CN114767881B - Preparation and application of tumor microenvironment response type diagnosis and treatment integrated nano probe - Google Patents
Preparation and application of tumor microenvironment response type diagnosis and treatment integrated nano probe Download PDFInfo
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Abstract
The invention relates to the fields of life medicine and inorganic nano materials, in particular to preparation and application of a tumor microenvironment response type diagnosis and treatment integrated nano probe. The probe takes a hexagonal palladium nano-sheet as a main body, and a near infrared two-region fluorescent small organic molecule BSA-CQ4T modified by bovine serum albumin is connected in an amidation mode through an amino acid short chain which can be identified and sheared by fibroblast activation protein, and is loaded with a reducing nano-silver, so that the effects of multi-mode imaging and treatment are achieved. The material cuts off the short chain of amino acid in the tumor microenvironment, separates BSA-CQ4T from the palladium nano-sheet, starts the fluorescence of BSA-CQ4T in the near infrared two regions, and illuminates the tumor area. Under the action of the hydrogen peroxide concentration environment with higher concentration in the tumor microenvironment, nano silver is oxidized to form silver ions, the photoacoustic signal of the near infrared first-area material is reduced, and silver ion treatment can be performed. And the photoacoustic signal of the palladium nano-sheet in the near infrared two regions is not changed, so that the proportional photoacoustic imaging effect is achieved.
Description
Technical Field
The invention relates to the fields of life medicine and inorganic nano materials, in particular to preparation and application of a tumor microenvironment response type diagnosis and treatment integrated nano probe.
Background
Tumors seriously endanger the life health of human beings and are also difficult to diagnose and treat in medicine. New, efficient and safe tumor diagnosis and treatment methods are required to be explored constantly. Due to the unique properties of the nano material, the nano material is continuously developed in the technical field of molecular imaging and the field of accurate medicine, and a new field of view is provided for diagnosis and treatment of tumors.
Compared with the traditional image development technology, the nano diagnosis and treatment system has remarkable advantages and great potential in the aspects of early diagnosis of tumors, tracking of distant metastasis, personalized treatment, curative effect monitoring and evaluation and the like. Photoinduced imaging such as photoacoustic imaging (Photoacoustic Imaging, PAI) and fluorescence imaging (fluorescence imaging), photoinduced therapeutic Light-induced imaging and treatment technology has been attracting attention in oncology. Near infrared region II (NIR-II, 1000-1700 nm) imaging has higher signal-to-noise ratio, better imaging quality and deeper tissue penetration than conventional optical imaging in the visible (400 nm-650 nm) and first near infrared window (NIR-I, 650nm-900 nm). Since the wavelength of light in the NIR-II region is longer, the scattering intensity of light decreases exponentially with increasing wavelength. NIR-II FL imaging can directly and rapidly display interesting dynamic biological tissues, and has higher space-time resolution and sensitivity. NIR-II-PA imaging combines the advantages of optical imaging and acoustic imaging. Dual mode optical imaging employing NIR-II-FL imaging and PA imaging may enable more comprehensive and accurate diagnostic information than single imaging modes. In addition, most nano diagnosis and treatment systems enter a tumor area through passive targeting or active targeting, weak development of materials remained in normal tissues and nonspecific background signals of healthy tissues are easy to interfere with accurate diagnosis, and detection sensitivity is reduced.
Early studies have shown that Tumor Microenvironment (TME) is closely related to the occurrence, progression and metastasis of tumors. The characteristic acidic, low-oxygen, redox disorder environment, a plurality of abnormally high-expressed cytokines and the like are helpful for developing various diagnosis and treatment systems depending on TME activation. By using TME as a switch to design the nano material, the sensitivity and accuracy of detection can be greatly improved. Therefore, from the sensitivity, accuracy and comprehensiveness of diagnosis, the nanoparticle which is required to be activated by specific tumor environment and has two modes of imaging is designed and developed, namely, inorganic palladium nano-sheets are used as main bodies, fluorescent molecules CQ4T are connected through short chains of amino groups recognized and sheared by fiber-forming activated proteins which can be specifically and highly expressed by the epithelial cancers, and the fluorescent molecules CQ4T are loaded with reduced nano-silver, so that two modes of fluorescence imaging and ratio photoacoustic imaging of tumor microenvironment activation are realized.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an intelligent response type near infrared two-region multi-mode imaging treatment integrated nano probe capable of aiming at a tumor microenvironment and a preparation method thereof. The material can be identified by tumor-specific high-expression tumor-associated Fibroblast Activation Protein (FAP), fluorescence is started in a near infrared two-region, proportional photoacoustic imaging is generated under the action of TME high-expression hydrogen peroxide, silver ions are released, and the excellent photo-thermal conversion efficiency of the material is matched while the tumor diagnosis effect with high sensitivity and high accuracy is achieved, so that photo-thermal treatment and ion treatment in the near infrared two-region are synchronously realized.
In order to solve the technical problems, the invention adopts the following technical scheme:
the tumor microenvironment response type diagnosis and treatment integrated nano probe comprises a palladium nano sheet, nano silver, fluorescent organic micromolecule BSA-CQ4T and an amino acid short chain; the palladium nano-sheet accounts for 43.4wt% of the nano-probe, the nano-silver accounts for 1.6wt% of the nano-probe, the fluorescent organic micromolecule BSA-CQ4T accounts for 40wt% of the nano-probe, and the amino acid short chain accounts for 15wt% of the nano-probe.
Further, the short chain of amino acids may be cleaved by fibroblast activation protein recognition; the fluorescent small organic molecule CQ4T is modified by bovine serum albumin; the nano silver adopts reducing nano silver; the nano probe takes palladium nano sheets as a main body; the amino acid short chain is connected with fluorescent small organic molecule BSA-CQ4T in an amidation mode.
Further, the edge length of the nano probe is 20-40nm.
A preparation method of tumor microenvironment response type diagnosis and treatment integrated nano-probe comprises the following steps:
s1, pd (acac) 2 Mixing powder, polyvinylpyrrolidone powder and sodium bromide powder, dissolving in a mixed solution of deionized water and N, N-dimethylformamide, placing into a glass pressure container, and stirring in a water bath at 30deg.C until the powder, the polyvinylpyrrolidone powder and the sodium bromide powder are dissolved to obtain a bright pale yellow transparent solution;
s2, introducing CO gas into the container where the solution obtained in the step S1 is located at the temperature of 4 ℃ and discharging clean air;
s3, placing the S2 container into an oil bath pot at 80 ℃, keeping CO continuously introduced and discharged, and stirring for 3 hours;
s4, removing the S3 container from the oil bath, closing the gas inlet and the gas outlet of CO gas, cooling to room temperature, and taking out the solution to obtain a dark blue near black solution;
s5, washing the solution obtained in the step S4 with acetone, and centrifuging at a high speed to obtain blue-black precipitate;
s6, precipitating the precipitate obtained in the step S5 by using ethanol: washing the mixed solution with the acetone volume ratio of 1:8, and centrifuging at a high speed to obtain blue-black precipitate;
s7, vacuum drying the precipitate obtained in the step S6, and preserving at room temperature to obtain solid palladium nano-sheets Pd NSs;
s8, dissolving fluorescent small organic molecule BSA-CQ4T and albumin in a mass ratio of 1:5-1:10 in a phosphate buffer solution, stirring at 70 ℃ to obtain a BSA-CQ4T solution, and storing at a dark place at 4 ℃;
s9, the mass ratio is 2:1-1:5, mixing the solid palladium nano-sheet Pd NSs obtained in the step 7 with an amino acid short-chain phosphate buffer solution, slowly stirring in a CO atmosphere at 4 ℃, ultrafiltering, centrifuging, and freeze-drying to obtain a dark blue near black solid Pd-pep;
s10, activating the solid Pd-pep obtained in the S9 in a phosphate buffer solution, performing ultrafiltration and centrifugation after activation, taking an upper solution, adding a BSA-CQ4T solution in the S8, and slowly stirring in an ice-water bath in a dark place under a CO atmosphere to obtain liquid Pd-pep-BSA-CQ4T;
s11, adding the liquid Pd-pep-BSA-CQ4T obtained in the S10 into a silver nitrate solution and a reducing agent in sequence under light-shielding and slow stirring, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
Further, pd (acac) as described in step S1 2 The mass ratio of the powder to the polyvinylpyrrolidone powder to the sodium bromide powder is 50:30:52.2, and the volume ratio of the deionized water to the dimethylformamide is 2:8; the polyvinylpyrrolidone mw=29000.
Further, the centrifugation speed in the step S5 and the step S6 is 10000rpm, and the centrifugation time is 8min; step S5, washing and centrifuging are repeated for 2 times; step S6 washing and centrifugation are repeated 3 times.
Further, the albumin in step S8 is bovine serum albumin.
Further, the amino acid sequence of the short chain amino acid in step S9 is Ac-Asp-Ala-Gly-Pro-Asn-Gln-Cys-NH 2 。
Further, the reducing agent in step S11 is 0.1M L-ascorbic acid, 0.1M NaOH; the mass concentration of the silver nitrate solution substance is 2.5-10 -3 M。
The application of the tumor microenvironment response type diagnosis and treatment integrated nano probe is that the nano probe or the nano probe prepared by the method is applied to photo-thermal combined ion therapy under the guidance of FAP response type near infrared two-region fluorescence imaging and proportional photo-acoustic imaging.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a simple synthesis method of a nano probe capable of intelligently responding to near infrared two-region activatable fluorescence imaging, photoacoustic imaging and photothermal treatment of a tumor microenvironment. The probe takes a uniform hexagonal palladium nano-sheet as a main body, and a near infrared two-region fluorescent organic small molecule BSA-CQ4T modified by Bovine Serum Albumin (BSA) is connected in an amidation mode through an amino acid short chain which can be identified and sheared by tumor-related fibroblast activation protein (Fibroblast Activation Protein, FAP), and is loaded with reducing nano-silver, so that the multi-modal imaging and treatment effects are achieved. When the material is used for normal biological tissues with the PH of 7.4, the fluorescence emission area of the organic fluorescent micromolecule is overlapped with the palladium nano-sheet absorption area, so that fluorescence is quenched; in tumor microenvironments (breast cancer, lung cancer and the like), namely when the tumor-associated fibroblasts are enriched, the amino acid short chain is cut off, BSA-CQ4T is separated from the palladium nano-sheet, and fluorescence of the BSA-CQ4T in a near infrared two-region is started to illuminate a tumor region. Meanwhile, in the tumor microenvironment, under the action of the hydrogen peroxide concentration (50-100 mu M) environment with higher concentration, nano silver is oxidized to form silver ions, the photoacoustic signal of the near infrared first-region material at 660nm is reduced, and the photoacoustic signal of the palladium nano sheet at the near 1050nm infrared second-region is unchanged, so that the proportional photoacoustic imaging effect is achieved. In addition, the palladium nano-sheet has excellent photo-thermal conversion efficiency, has high absorption peak in the near infrared two regions, can be used as an excellent photo-thermal initiator, and has the effect of photo-thermal treatment in the near infrared two regions. According to the method, the palladium nano-sheet with a proper size is designed and regulated, so that the palladium nano-sheet has high photo-thermal conversion efficiency and photo-acoustic imaging contrast effect in a near infrared two-region, an absorption region of the palladium nano-sheet is highly overlapped with a fluorescence emission region of BSA-CQ4T, and the palladium nano-sheet is firmly connected with the BSA-CQ4T through amidation, so that fluorescence of a product can be quenched in healthy tissues with a physiological condition PH of 7.4, and the palladium nano-sheet is separated from the BSA-CQ4T under the action of the fiber-forming activated protein with high specificity expressed by epithelial cancer, and the fluorescence of the near infrared two-region is started. In addition, silver ions are released under the action of hydrogen peroxide, so that proportional photoacoustic imaging is realized. I.e., under the guidance of activatable NIR-IIFL/proportional PA imaging, the 'localization timing' photo-thermal ion combined therapy is performed. The method is simple and easy to implement, and has the advantages that the proper size and the fusion proportion are found out, the detection sensitivity is improved, the diagnosis and treatment integration is achieved, and the effect is obvious.
Drawings
FIG. 1 is an EDX spectrum of a nanoprobe synthesized in example 1 of the present invention.
FIG. 2 is a TEM image of a nanoprobe synthesized in example 1 of the present invention.
FIG. 3 is an XPS image of the nano-probe synthesized in example 1 of the present invention, and the X-ray photoelectron spectrum of Pd element.
FIG. 4 is an XPS image of the nano-probe synthesized in example 1 of the present invention, and X-ray photoelectron spectrum of Ag element.
FIG. 5 shows the photo-thermal properties of the nanoprobe synthesized in example 1 of the present invention at different concentrations.
FIG. 6 is a fluorescence spectrum of the nanoprobe synthesized in example 1 of the present invention.
FIG. 7 is a study of cytotoxicity CCK-8 of the nanoprobe synthesized in example 1 of the present invention.
FIG. 8 is a fluorescence image of the synthesized nanoprobe of example 1 of the present invention at the cell level.
Fig. 9 is a proportional photoacoustic imaging of the nanoprobe synthesized in example 1 of the present invention at the cell level.
FIG. 10 is a fluorescence imaging of a nude mouse with a nanoprobe synthesized in example 1 of the present invention.
Fig. 11 is a photo-acoustic imaging diagram of a nude mouse living body of the nano probe synthesized in example 1 of the present invention.
FIG. 12 is a graph showing the photo-thermal treatment effect of the nano-probe synthesized in example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The tumor microenvironment responsive diagnosis and treatment integrated nano probe takes a uniform hexagonal palladium nano sheet as a main body, bovine serum albumin modified near infrared two-region fluorescent organic micromolecule BSA-CQ4T is connected in an amidation mode through an amino acid short chain which can be identified and sheared by fibroblast activation protein, and is loaded with reducing nano silver, wherein the palladium nano sheet accounts for 43.4wt% of the nano probe, the nano silver accounts for 1.6wt% of the nano probe, the amino acid short chain accounts for 30wt% of the nano probe, and the fluorescent organic micromolecule BSA-CQ4T accounts for 25wt% of the nano probe.
In this embodiment, the edge length of the nanoprobe is 20-40nm.
A preparation method of tumor microenvironment response type diagnosis and treatment integrated nano-probe comprises the following steps:
s1, pd (acac) 2 Mixing powder, polyvinylpyrrolidone powder and sodium bromide powder, dissolving in a mixed solution of deionized water and N, N-dimethylformamide, placing into a glass pressure container, and stirring in a water bath at 30deg.C until the powder, the polyvinylpyrrolidone powder and the sodium bromide powder are dissolved to obtain a bright pale yellow transparent solution;
s2, introducing CO gas into the container where the solution obtained in the step S1 is located at the temperature of 4 ℃ and discharging clean air;
s3, placing the S2 container into an oil bath pot at 80 ℃, keeping CO continuously introduced and discharged, and stirring for 3 hours;
s4, removing the S3 container from the oil bath, closing the gas inlet and the gas outlet of CO gas, cooling to room temperature, and taking out the solution to obtain a dark blue near black solution;
s5, washing the solution obtained in the step S4 with acetone, and centrifuging at a high speed to obtain blue-black precipitate;
s6, precipitating the precipitate obtained in the step S5 by using ethanol: washing the mixed solution with the acetone volume ratio of 1:8, and centrifuging at a high speed to obtain blue-black precipitate;
s7, vacuum drying the precipitate obtained in the step S6, and preserving at room temperature to obtain solid palladium nano-sheets Pd NSs;
s8, dissolving fluorescent small organic molecule BSA-CQ4T and albumin in a mass ratio of 1:5 to 1:10 in a phosphate buffer solution, stirring at 70 ℃ to obtain a BSA-CQ4T solution, and storing at a dark place at 4 ℃;
s9, the mass ratio is 2:1 to 1:5, mixing the solid palladium nano-sheet Pd NSs obtained in the step 7 with an amino acid short-chain phosphate buffer solution, slowly stirring in a CO atmosphere at 4 ℃, ultrafiltering, centrifuging, and freeze-drying to obtain a dark blue near black solid Pd-pep;
s10, activating the solid Pd-pep obtained in the S9 in a phosphate buffer solution, performing ultrafiltration and centrifugation after activation, taking an upper solution, adding a BSA-CQ4T solution in the S8, and slowly stirring in an ice-water bath in a dark place under a CO atmosphere to obtain liquid Pd-pep-BSA-CQ4T;
s11, adding the liquid Pd-pep-BSA-CQ4T obtained in the S10 into a silver nitrate solution and a reducing agent in sequence under light-shielding and slow stirring, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
Polyvinylpyrrolidone mw=29000 as described in step S1. The centrifugation speed in step S5 and step S6 is 10000rpm. The amino acid sequence of the amino acid short chain in the step S9 is Ac-Asp-Ala-Gly-Pro-Asn-Gln-Cys-NH 2 。
The reducing agent in the step S11 is 0.1M L-ascorbic acid and 0.1M NaOH; the mass concentration of the silver nitrate solution substance is 2.5-10 -3 M. The albumin is bovine serum albumin.
The nano probe has the functions of FAP response type near infrared two-region fluorescence imaging, tumor microenvironment proportion photoacoustic imaging and photo-thermal combined ion therapy.
In this example, BSA-CQ4T fluorescent molecules were derived from Monitoring the Real-Time Circulatory System-Related Physiological and Pathological Processes In Vivo Using a Multifunctional NIR-II probes.
Example 1
50mg of Pd (acac) 2 The powder, 30mg of PVP (mw=29000) powder, 52.2mg of NaBr powder were mixed, dissolved in a mixed solution of 2mL of deionized water and 8mL of n, n-dimethylformamide, placed in a glass pressure vessel, stirred in a 30 ℃ water bath for 30min, and the material was fully dissolved to obtain a bright orange-yellow transparent solution. And (3) rapidly introducing CO gas into the obtained solution at the temperature of 4 ℃ for 1min to clean the air in the container, rapidly placing the container into an oil bath pot at the temperature of 80 ℃ after CO is filled, keeping CO continuously introduced and discharged, and heating and stirring for 3h at the temperature of 80 ℃. And after 3h, closing the gas inlet and the gas outlet, rapidly cooling the ice water bath to room temperature, and taking out the solution to obtain the dark blue near black solution. 12mL of acetone was added and centrifuged at 10000rpm for 8min, and the supernatant was discarded to give a blue-black precipitate. This step was repeated 2 times. Ethanol is used for: the volume ratio of acetone is 1:8, centrifuging at 10000rpm for 10min, and discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was combined with bovine serum albumin at 1:10 in a phosphate buffer solution, and stirring at 70 ℃ for 5min. The obtained solution is BSA-CQ4T and is stored at 4 ℃ in dark.
Taking 10mg of the blue-black solid obtained in the above, dissolving in 20mL of phosphoric acid buffer solution, adding 10mg of customized amino acid short chain, slowly stirring for 24h in CO atmosphere at 4 ℃, centrifuging by using an ultrafiltration centrifuge tube, and freeze-drying to obtain a dark blue near-black solid, namely Pd-pep.
2mg of Pd-pep solid is taken and dissolved in phosphate buffer, and then stirred slowly in ice bath, 0.23mg of EDC and 0.14mg of NHS are added in turn, stirred slowly in CO atmosphere for 24 hours, centrifuged by an ultrafiltration centrifuge tube, the upper liquid is taken, 100 mu L of BSA-CQ4T (10 mg/mL) solution is added, and stirred slowly at 4 ℃ in dark place for 24 hours.
Adding silver nitrate aqueous solution (2.5×10) in sequence under slow stirring in the absence of light -3 M) 0.5mL, L-ascorbic acid aqueous solution (0.1M) 0.7mL, sodium hydroxide aqueous solution (0.1M) 0.3mL, stirring for 20min, and ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nano-probe.
The prepared tumor microenvironment response type diagnosis and treatment integrated nano probe is dissolved in PBS buffer solution, and is administrated through intravenous injection from a rat tail, so that the imaging performance and the treatment effect of the nano probe are detected.
Fig. 1 is an EDX spectrum of a tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. The figure shows that the synthesized bimodal imaging guided photothermal combined ion therapy nano probe has the energy spectrum analysis, and the Ag and S elements well dispersed are distributed in the Pd element-containing area, thus proving the successful preparation of the probe.
Fig. 2 is a TEM image of a tumor microenvironment responsive diagnosis and treatment integrated nano-probe synthesized in example 1 of the present invention. From the figure, the synthesized nano probe has a hexagonal shape and an average diameter of 51.83nm.
Fig. 3 and 4 are XPS diagrams of tumor microenvironment responsive diagnosis and treatment integrated nanoprobes synthesized in example 1 of the present invention. In the figure, 3 is the X-ray photoelectron spectrum of Pd element, and in the figure, 4 is the X-ray photoelectron spectrum of Ag element. As can be seen from FIG. 3, the two peaks at 335.2eV and 340.4eV represent the bond energies of Pd 3d5/2 and Pd 3d3/2 of the metal Pd (0). As can be seen from FIG. 4, the high resolution results for Ag 3d are at 642.33eV (Ag 3d 5/2) and 653.84eV (Ag 3d 3/2), which are related to the characteristic peaks of Ag (0).
Fig. 5 shows the photo-thermal properties of different concentrations of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in the embodiment 1 of the present invention. From the figure, the diagnosis and treatment integrated nano probe with different concentrations can be used for near infrared irradiation (1064 nm, 1W/cm) 2 ) The lower temperature rise curve shows that the higher the concentration, the faster the temperature rise, and the higher the temperature increase with irradiation time.
FIG. 6 is a fluorescence spectrum of a tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. From the figure, BSA can significantly enhance the fluorescence performance of CQ4T, and a product probe can quench the fluorescence of BSA-CQ 4T.
FIG. 7 shows the study of cytotoxicity CCK-8 of the tumor microenvironment responsive diagnosis and treatment integrated nano-probe synthesized in example 1 of the invention. From the figure, the effect of the bimodal imaging guided photothermal combined ion therapy nano probe with different concentrations on the proliferation inhibition rate of cells (normal cells HUVEC and tumor cells 4T 1) is not obvious, and the low toxicity is shown.
FIG. 8 is a fluorescence imaging diagram of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in the embodiment 1 of the present invention at a cell level. The probe group, the probe+2h group, the probe+4h group, the probe+FAP+2h group and the probe+FAP+4h group are arranged in sequence from left to right; from the figure, the fluorescence lighting effect of the NIR-II region after incubation with FAP is obvious compared with the pure probe group.
Fig. 9 is a cellular photoacoustic imaging diagram of a tumor microenvironment-responsive diagnosis and treatment integrated nanoprobe synthesized in embodiment 1 of the present invention. From the graph, the PA signal at 660nm (PA 660) shows a continuous decay and eventually vanishes, but the PA intensity at 1050nm (PA 1050) remains almost unchanged.
Fig. 10 is a graph of the in vivo fluorescence imaging effect of a nude mouse of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. As shown, after intravenous injection of the synthesized probes into tumor-bearing nude mice and healthy nude mice, the nude mice were photographed for different times using a near infrared two-zone fluorescence camera. Compared with a healthy nude mouse group, the tumor part of the tumor-bearing nude mouse group can be provided with obvious fluorescence of the NIR-II region, and the fluorescent lighting effect is good.
FIG. 11 shows the embodiment of the present inventionA photo-acoustic imaging effect diagram of a nude mouse living body of the tumor microenvironment response type diagnosis and treatment integrated nano probe synthesized in the embodiment 1. As shown, the synthesized probe was injected intravenously into the tumor-bearing nude tail, and dynamic changes in PA signal of tumor-bearing mice were recorded using MOST photoacoustic imaging system, shown in red and green, respectively. At 660nm, a significant PA signal (PA 660) was clearly observed in the tumor area within 2 hours after probe injection, PA660 intensity showed a slight decrease to some extent 4 hours after injection until PA660 intensity began to gradually increase again 6 hours after dosing. While PA1050 gradually increases over time until reaching the plateau at 8 hours. This data effectively demonstrates H in live TME 2 O 2 Activation of the synthesized nanoprobe.
Fig. 12 is a graph showing the photo-thermal therapeutic effect of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. As shown in the figure, the tumor part of the tumor-bearing nude mouse is subjected to photo-thermal treatment, the light is given to the nude mouse for 8h and 12h after the nano probe is injected into the vein of the nude mouse, the treatment temperature of the tumor part is obviously increased, and the photo-thermal treatment effect is good compared with the light given after PBS and the injection probe are injected for 4h and 48 h.
The preferred embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention, and the various changes are included in the scope of the present invention.
Example 2:
50mg of Pd (acac) 2 The powder, 30mg PVP (mw=29000) powder, 52.2mg NaBr powder were mixed, dissolved in a mixed solution of 2mL deionized water and 8mL N, N-dimethylformamide, placed in a glass pressure vessel, filled with argon, stirred in a 37 ℃ water bath for 30min, and the material was fully dissolved to give a bright orange-yellow transparent solution. And (3) rapidly introducing CO gas into the obtained solution at the temperature of 4 ℃ for 1min to exhaust other gases in the container, rapidly placing the container into an oil bath pot at the temperature of 80 ℃ after CO is filled, keeping CO continuously introduced and exhausted, and heating and stirring at the temperature of 80 ℃ for 3h. After 3 hours, the air inlet and the air outlet are closedThe solution was taken out after rapid cooling to room temperature in an ice-water bath to give a dark blue near black solution. 12mL of acetone was added and centrifuged at 10000rpm for 8min, and the supernatant was discarded to give a blue-black precipitate. This step was repeated 2 times. Ethanol is used for: the volume ratio of acetone is 1:8, centrifuging at 12000rpm for 10min, discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was combined with bovine serum albumin at 1:5 in a phosphate buffer solution, and stirring at 70 ℃ for 5min. The obtained solution is BSA-CQ4T and is stored at 4 ℃ in dark.
Taking 10mg of the blue-black solid obtained in the above, dissolving in 20mL of phosphoric acid buffer solution, adding 5mg of customized amino acid short chain, slowly stirring for 24h in CO atmosphere at 4 ℃, centrifuging by using an ultrafiltration centrifuge tube, and freeze-drying to obtain a dark blue near-black solid, namely Pd-pep.
2mg of Pd-pep solid was dissolved in phosphate buffer, stirred slowly in ice bath, 0.13mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.8mg of N-hydroxysuccinimide (NHS) were added sequentially, stirred slowly in CO for 24 hours, centrifuged with an ultrafiltration centrifuge tube, the supernatant was taken, 100. Mu.L of BSA-CQ4T solution was added, and stirred slowly at 4℃in the absence of light for 24 hours.
After 24h, add silver nitrate aqueous solution (2.5 x 10 in order with slow stirring in the dark -3 M) 0.5mL, L-ascorbic acid aqueous solution (0.1M) 0.7mL, sodium hydroxide aqueous solution (0.1M) 0.3mL, stirring for 20min, and ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nano-probe.
Example 3:
50mg of Pd (acac) 2 The powder, 30mg PVP (mw=29000) powder, 52.2mg NaBr powder were mixed, dissolved in a mixed solution of 2mL deionized water and 8mL N, N-dimethylformamide, placed in a glass pressure vessel, filled with argon, stirred in a 37 ℃ water bath for 30min, and the material was fully dissolved to give a bright orange-yellow transparent solution. Introducing CO gas into the solution at 4deg.C for 1min to remove other gases in the container, filling CO, and rapidly placing into an 80 deg.C oil bath pan to keep CO continuously introduced and removedAnd heating and stirring at 80 ℃ for 3 hours. And after 3h, closing the gas inlet and the gas outlet, rapidly cooling to room temperature in an ice water bath, and taking out the solution to obtain a dark blue and nearly black solution. 12mL of acetone was added and centrifuged at 10000rpm for 8min, and the supernatant was discarded to give a blue-black precipitate. This step was repeated 2 times. Ethanol is used for: the volume ratio of acetone is 1:8, centrifuging at 10000rpm for 10min, and discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was combined with bovine serum albumin at 1:8 in a phosphate buffer solution, and stirring at 70 ℃ for 5min. The obtained solution is BSA-CQ4T and is stored at 4 ℃ in dark.
1.5mg of the blue-black solid obtained above was taken and dissolved in 4mL of phosphate buffer, 3.0mg of a custom amino acid short chain was added, and the mixture was stirred slowly in a CO atmosphere at 4℃for 24 hours, centrifuged using an ultrafiltration centrifuge tube, then the supernatant was taken, stirred slowly in an ice bath, 0.69mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.42mg of N-hydroxysuccinimide (NHS) were added in sequence, the mixture was stirred slowly in a CO atmosphere for 24 hours, centrifuged using an ultrafiltration centrifuge tube, the supernatant was taken, 100. Mu.L of BSA-CQ4T solution was added, and the mixture was stirred slowly at 4℃in the absence of light for 24 hours.
After 24h, add silver nitrate aqueous solution (2.5 x 10 in order with slow stirring in the dark -3 M) 1.0mL, L-ascorbic acid aqueous solution (0.1M) 1.4mL, sodium hydroxide aqueous solution (0.1M) 0.6mL, stirring for 20min, and ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nano-probe.
Example 4:
50mg of Pd (acac) 2 Powder, 30mg of PVP (mw=29000) powder, dissolved in 8mL of N, N-dimethylformamide. 52.2mg of NaBr powder was taken and dissolved in 2mL of deionized water. This 2mL aqueous sodium bromide solution was slowly added dropwise to 8mL dimethylformamide solution under low-speed stirring to give a bright orange-yellow transparent solution. And (3) rapidly introducing CO gas into the obtained solution at the temperature of 4 ℃ for 1min to exhaust other gases in the container, rapidly placing the container into an oil bath pot at the temperature of 80 ℃ after CO is filled, keeping CO continuously introduced and exhausted, and heating and stirring at the temperature of 80 ℃ for 3h. After 3h, the air inlet and the air outlet are closed, and the ice water bath is carried outAnd (5) taking out the solution after being cooled to room temperature quickly, so as to obtain a dark blue and near black solution. 12mL of acetone was added and centrifuged at 10000rpm for 8min, and the supernatant was discarded to give a blue-black precipitate. This step was repeated 2 times. Ethanol is used for: the volume ratio of acetone is 1:8, centrifuging at 12000rpm for 10min, discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was combined with bovine serum albumin at 1:9 in a phosphate buffer solution, and stirring at 70 ℃ for 5min. The obtained solution is BSA-CQ4T and is stored at 4 ℃ in dark.
Taking 1mg of the blue-black solid obtained in the above, dissolving in 20mL of phosphoric acid buffer solution, adding 5mg of customized amino acid short chain, slowly stirring for 24h in CO atmosphere at 4 ℃, centrifuging by using an ultrafiltration centrifuge tube, and freeze-drying to obtain a dark blue near-black solid, namely Pd-pep.
2mg of Pd-pep solid was dissolved in phosphate buffer, stirred slowly in ice bath, 0.39mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.24mg of N-hydroxysuccinimide (NHS) were added sequentially, stirred slowly in CO for 24 hours, centrifuged with an ultrafiltration centrifuge tube, the supernatant was taken, 100. Mu.L of BSA-CQ4T solution was added, and stirred slowly at 4℃in the absence of light for 24 hours.
After 24h, add silver nitrate aqueous solution (2.5 x 10 in order with slow stirring in the dark -3 M) 0.5mL, L-ascorbic acid aqueous solution (0.1M) 0.7mL, sodium hydroxide aqueous solution (0.1M) 0.3mL, stirring for 20min, and ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nano-probe.
Example 5:
50mg of Pd (acac) 2 The powder, 30mg PVP (mw=29000) powder, 52.2mg NaBr powder were mixed, dissolved in a mixed solution of 2mL deionized water and 8mL N, N-dimethylformamide, placed in a glass pressure vessel, filled with argon, stirred in a 37 ℃ water bath for 30min, and the material was fully dissolved to give a bright orange-yellow transparent solution. Rapidly introducing CO gas into the obtained solution at 4deg.C for 1min to remove other gases in the container, rapidly placing into an 80 deg.C oil bath pot after CO is filled, keeping CO continuously introduced and discharged, adding at 80deg.CAnd (5) stirring for 3h. And after 3h, closing the gas inlet and the gas outlet, rapidly cooling to room temperature in an ice water bath, and taking out the solution to obtain a dark blue and nearly black solution. 12mL of acetone was added and centrifuged at 10000rpm for 8min, and the supernatant was discarded to give a blue-black precipitate. This step was repeated 2 times. Ethanol is used for: the volume ratio of acetone is 1:8, centrifuging at 12000rpm for 10min, discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was combined with bovine serum albumin at 1:6 in a phosphate buffer solution, and stirring at 70 ℃ for 5min. The obtained solution is BSA-CQ4T and is stored at 4 ℃ in dark.
Taking 1mg of the blue-black solid obtained in the above, dissolving in 20mL of phosphoric acid buffer solution, adding 3mg of customized amino acid short chain, slowly stirring for 24h in CO atmosphere at 4 ℃, centrifuging by using an ultrafiltration centrifuge tube, and freeze-drying to obtain a dark blue near-black solid, namely Pd-pep.
2mg of Pd-pep solid was dissolved in phosphate buffer, stirred slowly in ice bath, 0.35mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.21mg of N-hydroxysuccinimide (NHS) were added sequentially, stirred slowly in CO for 24 hours, centrifuged with an ultrafiltration centrifuge tube, the supernatant was taken, 200. Mu.L of BSA-CQ4T solution was added, and stirred slowly at 4℃in the absence of light for 24 hours.
After 24h, add silver nitrate aqueous solution (2.5 x 10 in order with slow stirring in the dark -3 M) 0.5mL, L-ascorbic acid aqueous solution (0.1M) 0.7mL, sodium hydroxide aqueous solution (0.1M) 0.3mL, stirring for 20min, and ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nano-probe.
The preferred embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention, and the various changes are included in the scope of the present invention.
Claims (6)
1. The utility model provides an integrative nanoprobe is diagnose to tumour microenvironment response type which characterized in that: the nano probe comprises a palladium nano sheet, nano silver, fluorescent organic micromolecule BSA-CQ4T and amino acid short chains; the palladium nano-sheet accounts for 43.4wt% of the nano-probe, the nano-silver accounts for 1.6wt% of the nano-probe, the fluorescent small organic molecule BSA-CQ4T accounts for 40wt% of the nano-probe, and the amino acid short chain accounts for 15wt% of the nano-probe;
the short chain of amino acids may be sheared by fibroblast activation protein recognition; the fluorescent small organic molecule BSA-CQ4T is modified by bovine serum albumin; the nano silver adopts reducing nano silver; the nano probe takes palladium nano sheets as a main body; the amino acid short chain is connected with the fluorescent small organic molecule BSA-CQ4T in an amidation mode;
the edge length of the nano probe is 20-40nm;
the amino acid sequence of the amino acid short chain is Ac-Asp-Ala-Gly-Pro-Asn-Gln-Cys-NH 2 。
2. The preparation method of the tumor microenvironment response type diagnosis and treatment integrated nano probe according to claim 1, which is characterized by comprising the following steps:
s1, pd (acac) 2 Mixing powder, polyvinylpyrrolidone powder and sodium bromide powder, dissolving in a mixed solution of deionized water and N, N-dimethylformamide, placing into a glass pressure container, and stirring in a water bath at 30deg.C until the powder, the polyvinylpyrrolidone powder and the sodium bromide powder are dissolved to obtain a bright pale yellow transparent solution;
s2, introducing CO gas into the container where the solution obtained in the step S1 is located at the temperature of 4 ℃ and discharging clean air;
s3, placing the S2 container into an oil bath pot at 80 ℃, keeping CO continuously introduced and discharged, and stirring for 3 hours;
s4, removing the S3 container from the oil bath, closing the gas inlet and the gas outlet of CO gas, cooling to room temperature, and taking out the solution to obtain a dark blue near black solution;
s5, washing the solution obtained in the step S4 with acetone, and centrifuging at a high speed to obtain blue-black precipitate;
s6, precipitating the precipitate obtained in the step S5 by using ethanol: washing the mixed solution with the acetone volume ratio of 1:8, and centrifuging at a high speed to obtain blue-black precipitate;
s7, vacuum drying the precipitate obtained in the step S6, and preserving at room temperature to obtain solid palladium nano-sheets Pd NSs;
s8, dissolving fluorescent small organic molecule BSA-CQ4T and albumin in a mass ratio of 1:5-1:10 in a phosphate buffer solution, stirring at 70 ℃ to obtain a BSA-CQ4T solution, and storing at a dark place at 4 ℃;
s9, the mass ratio is 2:1-1:5, mixing the solid palladium nano-sheet Pd NSs obtained in the step 7 with an amino acid short-chain phosphate buffer solution, slowly stirring in a CO atmosphere at 4 ℃, ultrafiltering, centrifuging, and freeze-drying to obtain a dark blue near black solid Pd-pep;
s10, activating the solid Pd-pep obtained in the S9 by using N-hydroxysuccinimide/1-3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride NHS/EDC in a phosphate buffer, ultrafiltering and centrifuging after activation, adding a BSA-CQ4T solution in the S8, keeping away from light in a CO atmosphere, and slowly stirring in an ice-water bath to obtain liquid Pd-pep-BSA-CQ4T;
s11, adding the liquid Pd-pep-BSA-CQ4T obtained in the S10 into a silver nitrate solution and a reducing agent in sequence under light-shielding and slow stirring, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
3. The method for preparing the tumor microenvironment response type diagnosis and treatment integrated nano probe according to claim 2, which is characterized by comprising the following steps: pd (acac) as described in step S1 2 The mass ratio of the powder to the polyvinylpyrrolidone powder to the sodium bromide powder is 50:30:52.2, and the volume ratio of the deionized water to the dimethylformamide is 2:8; the polyvinylpyrrolidone mw=29000.
4. The method for preparing the tumor microenvironment response type diagnosis and treatment integrated nano probe according to claim 2, which is characterized by comprising the following steps: the centrifugal speed in the step S5 and the step S6 is 10000rpm, and the centrifugal time is 8min; step S5, washing and centrifuging are repeated for 2 times; step S6 washing and centrifugation are repeated 3 times.
5. The method for preparing the tumor microenvironment response type diagnosis and treatment integrated nano probe according to claim 2, which is characterized by comprising the following steps: the albumin in step S8 is bovine serum albumin.
6. The method for preparing the tumor microenvironment response type diagnosis and treatment integrated nano probe according to claim 2, which is characterized by comprising the following steps: the reducing agent in the step S11 is 0.1M L-ascorbic acid, 0.1MNaOH; the mass concentration of the silver nitrate solution substance is 2.5-10 -3 M。
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| CN111888487A (en) * | 2020-09-04 | 2020-11-06 | 山西医科大学 | Tumor targeted photoacoustic imaging guided multi-stage treatment nano probe and preparation method thereof |
| CN112370534A (en) * | 2020-11-26 | 2021-02-19 | 山西医科大学 | Tumor microenvironment response degradation type diagnosis and treatment integrated nanoprobe and preparation method thereof |
| CN112451665A (en) * | 2020-11-26 | 2021-03-09 | 山西医科大学 | Novel NIR-II diagnosis and treatment integrated silicon-carbon nanoprobe and preparation method thereof |
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| CN111888487A (en) * | 2020-09-04 | 2020-11-06 | 山西医科大学 | Tumor targeted photoacoustic imaging guided multi-stage treatment nano probe and preparation method thereof |
| CN112370534A (en) * | 2020-11-26 | 2021-02-19 | 山西医科大学 | Tumor microenvironment response degradation type diagnosis and treatment integrated nanoprobe and preparation method thereof |
| CN112451665A (en) * | 2020-11-26 | 2021-03-09 | 山西医科大学 | Novel NIR-II diagnosis and treatment integrated silicon-carbon nanoprobe and preparation method thereof |
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