 4 The negative logarithm of the cooling curve temperature is fitted to obtain Figure 5. According to the relevant reported method, we measured the photothermal conversion efficiency (η) of PDA-RGD, and the η value of PDA-RGD was 54.27%. Compared with the widely used and reported photothermal materials, such as gold nanorods (η=22%), black phosphorus (η=30.84%), MoS two (η=35%) and ICG (η=28.4%), the photothermal conversion performance of PDA-RGD is superior, indicating that it has the potential for photothermal imaging.

[0085] The biocompatibility of PDA-RGD was tested by CCK-8 method: 3T3 cells were cultured at a density of 1×10 5 Cells / well were cultured in a 96-well plate at 37°C and 5% CO2 in an incubator overnight. After treatment with fresh medium containing different concentrations (10, 20, 50, 100, 200, 500 and 1000 μg / mL) of PDA-RGD for 24 h, each group of cells was washed three times with PBS. Then, the cells were detected using the CCK-8 detection kit according to the manufacturer's instructions ( Figure 6 ), CCK-8 assay was performed on 3T3 cells after treatment with different concentrations of PDA-RGD for 24 h. The results showed that even when the PDA-RGD concentration reached 100 μg / mL, the cell viability of 3T3 was still maintained above 85%.

[0086] Photothermal performance of PDA-RGD at different concentrations: PDA-RGD suspensions of different concentrations (10, 20, 50, 100, 200, 500 and 1000 μg / mL) were prepared, and pure water was used as a negative control. After the suspension was evenly dispersed by ultrasound, 100 μL of PDA-RGD was added to a 96-well plate and irradiated with an 808 nm near-infrared laser at a power of 3 W / cm2 for 6 minutes. The photothermal images and temperature values ​​were recorded every 30 seconds using an infrared thermal imager ( Figure 7 ), the results showed that with the increase of PDA-RGD concentration, the stable temperature of photothermal conversion increased. After 6 minutes of irradiation, the temperature of PDA-RGD solution increased by 79.1℃ at 1000μg / mL, indicating excellent photothermal performance. In contrast, pure water only increased by 2.1℃. The results show that under near-infrared laser irradiation, the temperature measured by PDA-RGD thermal imaging has a significant concentration dependence. Therefore, we can quantify the concentration and location distribution of PDA-RGD by photothermal imaging.

[0087] Optimization of laser power and irradiation time: The laser was irradiated with different powers (1.5, 3, 4.5 and 6 W / cm 2 ) was used to irradiate 100 μL of PDA-RGD suspension for 10 min. The temperature change was recorded every 30 s using an infrared thermal imager to select the optimal laser power and irradiation time. Figure 8 As shown, when the laser power is less than 3W / cm 2 The temperature rise of twoPDA-RGD (100 μg / mL) did not exceed the temperature safety limit (45°C).

[0088] Stability and reversibility of PDA-RGD: 100 μg / mL PDA-RGD suspension was prepared and used for repeated heating-cooling cycle experiments. The power used for the sample was 3 W / cm 2 The 808nm near-infrared laser was continuously irradiated for 6 minutes, and then cooled to room temperature. The above operation was repeated 5 times. The infrared thermal imager was used to record the photothermal image and temperature value every 30 seconds. Fig. 9 As shown in the figure, after 6 minutes of laser irradiation, the temperature of PDA-RGD (100 μg / mL) increased by 25°C and then returned to room temperature. Under the same conditions, after five heating / cooling cycles, the peak temperature rise of PDA-RGD remained at 25°C. The relative standard deviation (RSD) of the reversibility of PDA-RGD was about 1.09%. This shows that PDA-RGD has good stability and repeatability as a temperature sensor for thermal imaging detection.

[0089] Example 3: Tumor cell-specific effects of PDA-RGD and overexpressed αvβ3 integrin

[0090] To evaluate the targeting ability of PDA-RGD-assisted hyperthermia and the feasibility of applying this product, tumor cells overexpressing ανβ3 integrin were identified from normal cells. Mouse melanoma cells (B16), mouse breast cancer cells (4T1), and human umbilical vein endothelial cells (HUVEC) overexpressing ανβ3 integrin were used as integrin-positive cells, while human breast cancer cells (MCF-7) and mouse fibroblasts (3T3) were used as negative controls. The cell gradient density of each group of cells (B16 cells, 4T1 cells, MCF-7 cells, HUVEC cells, and 3T3 cells) was 1×10 3 ,2×10 3 ,5×10 3 ,1×10 4 and 2×10 4 Cells / well were cultured in 96-well plates in a 37°C and 5% CO2 incubator overnight. After being treated with 100 μL of fresh culture medium containing 100 μg / mL PDA-RGD for 30 minutes, each group of cells was washed 3 times with PBS to remove free PDA-RGD, and then 100 μL of new culture medium was added again. The cells were irradiated with an 808nm near-infrared laser with a power of 3W / cm2 for 6 minutes, and the photothermal images and the temperature value of each well were recorded every 30 seconds with an infrared thermal imager. The number of cells in the 96-well plate was counted using a hemocytometer. Fig.10As shown, as the cell concentration increases, the temperature of integrin-overexpressing cells rises faster than that of normal cells, and the slope of the fitting line in the cell group with ανβ3 integrin overexpression is between 1.4 and 1.8 (K>1); the fitting line of the normal cell group with normal integrin expression is between 0.3 and 0.6 (K<1). Therefore, the ΔT of the ανβ3 integrin-overexpressing cell group is more than 2.3 times that of the ΔT of the ανβ3 integrin-normal expressing cell group. Specifically, the slope of the fitting line for 4T1 cells is 1.4393 (the temperature increases by 1.4393°C for every 1×104 4T1 cells added), while the slope of the fitting curve for 3T3 cells is 0.3354. Under the same irradiation, the temperature rise of 4T1 cells is 4.29 times that of 3T3 cells. All these measurements well demonstrate the selective binding of PDA-RGD to ανβ3 integrin-overexpressing cells. The results showed that PDA-RGD-assisted thermal imaging was able to qualitatively distinguish different cell types, which provided the possibility for thermal imaging to identify tumor cells overexpressing ανβ3 integrin from normal cells during resection surgery.

[0091] To further explore the evidence that PDA-RGD specifically binds to the ανβ3 integrin of tumor cells but not normal cells, we performed flow cytometry experiments of FAM fluorescently labeled PDA-RGD on 4T1 and 3T3 cells. Each group of cells (4T1 and 3T3) was cultured in a 24-well plate at a density of 5×104 cells / well. After treatment with 500 μL of fresh culture medium containing 100 μg / mL PDA-RGD for 30 minutes, each group of cells was washed 3 times with PBS to remove free PDA-RGD, and then 500 μL of new culture medium was added again. The cells were trypsinized, then centrifuged to remove the supernatant, and then resuspended in 200 μL PBS solution. Subsequently, the cell suspension was tested using a flow cytometer in the 488 nm fluorescence channel, and the data was analyzed using Flowjo7.6.1 software. Fig.11 As shown, 4T1 cells (integrin overexpression) have stronger fluorescence intensity compared with 3T3 cells (normal ανβ3 integrin expressing cells), indicating that overexpression of ανβ3 integrin on 4T1 cells promotes the binding of cells to PDA-RGT nanospheres. The results show that PDA-RGD can selectively accumulate in tumor areas where ανβ3 integrin is overexpressed.

[0092] Example 4: PDA-RGD in vivo tumor imaging effect

[0093] Each group of cells (4T1, 3T3, MCF-7 and B16) was cultured at a density of 5×10 4Cells / well were seeded in four adjacent wells of a 96-well plate. After being treated with 100 μL of fresh medium containing 100 μg / mL PDA-RGD for 30 min, each group of cells was washed 3 times with PBS to remove free PDA-RGG, and then 100 μL of new medium was added again. The 96-well plate was irradiated with an 808 nm near-infrared laser at a power of 3 W / cm2 for 6 minutes, and the thermal image and temperature value of the 96-well plate were recorded every 30 seconds with an infrared thermal imager. Fig.12 , Fig.13 As shown, the temperature of the wells inoculated with normal ανβ3 integrin-expressing cells (MCF-7 cells and 3T3 cells) increased by 5.4°C and 5.1°C, respectively, while the temperature of the wells inoculated with ανβ3 integrin-overexpressing cells (4T1 cells and B16 cells) increased by 11°C and 10.2°C, respectively. Thermal imaging detected that the temperature of ανβ3 integrin-overexpressing cells was higher than that of normal cells. The results demonstrated the qualitative and quantitative visualization performance of PDA-RGD-assisted thermal imaging for cell identification.

[0094] The middle of the wells in the 24-well plate were separated by a silicone plate, and the density of 3T3 and 4T1 cells was 1×10 5 Cells / well were cultured in two semicircular areas of these wells. After treatment with 500 μL fresh medium containing 100 μg / mL PDA-RGD for 30 min, each group of cells was washed 3 times with PBS to remove free PDA-RGD, and then 500 μL of new medium was added again. The power was 3 W / cm 2 The 808nm near-infrared laser was continuously irradiated on the 24-well plate for 6 minutes, and the infrared thermal imager was used to record the photothermal image and the temperature value of each well every 30 seconds. The thermal imaging results are shown in the figure below. Fig.14 , Fig.15 As shown in the figure, after PDA-RGD treatment, the temperature rise of the tumor cell 4T1 cell inoculation area was significantly higher than that of the normal cell 3T3 cell inoculation area, and the edges of the two areas were clearly visible. Therefore, the ability of PDA-RGD-assisted thermal imaging to identify the boundary line between tumor cells (4T1) overexpressing ανβ3 integrin and normal ανβ3 integrin expressing cells (3T3) was verified in vitro, rather than just the cell recognition ability. The results show that the resolution of this thermal imaging technology can distinguish the boundary between the two cell populations, making it possible to accurately remove tumor tissue from normal tissue.

[0095] To detect thermal imaging in tumors, BALB / C nude mice (female, 4-5 weeks old, 16-18 g) were purchased from Southern Medical University Laboratory Animal Technology Development Co., Ltd. (Guangzhou, China) and were placed at 5 × 10 5200 μL of 4T1 cells were injected into the leg muscle at a density of 10 cells / mL. After 2 weeks of normal feeding, the tumor grew into a small tumor (about 5 mm in diameter). After isolating the leg muscle containing the tumor, it was immediately immersed in a complete culture medium suspension containing 100 μg / mL PDA-RGD and incubated for 10 minutes. After incubation, the sample was washed 3 times with PBS solution to remove unbound PDA-RGT. The sample was then irradiated with an 808 nm near-infrared laser at a power of 3 W / cm2 for 6 minutes, and the photothermal images and temperature values ​​of the leg muscles were recorded with an infrared thermal imager. In addition, different areas (tumor area, edge area, and normal tissue area) were marked in the thermal image to intuitively represent the obvious temperature difference and quantify the corresponding temperature changes in different areas. Fig.16 And its quantitative results Fig.18 As shown, the temperature rise of the tumor tissue area is significantly higher than that of the edge area and normal tissue area. Within the safe temperature range (below 45°C), the outline of the tumor can be clearly identified from the surrounding background tissue, so that the tumor can be further removed under the guidance of thermal imaging.

[0096] Example 5: Effect of PDA-RGD intraoperative photothermal imaging to guide tumor resection in vivo

[0097] 5 × 10 cells were cultured in 100 μL of 4T1 cells. 5 The peritoneal epithelial cells of mice were injected at a density of 10 cells / mL to establish a peritoneal tumor model. When the diameter of the suspected tumor area reached 2-3mm, it was used to simulate intraoperative thermal imaging to guide tumor resection. After cutting the abdominal skin to expose the tumor, a spray (1mL) containing 100μg / mL PDA-RGD was sprayed into the suspected tumor area and incubated for 10 minutes. After incubation, unbound PDA-RGD was removed with saline. Then, the power was 3W / cm 2 The suspected tumor area was irradiated with an 808nm near-infrared laser, and the photothermal images and temperature values ​​of the tumor-bearing mice were recorded with an infrared thermal imager. By marking different areas (tumor area, edge area, and normal tissue area) in the thermal image, the obvious temperature difference was visually represented and the corresponding temperature changes in different areas were quantified. Fig.17 As shown, thermal imaging results can clearly display and specifically locate tumors with a diameter less than 3 mm, making it possible to remove tiny tumors under the guidance of real-time thermal imaging. Fig.17 Quantification of thermal imaging results Fig.19 It was also confirmed that the temperature of the tumor was higher than that of the surrounding normal tissue.

[0098] Subsequently, the tumor was removed under the guidance of photothermal imaging, and the surgical process was recorded by video under the infrared thermal imager. Finally, H&E staining was used to further compare and analyze the tumor removal results in the above surgeries without and with photothermal imaging guidance. Fig. 20The results showed that under the guidance of PDA-RGD-mediated thermal imaging, the two tumors in the abdomen could be clearly and conveniently located, and the boundaries of normal tissue and tumor tissue could be intuitively identified. Subsequently, real-time thermal imaging-guided precise tumor resection surgery was performed in vivo. It is worth noting that under the guidance of thermal imaging, the lesions were completely removed. The results confirmed that under the guidance of nanospray-assisted thermal imaging, the location and boundaries of the tumor can be accurately identified, thereby facilitating radical resection.

[0099] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation, characterized in that: The steps include: (1) Synthesis of polydopamine / polyethyleneimine polymer dot composite nanospheres; (2) Grafting tumor targeting peptides to obtain a sprayable imaging agent with photothermal / fluorescence dual-modality imaging and capable of targeting tumors, namely the sprayable surgical auxiliary imaging agent based on in situ adaptation.

2. The method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 1, characterized in that: The synthesis of polydopamine / polyethyleneimine polymer dot composite nanospheres described in step (1) comprises the following steps: A. Dissolve dopamine in water to obtain solution A; B. mixing polyethyleneimine carbonized polymer dots, water, and an organic solvent to obtain a solution B; C. Add solution B to solution A, add ammonia solution, mix well, and react; collect the obtained reaction product by centrifugation, wash the obtained precipitate, and dry it to obtain polydopamine nanospheres.

3. The method for preparing the spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 2, characterized in that: The preparation method of polyethyleneimine carbonized polymer dots described in step B comprises the following steps: taking 50-300 mg of citric acid and 10-200 mg of polyethyleneimine polymer dots and dissolving them in 5 mL of 0.05M HCl aqueous solution, stirring evenly and placing them in a microwave reactor at 100-500W for 1-10 minutes; after the reaction is completed, cooling to room temperature, dialyzing, and freeze-drying to obtain polyethyleneimine carbonized polymer dots with fluorescent properties.

4. The method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 2, characterized in that: The dopamine described in step A is dopamine hydrochloride; The concentration of solution A described in step A is 45-55 mg / mL; The organic solvent in step B is at least one of methanol and ethanol; The concentration of the polyethyleneimine carbonized polymer dots in step B is 0.2-5 mg / mL; The volume ratio of water to organic solvent in step B is 5:8-10; The concentration of the ammonia solution in step C is 26% to 30% by volume; The amount of the ammonia solution described in step C is calculated based on the volume ratio of ammonia solution: solution A = 1:6-7.

5. The method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 4, characterized in that: The concentration of solution A described in step A is 50 mg / mL; The organic solvent described in step B is anhydrous ethanol; The concentration of the polyethyleneimine carbonized polymer dots in step B is 0.2-5 mg / mL; The volume ratio of water to organic solvent in step B is 5:9; The concentration of the ammonia solution described in step C is 28% by volume; The amount of the ammonia solution in step C is calculated as follows: ammonia solution: solution A = volume ratio 1:6.6; The reaction time in step C is 20 to 28 hours; The centrifugal conditions described in step C are 10000-14000 rpm for 15-30 min; The washing times described in step C are 2 to 4 times; The drying described in step C is vacuum drying.

6. The method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 1, characterized in that: The grafting of tumor targeting peptides in step (2) comprises the following steps: D. dispersing polydopamine / polyethyleneimine polymer dot composite nanospheres in an aqueous solution to obtain a PDA solution; E. Add RGD targeting peptide, lactose, glycoprotein, folic acid or biotin OC12 ligand molecules to the polydopamine solution for reaction; F. Centrifuge the obtained product, take the precipitate, wash, and dry to obtain the nanoaptamer PDA-ligands in the photothermal imaging agent.

7. The method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 6, characterized in that: The concentration of the PDA solution described in step D is 45-55 mg / mL; The mass of the ligand described in step E is calculated based on the mass of polydopamine at a mass ratio of polydopamine:ligand molecule=5:0.8-1.

2.

8. The method for preparing a spray-type surgical auxiliary imaging agent based on in-situ adaptation according to claim 6, characterized in that: The concentration of the PDA solution described in step D is 50 mg / mL; The mass of the ligand described in step E is calculated based on the mass of polydopamine at a mass ratio of polydopamine:ligand molecules = 5:1; The centrifugal conditions described in step F are 10000-14000 rpm for 15-30 min; The drying described in step F is vacuum drying.

9. A spray-type surgical auxiliary imaging agent based on in-situ adaptation, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the spray-type surgical auxiliary imaging agent based on in situ adaptation according to claim 9 in the preparation of anti-tumor drugs and tumor diagnostic agents.

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