Carboxylesterase-responsive theranostic probe, preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a diagnostic and therapeutic integrated probe responsive to carboxylesterase, and a preparation method and application thereof. The present invention discloses a small molecule diagnostic and therapeutic probe represented by the following structural general formula. This small molecule diagnostic and therapeutic probe is obtained by reacting a near-infrared fluorescent dye, chlorambucil, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent. The present invention encapsulates the above small molecule diagnostic and therapeutic probe with poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) to obtain a nano diagnostic and therapeutic integrated probe. The fluorescence wavelength of the obtained nano diagnostic and therapeutic integrated probe is located in the near-infrared region, which can realize near-infrared fluorescence imaging diagnosis of tumors, and has combined therapeutic properties of chemotherapy, photodynamic therapy, and sonodynamic therapy, and can significantly kill tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a carboxylesterase-responsive integrated diagnosis and treatment probe and a preparation method and application thereof. Background Art

[0002] The morbidity and mortality of malignant tumors continue to rise, seriously threatening human life and health, and also bringing a heavy economic burden to families and society. Therefore, it is urgent to develop early detection methods and new effective treatment methods for tumors. The integrated diagnosis and treatment probe based on molecular imaging integrates molecular imaging modules and tumor treatment modules at the same time, which can not only meet the needs of diagnosis and treatment at the same time, but also visualize the distribution and release process of drugs in the body. This imaging-guided treatment mode is conducive to real-time monitoring of treatment effects and timely optimization of treatment plans, and is expected to provide new solutions for achieving precise and personalized treatment of tumors. Carboxylesterase is overexpressed in many malignant tumors and is a potential tumor marker. Therefore, the development of integrated diagnosis and treatment probes responsive to carboxylesterase is of great significance for the early diagnosis and effective treatment of malignant tumors.

[0003] In recent years, near-infrared fluorescence imaging based on near-infrared fluorescent probes can effectively avoid background fluorescence interference from organisms, and has strong tissue penetration ability and little light damage to living organisms. It has been widely used in the research of early diagnosis of malignant tumors. Since chemical, photodynamic, and sonodynamic treatment methods all have their inherent shortcomings, multi-modality combined treatment can achieve better tumor inhibition effects. Therefore, the development of molecular probes that combine near-infrared fluorescence imaging and multi-modality combined treatment will provide new technical means for the integrated diagnosis and treatment of tumors.

[0004] However, there are few reports on near-infrared fluorescent probes responsive to carboxylesterase and their integrated applications in tumor diagnosis and treatment, which is a technical problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the technical problems existing in the above-mentioned prior art, the present invention provides a carboxylesterase-responsive nano-diagnostic and therapeutic integrated probe, the fluorescence wavelength of which is in the near-infrared region, which can realize near-infrared fluorescence imaging diagnosis of tumors, and has the performance of chemical, photodynamic, and sonodynamic combined treatment, which can significantly kill tumor cells.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a carboxylesterase-responsive small molecule diagnostic probe, the general structural formula of which is:

[0008]

[0009] In the formula, R is any one of methyl, ethyl and propyl.

[0010] In a second aspect, the present invention provides a method for preparing a small molecule diagnostic and therapeutic probe responsive to carboxylesterase, and the preparation method includes the following steps:

[0011] Step 1: Add a near-infrared fluorescent dye, chlorambucil, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) to an organic solvent for reaction;

[0012] Step 2: After the reaction ends, perform separation and purification to obtain the small molecule diagnostic and therapeutic probe.

[0013] Further, in the Step 1, the organic solvent is at least one of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, chloroform, acetonitrile, and methanol; the structural formula of the near-infrared fluorescent dye is:

[0014]

[0015] In the formula, R is any one of methyl, ethyl, and propyl.

[0016] Further, in the Step 1, the volume of the organic solvent is 1 - 20 mL; the molar ratio of the near-infrared fluorescent dye, chlorambucil, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine is 1:(1 - 5):(1 - 5):(0.005 - 0.05); the reaction time is 10 - 200 min, and the reaction temperature is -10 - 50 °C.

[0017] In a third aspect, the present invention provides a nano diagnostic and therapeutic integrated probe responsive to carboxylesterase, and the nano diagnostic and therapeutic integrated probe is obtained by encapsulating the aforementioned small molecule diagnostic and therapeutic probe with poly(lactic acid - co - glycolic acid)-polyethylene glycol (PLGA-PEG) through a microemulsion method.

[0018] In a fourth aspect, the present invention provides a method for preparing a nano diagnostic and therapeutic integrated probe responsive to carboxylesterase, and the preparation method includes the following steps:

[0019] Step 1: Add the aforementioned small molecule diagnostic and therapeutic probe and poly(lactic acid - co - glycolic acid)-polyethylene glycol to an organic solvent, perform ultrasonic treatment, and then add an aqueous solution of polyvinyl alcohol and continue the reaction;

[0020] Step 2: After the reaction ends, perform centrifugation to obtain the nano diagnostic and therapeutic integrated probe.

[0021] Further, in the Step 1, the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform, and cyclohexane.

[0022] Further, in the step 1, the volume of the organic solvent is 1 to 20 mL; the mass ratio of the small molecule diagnostic and therapeutic probe to poly(lactic-co-glycolic acid)-polyethylene glycol is 1:(1 to 15); the aqueous solution of polyvinyl alcohol is formed by dissolving 100 to 500 mg of polyvinyl alcohol in 5 to 50 mL of water; the ultrasonic time is 2 to 30 min, and the reaction time is 10 to 48 h.

[0023] Fifthly, the present invention provides an application of the aforementioned carboxylesterase-responsive nano diagnostic and therapeutic integrated probe in the preparation of a reagent for tumor diagnosis and / or tumor treatment.

[0024] Further, the tumor diagnosis is tumor near-infrared fluorescence imaging diagnosis; the tumor treatment is combined treatment of chemotherapy, photodynamic therapy and sonodynamic therapy.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. The analysis wavelength of the present invention is located in the near-infrared region, which can effectively avoid the interference of biological background fluorescence, has strong tissue penetration, and causes little light damage to the living body.

[0027] 2. The carboxylesterase-responsive nano diagnostic and therapeutic integrated probe prepared by the present invention can show a near-infrared fluorescence enhancement response to the carboxylesterase highly expressed in tumor cells, and can realize the near-infrared fluorescence imaging diagnosis of tumors.

[0028] 3. The carboxylesterase-responsive nano diagnostic and therapeutic integrated probe prepared by the present invention has the combined treatment performance of chemotherapy, photodynamic therapy and sonodynamic therapy, can significantly kill tumor cells and inhibit the growth of tumors in nude mice, and is an excellent diagnostic and therapeutic reagent.

[0029] 4. The carboxylesterase-responsive nano diagnostic and therapeutic integrated probe prepared by the present invention has a determined composition, has good biosafety, has no obvious toxicity to nude mice, and meets the basic requirements of clinical drug use.

[0030] 5. The method of the present invention has the advantages of simple operation, low cost, rapidity and sensitivity, and is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 1H NMR spectrum of the small molecule diagnostic and therapeutic probe HC synthesized in Example 1 1 1H NMR spectrum.

[0032] Figure 2 13C NMR spectrum of HC synthesized in Example 1 13 13C NMR spectrum.

[0033] Figure 3 High-resolution mass spectrum of HC synthesized in Example 1.

[0034] Figure 4Transmission electron microscopy image of the integrated nanodiagnostic probe PLGA-PEG@HC after encapsulation in Example 1.

[0035] Figure 5 Particle size distribution diagram of the integrated nanodiagnostic probe PLGA-PEG@HC after encapsulation in Example 1.

[0036] Figure 6 Absorption spectrum response diagram of the integrated nanodiagnostic probe PLGA-PEG@HC to carboxylesterase in Example 2, where curve a is the absorption spectrum of PLGA-PEG@HC (0.2 mg / mL), and curve b is the absorption spectrum after the reaction of PLGA-PEG@HC (0.2 mg / mL) with carboxylesterase (100 U / L).

[0037] Figure 7 Fluorescence response diagram of the integrated nanodiagnostic probe PLGA-PEG@HC to different concentrations of carboxylesterase in Example 2. The concentrations of carboxylesterase in the figure are 0, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 U / L from bottom to top.

[0038] Figure 8 Confocal imaging diagram of the integrated nanodiagnostic probe PLGA-PEG@HC on HeLa and HcerEpic cells in Example 3. The first column in the figure is the white light channel, and the second column is the fluorescence channel.

[0039] Figure 9 Confocal imaging diagram of the integrated nanodiagnostic probe PLGA-PEG@HC for pre-treating HeLa cells with the carboxylesterase inhibitor triphenyl phosphate in Example 3. The first row in the figure is the white light channel, and the second row is the fluorescence channel.

[0040] Figure 10 In vivo fluorescence imaging at different time points after tail vein injection of PLGA-PEG@HC into tumor-bearing nude mice in Example 4. The left side of each figure is the control group, and the right side is the experimental group.

[0041] Figure 11 Change in fluorescence intensity at 525 nm after 660 nm laser irradiation of DCFH-DA in PBS (curve a) and the reaction solution of PLGA-PEG@HC (0.2 mg / mL) with carboxylesterase (100 U / L) (curve b) in Example 5.

[0042] Figure 12 Change in absorbance at 425 nm after ultrasonic stimulation of DPBF in PBS (curve a) and the reaction solution of PLGA-PEG@HC (0.2 mg / mL) with carboxylesterase (100 U / L) (curve b) in Example 5.

[0043] Figure 13 Survival rates of HeLa cells (Figure A) and HcerEpic cells (Figure B) treated by different methods in Example 6.

[0044] Figure 14 Tumor volume change curves of nude mice in different treatment groups in Example 7.

[0045] Figure 15 Tumor pictures of nude mice in different treatment groups in Example 7 after 14 days of treatment.

[0046] Figure 16 Body weight change curves of nude mice in different treatment groups in Example 8.

[0047] Figure 17 Main serum biochemical indexes of nude mice in different treatment groups in Example 8 after 14 days of treatment.

[0048] Figure 18 Pathological section analysis of main organs of nude mice in different treatment groups in Example 8 after 14 days of treatment. Detailed implementation manners

[0049] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0050] Example 1: Preparation of carboxylesterase-responsive nano-theranostic probe PLGA-PEG@HC

[0051] The small molecule theranostic probe HC was synthesized according to the following route:

[0052]

[0053] Specific steps: Near-infrared fluorescent dye (410 mg, 1 mmol), chlorambucil (450 mg, 1.5 mmol), EDC (230 mg, 1.2 mmol), and DMAP (1.8 mg, 0.01 mmol) were mixed evenly in dichloromethane (5 mL) and reacted at room temperature for 90 min. After the mixed solution was concentrated under reduced pressure, it was purified by column chromatography to obtain the small molecule probe HC, which was a purple powder.

[0054] Encapsulation of the small molecule theranostic probe HC:

[0055] Weigh 1 mg of HC and 5 mg of PLGA-PEG, dissolve them separately in 1 mL of dichloromethane. At room temperature, after ultrasonic treatment for 5 min, mix the two, add dichloromethane to make the volume up to 4 mL, and continue ultrasonic treatment for 5 min. Dissolve polyvinyl alcohol (200 mg) in 10 mL of water and add the above organic phase, stir at room temperature for 24 h, and then perform centrifugation. The supernatant is the encapsulated nano-theranostic probe PLGA-PEG@HC.

[0056] The structure of the small molecule probe HC was confirmed as follows:

[0057] 1 1H NMR and 13 13C NMR spectra are shown in Figure 1 and 2 . 1 1H NMR (400 MHz, 298 K, CDCl3): δ 8.63 (d, 1H), 7.49 (m, 2H), 7.40 (d, 2H), 7.27 (s, 1H), 7.12 (d, 3H), 7.08 (s, 1H), 6.98 (m, 1H), 6.77 (d, 1H), 6.66 (d, 2H), 4.57 (t, 2H), 3.72 (t, 4H), 3.64 (t, 4H), 2.76 (d, 4H), 2.65 (m, 4H), 2.07 (t, 4H), 1.98 (t, 2H), 1.80 (s, 6H), 1.08 (t, 3H). 13 13C NMR (100 MHz, 298 K, CDCl3): δ 178.8, 171.7, 160.0, 153.1, 152.8, 146.4, 144.7, 142.2, 141.5, 131.1, 130.3, 130.2, 129.9, 129.5, 128.3, 128.0, 122.6, 119.8, 119.1, 115.6, 113.5, 112.4, 112.4, 109.5, 106.5, 53.7, 51.1, 47.7, 40.7, 34.0, 33.8, 29.6, 28.3, 28.3, 26.7, 24.3, 21.7, 20.3, 11.6.

[0058] High-resolution mass spectrometry: C 42 1 47 2 3+ H + Cl2N2O Figure 3 , M ; calculated value: 697.2958; measured value: 697.2954 (

[0059] ). Figure 4 Figure 5)It was confirmed that the encapsulation of the small molecule probe was successful, and the obtained nano-theranostic probe PLGA-PEG@HC was a uniformly sized sphere with a particle size of about 100 nm and good dispersibility.

[0060] Example 2. Optical response of the nano-theranostic probe PLGA-PEG@HC to carboxylesterase

[0061] Weigh 0.2 g of the nano-theranostic probe PLGA-PEG@HC and dissolve it in 10 mL of ultrapure water to prepare a probe stock solution (also called the mother liquor, with a concentration of 20 mg / mL); add 50 μL of the probe mother liquor to a certain amount of phosphate buffer solution (PBS, 0.01 M), then add carboxylesterase solutions with different concentrations, and finally make up the volume to 5 mL with PBS. After reacting at 37 °C for 10 minutes, measure the absorption spectrum and fluorescence emission spectrum. When measuring the fluorescence emission spectrum, it is excited at 670 nm, and the excitation and emission slit widths are 10 nm and 5 nm respectively.

[0062] Figure 6 The absorption spectrum response diagram of the nano-theranostic probe PLGA-PEG@HC to carboxylesterase is shown. Figure 6 In it, a is the absorption spectrum of PLGA-PEG@HC (0.2 mg / mL), and b is the absorption spectrum after the reaction of PLGA-PEG@HC (0.2 mg / mL) with carboxylesterase (100 U / L). Figure 7 The fluorescence response diagram of the nano-theranostic probe PLGA-PEG@HC to carboxylesterase is shown, and the concentration range of carboxylesterase is 0 - 100 U / L. Figure 7 The concentrations of carboxylesterase from bottom to top are 0, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 U / L respectively. The above experiments show that the nano-theranostic probe PLGA-PEG@HC exhibits a significant fluorescence enhancement response to carboxylesterase.

[0063] Example 3. Application of the nano-theranostic probe PLGA-PEG@HC in tumor cell imaging

[0064] (1) Under the conditions of 37 °C and 5% CO2, culture human cervical cancer cells HeLa and human normal cervical epithelial cells HcerEpic with DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0065] (2) Inoculate logarithmic-phase HeLa and HcerEpic cells into a confocal dish. After culturing for 24 h, discard the culture medium and wash three times with PBS (pH 7.4). Then co-incubate with PLGA-PEG@HC (1 mg / mL) prepared by the present invention for 30 min and place it for confocal microscopy imaging. The imaging excitation wavelength is 635 nm, and the emission wavelength collection range is 650 - 750 nm.

[0066] Figure 8 The confocal imaging diagrams of HeLa and HcerEpic cells are shown. It can be seen that HeLa cells show bright red fluorescence, while normal cells HcerEpic show weak fluorescence, indicating that PLGA-PEG@HC can perform selective fluorescence imaging on tumor cells.

[0067] Figure 9 The confocal imaging diagram of HeLa cells pre-incubated with PLGA-PEG@HC for the carboxylesterase inhibitor triphenyl phosphate is shown. The fluorescence of the cells pre-treated with triphenyl phosphate in the inhibitor group is significantly reduced, indicating that the enhanced fluorescence response of PLGA-PEG@HC in tumor cells is caused by the overexpressed carboxylesterase in tumor cells.

[0068] Example 4: In Vivo Imaging of Tumors in Nude Mice Using the Nanotheranostic Probe PLGA-PEG@HC

[0069] (1) This experiment was approved by the Animal Ethics and Use Committee of Shanxi Medical University. The experimental animals were 5-week-old BALB / c nude mice, with a body weight of 15 g ± 1 g per mouse, and all were raised in a sterile environment. A tumor model was established using HeLa cells. The cells were diluted at a density of 5×10 6 cells / mL, placed on ice, and after anesthetizing the nude mice with 6% chloral hydrate, 200 μL of the cell suspension was subcutaneously injected into the right axilla of the nude mice. The body weight of the nude mice was weighed every other day and the tumor size was measured. The tumor volume was calculated according to the formula V = a*b 2 / 2 (a is the major axis and b is the minor axis), and the imaging experiment was carried out when the volume exceeded 50 mm 3 .

[0070] (2) Take the nude mice with successful modeling, and inject PLGA-PEG@HC (final concentration 10 mg / mL) via the tail vein. Perform fluorescence imaging on the nude mice at different time points to observe the distribution of fluorescence in vivo. The excitation wavelength during imaging is 670 nm, and the emission wavelength is 750 nm.

[0071] Figure 10The in vivo fluorescence imaging of nude mice at different time points is shown. In the control group (PBS was injected via the tail vein), there was no fluorescence throughout. However, the tumors of nude mice injected with PLGA-PEG@HC emitted fluorescence at 2 h, reached the maximum at 8 h, and the fluorescence intensity at the tumor site was much higher than that at other sites. This indicates that PLGA-PEG@HC can passively target the tumor site of nude mice and specifically turn on the fluorescence signal, showing potential application value in tumor imaging diagnosis.

[0072] Example 5, In vitro photodynamic and sonodynamic effects of the nano-theranostic probe PLGA-PEG@HC

[0073] To evaluate the photodynamic and sonodynamic effects of PLGA-PEG@HC, the commercial reactive oxygen species probes 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and 1,3-diphenylisobenzofuran (DPBF) were used to evaluate the performance of the system in generating reactive oxygen species under laser irradiation and ultrasonic stimulation, respectively. The fluorescence intensity of DCFH-DA at 525 nm is positively correlated with the concentration of reactive oxygen species, and the absorbance of DPBF at 425 nm is negatively correlated with the concentration of singlet oxygen. PLGA-PEG@HC (0.2 mg / mL) was reacted with carboxylesterase (100 U / L) in a shaker at 37 °C for 20 min. Activated DCFH-DA (5 nM) was added to the reaction system, and then the reaction solution was irradiated with a 660 nm laser for a certain time, and the fluorescence spectrum was measured to evaluate its photodynamic performance. The fluorescence excitation wavelength was 490 nm. DPBF (20 μg / mL) was added to the reaction system, and after ultrasonic treatment (1 W / cm 2 ) at different times, the absorbance at 425 nm was recorded to evaluate its sonodynamic performance.

[0074] Figure 11 The figure shows the change of fluorescence at 525 nm with laser irradiation time for different systems (a is DCFH-DA, b is the reaction solution of DCFH-DA, PLGA-PEG@HC and carboxylesterase). It can be seen that with the extension of laser irradiation time, the fluorescence of DCFH-DA at 525 nm in the reaction system of PLGA-PEG@HC and carboxylesterase gradually increases, while laser irradiation hardly affects the fluorescence of DCFH-DA itself. This indicates that the reaction system of PLGA-PEG@HC and carboxylesterase can generate reactive oxygen species under 660 nm laser irradiation and has the potential for photodynamic therapy.

[0075] Figure 12The figure shows the change in absorbance at 425 nm of different systems (a is DPBF, b is the reaction solution of DPBF with PLGA-PEG@HC and carboxylesterase) with ultrasonic time. It can be seen that as the ultrasonic time extends, the absorbance of DPBF at 425 nm in the reaction system of PLGA-PEG@HC and carboxylesterase gradually decreases, while the absorbance of DPBF itself is hardly affected by ultrasound. This indicates that the reaction system of PLGA-PEG@HC and carboxylesterase has the ability to generate singlet oxygen under ultrasonic stimulation and can be used for sonodynamic therapy.

[0076] Example 6: The nano-diagnosis and treatment integrated probe PLGA-PEG@HC is used for selective killing of tumor cells

[0077] The killing performance of PLGA-PEG@HC on different cells was evaluated by the MTT method. Logarithmic growth phase HeLa and HcerEpic cells were seeded in 96-well plates. After adherent culture for 24 h, different concentrations of PLGA-PEG@HC were added and incubated for 24 h. The culture medium was discarded, and after washing 3 times with PBS, the laser group was irradiated with 660 nm laser (0.25 W / cm 2 ) for 10 min, the ultrasound group was treated with intermittent ultrasound (0.75 W / cm 2 ) for 5 min (i.e., ultrasound for 15 s, intermittent for 15 s, a total of 5 min), the combined group was treated with the superposition of laser and ultrasound, and the control group was placed in a dark environment. After the cells were washed 3 times with PBS, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and after continuing to incubate for 4 h, the MTT was discarded. After washing with PBS, 150 μL of DMSO was added. Finally, the absorbance at 490 nm was measured using an enzyme-labeling instrument to calculate the cell survival rate.

[0078] Figure 13Shown are the survival rates of HeLa (Figure A) and HcerEpic (Figure B) cells after being treated in different ways. It can be seen that as the concentration of PLGA-PEG@HC increases, the survival rate of HeLa cells gradually decreases. When the concentration is 2.0 mg / mL, the survival rate of HeLa cells is only 66%, while the survival rate of HcerEpic cells is still as high as 86%. This is because the overexpressed carboxylesterase in HeLa cells triggers the hydrolysis of the probe, and the released chemotherapeutic drug chlorambucil has a killing effect on the cells. In addition, in the presence of 660 nm laser or ultrasound stimulation alone, the survival rate of HeLa cells incubated with PLGA-PEG@HC further decreases, indicating that PLGA-PEG@HC has sonodynamic and photodynamic killing abilities at the cellular level; when both ultrasound and laser are present, the survival rate of HeLa cells is the lowest, only 18%, while the survival rate of HcerEpic cells is still above 60% under the same conditions. These results show that the probe PLGA-PEG@HC can kill tumor cells through the combination of chemotherapy, photodynamic therapy and sonodynamic therapy, and has low toxicity and side effects on normal cells.

[0079] Example 7: Application of the integrated nano-diagnosis and treatment probe PLGA-PEG@HC in anti-tumor treatment of nude mice

[0080] The tumor model nude mice were established in the same way as in Example 4. Treatment started when the tumor volume exceeded 100 mm 3 . The nude mice were randomly divided into 7 groups (n = 3) and corresponding treatment methods were adopted. The grouping is as follows: PBS group, PBS + laser (L) + ultrasound (US) group, HC + L + US group, PLGA-PEG@HC group, PLGA-PEG@HC + US group, PLGA-PEG@HC + L group and PLGA-PEG@HC + L + US group. During treatment, the probe was injected into the nude mice through the tail vein (100 μL, where the concentration of PLGA-PEG@HC was 10 mg / mL and the concentration of HC was 13.95 μg / mL). 8 h after injection, the laser group irradiated the tumor site with a 660 nm laser (0.75 W / cm 2 ) for 10 min, and the ultrasound group treated the tumor with an ultrasonic instrument (1.5 W / cm 2 ). The combined group was treated with the superposition of laser and ultrasound. Treatment was carried out once every two days for a total of 3 times. During treatment, the long and short diameters of the tumor were measured every other day, and the tumor volume was calculated according to the formula V = a * b 2 / 2 (a is the long diameter and b is the short diameter). After the treatment was completed, the tumors were dissected and photographed.

[0081] Figure 14 Shown are the tumor volume change curves of nude mice in different groups, Figure 15Tumor pictures of nude mice in different groups after 14 days of treatment. These results indicate that PLGA-PEG@HC can exert anti-tumor effects by combining chemotherapy, photodynamic therapy and sonodynamic therapy.

[0082] Example 8. Biosafety of the nanotheranostic probe PLGA-PEG@HC

[0083] In Example 7, the body weights of nude mice were recorded every other day during the 14-day treatment period. After the 14-day treatment period, the nude mice were anesthetized and blood was collected from the orbital cavity. The collected blood samples were centrifuged at 3000 rpm / min for 10 min, and the upper serum was taken to detect the liver function indexes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the kidney function indexes blood urea nitrogen (BUN) and creatinine (CRE) using a biochemical analyzer; after the above operations, the nude mice were sacrificed, and the heart, liver, spleen, lungs and kidneys were taken out, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin, and observed under a fluorescence microscope.

[0084] Figure 16 The body weight change curves of nude mice in different groups are shown. The body weights of nude mice in each group all increased slightly; Figure 17 The main serum biochemical indexes of nude mice in different groups after 14 days of treatment are shown. It can be seen that there are no significant differences in the liver and kidney function indexes of nude mice in each treatment group compared with the control group; Figure 18 The pathological section analysis of the main organs of nude mice in different groups after 14 days of treatment is shown. The results show that there are no obvious abnormalities in the main organs. These results all indicate that the nanotheranostic probe PLGA-PEG@HC has good biosafety, and there are no obvious side effects in tumor treatment based on this probe.

[0085] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of a carboxylesterase-responsive nano-theranostic probe in the preparation of a reagent for tumor diagnosis and / or tumor treatment, characterized in that, The nano-theranostic probe is obtained by encapsulating a small molecule theranostic probe represented by the following formula with poly(lactic acid-co-glycolic acid)-polyethylene glycol through the microemulsion method; In the formula, R is any one of methyl, ethyl, and propyl; The tumor diagnosis is tumor near-infrared fluorescence imaging diagnosis; the tumor treatment is combined treatment of chemotherapy, photodynamic therapy, and sonodynamic therapy.

2. Use of a nano-diagnosis and treatment integrated probe responsive to carboxylesterase according to claim 1, characterized in that, The preparation method of the small molecule theranostic probe includes the following steps: Step 1: React near-infrared fluorescent dye, chlorambucil, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine in an organic solvent; Step 2: After the reaction is completed, separate and purify to obtain the small molecule theranostic probe.

3. Use of a nano-theranostic probe responsive to carboxylesterase according to claim 2, characterized in that, In Step 1, the organic solvent is at least one of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, chloroform, acetonitrile, and methanol; the structural formula of the near-infrared fluorescent dye is: In the formula, R is any one of methyl, ethyl, and propyl.

4. Use of a nano-theranostic probe responsive to carboxylesterase according to claim 2, characterized in that, In Step 1, the volume of the organic solvent is 1-20 mL; the molar ratio of the near-infrared fluorescent dye, chlorambucil, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine is 1:(1-5):(1-5):(0.005-0.05); the reaction time is 10-200 min, and the reaction temperature is -10-50 °C.

5. Use of a nano-diagnosis and treatment integrated probe responsive to carboxylesterase according to claim 1, characterized in that, The preparation method of the nano-theranostic probe includes the following steps: Step 1: Add the small molecule theranostic probe and poly(lactic acid-co-glycolic acid)-polyethylene glycol in an organic solvent, perform ultrasonic treatment, and then add an aqueous solution of polyvinyl alcohol and continue the reaction; Step 2: After the reaction is completed, perform centrifugation to obtain the nano-theranostic probe.

6. Use of a nanotheranostic probe responsive to carboxylesterase according to claim 5, characterized in that, In Step 1, the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform, and cyclohexane.

7. Use of a nano-theranostic probe responsive to carboxylesterase according to claim 5, characterized in that, In Step 1, the volume of the organic solvent is 1-20 mL; the mass ratio of the small molecule theranostic probe to poly(lactic acid-co-glycolic acid)-polyethylene glycol is 1:(1-15); the aqueous solution of polyvinyl alcohol is formed by dissolving 100-500 mg of polyvinyl alcohol in 5-50 mL of water; the ultrasonic time is 2-30 min, and the reaction time is 10-48 h.

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