CN114767881A - Preparation and application of tumor microenvironment response type diagnosis and treatment integrated nanoprobe - Google Patents
Preparation and application of tumor microenvironment response type diagnosis and treatment integrated nanoprobe Download PDFInfo
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Abstract
The invention relates to the field of biomedicine and inorganic nano materials, in particular to preparation and application of a tumor microenvironment responsive diagnosis and treatment integrated nano probe. The probe takes a hexagonal palladium nanosheet as a main body, connects near-infrared two-region fluorescent organic micromolecules BSA-CQ4T modified by bovine serum albumin in an amidation mode through an amino acid short chain which can be recognized and sheared by fibroblast activation protein, carries reductive nano silver, and achieves the effects of multi-modal imaging and treatment. The short chain of amino acid in a tumor microenvironment is cut off, BSA-CQ4T is separated from palladium nanosheets, and the fluorescence of BSA-CQ4T in a near infrared two-region is turned on to illuminate a tumor area. Under the action of hydrogen peroxide with higher concentration in a tumor microenvironment, the nano silver is oxidized to form silver ions, the photoacoustic signal of the near-infrared material is reduced, and silver ion treatment can be carried out. And the photoacoustic signals of the palladium nanosheets in the near-infrared region II are not changed greatly, so that the proportional photoacoustic imaging effect is achieved.
Description
Technical Field
The invention relates to the field of biomedicine and inorganic nano materials, in particular to preparation and application of a tumor microenvironment responsive diagnosis and treatment integrated nano probe.
Background
The tumor seriously harms the life health of human beings and is also a medical diagnosis and treatment difficulty. A new, efficient and safe tumor diagnosis and treatment method needs to be continuously explored. 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 precise medicine, and provides a new visual field for diagnosis and treatment of tumors.
Compared with the traditional image development technology, the nano diagnosis and treatment system has remarkable advantages and great potentials in the aspects of early diagnosis of tumors, tracking of distant metastasis, personalized treatment, curative effect monitoring and evaluation and the like. Light-induced Imaging such as Photoacoustic Imaging (PAI) and fluorescence Imaging (fluoroscope Imaging), Light-induced therapy Light-induced Imaging and treatment technology are of interest in oncology. Compared with the conventional optical imaging in visible light (400nm-650nm) and the first near infrared window (NIR-I, 650nm-900nm), the near infrared II region (NIR-II, 1000-1700nm) imaging has higher signal-to-noise ratio, better imaging quality and deeper tissue penetration. Since the wavelength of light in the NIR-II region is longer, the scattering intensity of light decreases exponentially as the wavelength increases. NIR-II FL imaging can directly and quickly display interested dynamic biological tissues, and has higher space-time resolution and sensitivity. NIR-II-PA imaging combines the advantages of optical imaging and acoustic imaging. Compared with a single imaging mode, the dual-mode optical imaging adopting NIR-II-FL imaging and PA imaging is possible to make the diagnostic information more comprehensive and accurate. In addition, most of nano diagnosis and treatment systems enter a tumor region through passive targeting or active targeting, and the weak development of materials remained in normal tissues and nonspecific background signals of healthy tissues are easy to interfere with accurate diagnosis, so that the detection sensitivity is reduced.
Early studies have shown that the Tumor Microenvironment (TME) is closely related to the development, progression and metastasis of tumors. Its characteristic acidity, hypoxia, redox-disturbed environment and many abnormally highly expressed cytokines, etc., contribute to the development of various diagnostic systems that rely on TME activation. The nano material is designed by taking TME as a switch, so that the sensitivity and the accuracy of detection can be greatly improved. Therefore, from the aspects of sensitivity, accuracy and comprehensiveness of diagnosis, a nano particle which needs to be activated by a specific tumor environment and has two-mode imaging is designed and developed, namely, an inorganic palladium nanosheet is used as a main body, fluorescent molecules CQ4T are connected through a fiber-forming activated protein recognition shearing amino short chain which can be specifically and highly expressed by epithelial cancer, and reducing nano silver is loaded, so that two-mode imaging of tumor microenvironment activated fluorescent imaging and ratio photoacoustic imaging is realized.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an intelligent response type near-infrared two-zone multi-modal imaging treatment integrated nano probe for a tumor microenvironment and a preparation method thereof. The material can be identified by tumor-associated Fibroblast Activation Protein (FAP) with high tumor specificity expression, fluorescence is started in a near-infrared region II, proportional photoacoustic imaging is generated under the action of hydrogen peroxide with high TME expression, silver ions are released, and photo-thermal treatment and ion treatment in the near-infrared region II are synchronously realized by matching with excellent photo-thermal conversion efficiency of the material while the tumor diagnosis effect with high sensitivity and high accuracy is achieved.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a tumor microenvironment response type diagnosis and treatment integrated nanoprobe comprises palladium nanoplates, nano silver, fluorescent organic micromolecules BSA-CQ4T and amino acid short chains; the palladium nanosheet accounts for 43.4 wt% of the nano probe, the nano silver accounts for 1.6 wt% of the nano probe, the fluorescent organic micromolecule BSA-CQ4T accounts for 40 wt% of the nano probe, and the amino acid short chain accounts for 15 wt% of the nano probe.
Further, the short chain of amino acids can be cleaved by recognition of fibroblast activation protein; the fluorescent organic small molecule CQ4T is modified by bovine serum albumin; the nano silver is reductive nano silver; the nano probe takes a palladium nano sheet as a main body; the short chain of the amino acid is connected with a fluorescent organic small molecule BSA-CQ4T in an amidation mode.
Further, the edge length of the nanoprobe is 20-40 nm.
A preparation method of a tumor microenvironment response type diagnosis and treatment integrated nanoprobe comprises the following steps:
s1 general formula Pd (acac)2Mixing the powder, polyvinylpyrrolidone powder and sodium bromide powder, dissolving the mixture into a mixed solution of deionized water and N, N-dimethylformamide, putting the mixture into a glass pressure container, and stirring the mixture in water bath at 30 ℃ until the mixture is dissolved to obtain a bright light yellow transparent solution;
s2, introducing CO gas into the container in which the solution obtained in the S1 is located at the temperature of 4 ℃, and exhausting air;
s3, placing the S2 container in an oil bath kettle at the temperature of 80 ℃, keeping the continuous introduction and discharge of CO, and stirring for 3 hours;
s4, moving the container S3 out of the oil bath pan, closing the gas inlet and outlet of CO gas, cooling to room temperature, and taking out the solution to obtain a dark blue nearly black solution;
s5, washing the solution obtained in the step S4 with acetone, and centrifuging at a high speed to obtain a blue-black precipitate;
s6, precipitation of S5 with ethanol: washing with a mixed solution with the volume ratio of acetone of 1:8, and centrifuging at a high speed to obtain a blue-black precipitate;
s7, carrying out vacuum drying on the precipitate obtained in the step S6, and storing at room temperature to obtain solid palladium nanosheet Pd NSs;
s8, dissolving fluorescent organic micromolecules BSA-CQ4T and albumin in phosphate buffer solution according to the mass ratio of 1:5-1:10, stirring at 70 ℃ to obtain BSA-CQ4T solution, and storing at 4 ℃ in a dark place;
s9, mixing the raw materials in a mass ratio of (2: 1-1): 5, mixing the solid palladium nanosheet Pd NSs obtained from S7 with a phosphate buffer solution with short chain of amino acid, slowly stirring in a CO atmosphere at 4 ℃, performing ultrafiltration centrifugation, and freeze-drying to obtain dark blue and nearly black solid Pd-pep;
s10, activating the solid Pd-pep obtained from S9 in phosphate buffer solution by NHS/EDC, ultrafiltering and centrifuging after activation, taking supernatant, adding BSA-CQ4T solution in S8, keeping out of the sun in CO atmosphere, and slowly stirring in an ice water bath to obtain liquid Pd-pep-BSA-CQ 4T;
and S11, adding a silver nitrate solution and a reducing agent into the liquid Pd-pep-BSA-CQ4T obtained in the S10 in sequence under the conditions of keeping out of the sun and slowly stirring, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
Further, Pd (acac) in step S12The 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 is 29000.
Further, the centrifugation speed in the steps S5 and S6 is 10000rpm, and the centrifugation time is 8 min; step S5 washing, centrifuging and repeating for 2 times; step S6 washing and centrifugation were repeated 3 times.
Further, in step S8, the albumin is bovine serum albumin.
Further, the amino acid short chain in step S9 has the amino acid sequence Ac-Asp-Ala-Gly-Pro-Asn-Gln-Cys-NH2。
Further, the reducing agent in step S11 is 0.1M L-ascorbic acid, 0.1M NaOH; the mass concentration of the silver nitrate solution is 2.5 x 10-3M。
The application of the nano-probe or the nano-probe prepared by the method in the photo-thermal combined ion therapy under the guidance of FAP response type near-infrared two-region fluorescence imaging and proportional photo-acoustic imaging is disclosed.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a simple synthesis method of a nano probe capable of intelligently responding to near-infrared two regions of a tumor microenvironment and activating fluorescence imaging, photoacoustic imaging and photothermal therapy. The probe takes a uniform hexagonal palladium nanosheet as a main body, connects a near-infrared two-region fluorescent organic micromolecule BSA-CQ4T modified by Bovine Serum Albumin (BSA) in an amidation mode through an amino acid short chain which can be recognized and sheared by tumor-related Fibroblast Activation Protein (FAP), and carries reductive nano-silver, so that the multi-modal imaging and treatment effects are achieved. When the material is in a normal biological tissue with the pH of 7.4, the fluorescence is quenched due to the overlapping of a fluorescence emission region of the organic fluorescent micromolecule and an absorption region of the palladium nanosheet; in a tumor microenvironment (breast cancer, lung cancer and the like), namely when the tumor-associated fibroblasts are rich, the short chain of the amino acid is cut off, BSA-CQ4T is separated from the palladium nanosheet, and the fluorescence of BSA-CQ4T in a near-infrared two-region is turned on to illuminate a tumor region. Meanwhile, in a tumor microenvironment, under the environment action of a hydrogen peroxide concentration (50-100 mu M) with higher concentration, nano silver is oxidized to form silver ions, photoacoustic signals of the material in a first near infrared region of 660nm are reduced, photoacoustic signals of palladium nanosheets in a second near infrared region of 1050nm are unchanged, and a proportional photoacoustic imaging effect is achieved. In addition, the palladium nanosheet has excellent photothermal conversion efficiency, has high absorption peaks in the near-infrared two regions, and can be used as an excellent photothermal initiator to achieve the near-infrared two-region photothermal treatment effect. According to the method, the palladium nanosheet with a proper size is designed and adjusted, so that the palladium nanosheet has high photo-thermal conversion efficiency and a photo-acoustic imaging contrast effect in a near-infrared two-region, an absorption region of the palladium nanosheet is highly overlapped with a fluorescence emission region of BSA-CQ4T, the palladium nanosheet is firmly connected with BSA-CQ4T through amidation, the fluorescence of the product can be quenched in a healthy tissue with a physiological condition PH of 7.4, and under the action of fibroblast activation protein with high epithelial cancer specificity expression, the palladium nanosheet is separated from BSA-CQ4T, and the near-infrared two-region fluorescence is started. In addition, silver ions are released under the action of hydrogen peroxide, and proportional photoacoustic imaging is realized. Namely, under the guidance of activatable NIR-IIFL/ratio PA imaging, the photo-thermal ion combination therapy of 'positioning timing' is carried out. The method is simple and easy to implement, suitable size and fusion proportion are found out, the detection sensitivity is improved, 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 diagram of a nanoprobe synthesized in example 1 of the present invention, and an X-ray photoelectron spectrum of Pd element.
FIG. 4 is an XPS plot of nanoprobes synthesized in example 1 of the present invention, an X-ray photoelectron spectrum of Ag element.
FIG. 5 shows the photothermal properties of nanoprobes 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 CCK-8 study of cytotoxicity of nanoprobes synthesized in example 1 of the invention.
FIG. 8 is a fluorescent image at the cell level of the nanoprobe synthesized in example 1 of the present invention.
FIG. 9 is a proportional photoacoustic imaging at the cell level for the nanoprobes synthesized in example 1 of the present invention.
FIG. 10 is a diagram illustrating in vivo fluorescence imaging of nude mice using the synthesized nanoprobe of example 1 of the present invention.
FIG. 11 is a photoacoustic imaging plot of a nude mouse in vivo of a nano-probe synthesized in example 1 of the present invention.
FIG. 12 is a graph showing the photothermal treatment effect of the nanoprobe synthesized in example 1 of the present invention in a living body of a nude mouse.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The probe takes uniform hexagonal palladium nanosheets as a main body, connects near-infrared two-region fluorescent organic micromolecules BSA-CQ4T modified by bovine serum albumin in an amidation mode through amino acid short chains capable of being recognized and sheared by fibroblast activation protein, and carries reductive nano silver, wherein the palladium nanosheets account for 43.4 wt% of the nano probe, the nano silver accounts for 1.6 wt% of the nano probe, the amino acid short chains account for 30 wt% of the nano probe, and the fluorescent organic micromolecules BSA-CQ4T account for 25 wt% of the nano probe.
In this embodiment, the nanoprobe has an edge length of 20-40 nm.
A preparation method of a tumor microenvironment response type diagnosis and treatment integrated nanoprobe comprises the following steps:
s1 general formula Pd (acac)2Mixing the powder, polyvinylpyrrolidone powder and sodium bromide powder, dissolving the mixture in a mixed solution of deionized water and N, N-dimethylformamide, putting the mixture into a glass pressure container, and stirring the mixture in a water bath at 30 ℃ until the mixture is dissolved to obtain a bright light yellow transparent solution;
s2, introducing CO gas into the container in which the solution obtained in the S1 is positioned at 4 ℃, and exhausting air;
s3, placing the S2 container in an oil bath kettle at the temperature of 80 ℃, keeping CO continuously introduced and discharged, and stirring for 3 hours;
s4, moving the container S3 out of the oil bath pan, closing the gas inlet and outlet of CO gas, cooling to room temperature, and taking out the solution to obtain a dark blue nearly black solution;
s5, washing the solution obtained in the step S4 with acetone, and centrifuging at a high speed to obtain a blue-black precipitate;
s6, precipitation of S5 with ethanol: washing with a mixed solution of acetone in a volume ratio of 1:8, and centrifuging at a high speed to obtain a blue-black precipitate;
s7, carrying out vacuum drying on the precipitate obtained in the S6, and storing at room temperature to obtain solid palladium nanosheets (Pd NSs);
s8, dissolving fluorescent organic micromolecules BSA-CQ4T and albumin in phosphate buffer solution according to the mass ratio of 1:5-1:10, stirring at 70 ℃ to obtain BSA-CQ4T solution, and storing at 4 ℃ in a dark place;
s9, mixing the raw materials in a mass ratio of 2:1 to 1:5, mixing the solid palladium nanosheet Pd NSs obtained in the step S7 with a phosphate buffer solution with a short chain of amino acid, slowly stirring in a CO atmosphere at 4 ℃, performing ultrafiltration centrifugation, and freeze-drying to obtain a dark blue and nearly black solid Pd-pep;
s10, activating the solid Pd-pep obtained in the S9 in a phosphate buffer solution by NHS/EDC, performing ultrafiltration and centrifugation after activation, taking supernatant, adding BSA-CQ4T solution in S8, keeping out of the sun in a CO atmosphere, and slowly stirring in an ice water bath to obtain liquid Pd-pep-BSA-CQ 4T;
s11, adding the liquid Pd-pep-BSA-CQ4T obtained from S10 into a silver nitrate solution and a reducing agent in sequence under the conditions of keeping out of the sun and slowly stirring, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
In step S1, polyvinylpyrrolidone Mw is 29000. The centrifugal speed in step S5 and step S6 is 10000 rpm. The amino acid short chain in step S9 has the amino acid sequence Ac-Asp-Ala-Gly-Pro-Asn-Gln-Cys-NH2。
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 x 10-3And 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 photothermal combined ion therapy.
The BSA-CQ4T fluorescent molecule In this example was obtained from Monitoring the Real-Time circulation System-Related Physiological and clinical Processes In Vivo Using a multifunctionalal NIR-II Probe.
Example 1
50mg of Pd (acac)2The powder, 30mg of PVP (Mw 29000) powder, and 52.2mg of NaBr powder were mixed, and dissolved in a mixed solution of 2mL of deionized water and 8mL of n.n-dimethylformamide,placing in a glass pressure container, stirring in water bath at 30 deg.C for 30min to dissolve the materials sufficiently to obtain bright orange yellow transparent solution. Introducing CO gas into the obtained solution at 4 deg.C for 1min to remove air, rapidly placing into 80 deg.C oil bath pan after CO is full, keeping CO continuously introduced and removed, heating at 80 deg.C and stirring for 3 hr. And after 3h, closing the gas inlet and the gas outlet, rapidly cooling the solution to room temperature in an ice water bath, and taking out the solution to obtain a dark blue nearly black solution. Adding 12mL of acetone, centrifuging at 10000rpm for 8min, and discarding the supernatant to obtain a blue-black precipitate. This step was repeated 2 times. Using ethanol: the volume ratio of acetone is 1:8, washing the mixed solution, centrifuging the solution at the speed of 10000rpm for 10min, discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum drying oven and stored.
CQ4T was mixed with bovine serum albumin at a ratio of 1:10 in phosphate buffer, and stirring at 70 ℃ for 5 min. The resulting solution, BSA-CQ4T, was stored at 4 ℃ in the dark.
And (3) dissolving 10mg of the obtained blue-black solid in 20mL of phosphate buffer, adding 10mg of the customized amino acid short chain, slowly stirring for 24h at 4 ℃ in a CO atmosphere, centrifuging by using an ultrafiltration centrifugal tube, and freeze-drying to obtain the deep blue near-black solid, namely the Pd-pep.
Dissolving 2mg of Pd-pep solid in a phosphate buffer solution, slowly stirring in an ice bath, sequentially adding 0.23mg of EDC and 0.14mg of NHS, slowly stirring in a CO atmosphere for 24h, centrifuging by using an ultrafiltration centrifugal tube, taking the supernatant, adding 100 mu L of BSA-CQ4T (10mg/mL) solution, and slowly stirring at 4 ℃ in the dark for 24 h.
Aqueous silver nitrate solution (2.5 x 10) was added in sequence under slow stirring in the dark-3M)0.5mL, L-ascorbic acid aqueous solution (0.1M)0.7mL, sodium hydroxide aqueous solution (0.1M)0.3mL, stirring for 20min, ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nanoprobe.
The tumor microenvironment responsive diagnosis and treatment integrated nano probe prepared by the method is dissolved in PBS buffer solution, and is administrated by rat tail intravenous injection, so that the imaging performance and the treatment effect of the nano probe are detected.
Fig. 1 is an EDX spectrogram of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. According to the energy spectrum analysis of the photo-thermal combined ion treatment nano probe guided by the synthesized bimodal imaging, Ag and S elements with good dispersion are distributed in a region containing Pd elements, and the successful preparation of the probe is proved.
Fig. 2 is a TEM image of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in embodiment 1 of the present invention. As can be seen, the synthesized nanoprobes have hexagonal shapes and an average diameter of 51.83 nm.
Fig. 3 and 4 are XPS charts of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. In the figure, 3 is an X-ray photoelectron spectrum of Pd element, and in the figure, 4 is an 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 for metallic Pd (0). As can be seen from FIG. 4, the high resolution results for Ag 3d are located at 642.33eV (Ag 3d5/2) and 653.84eV (Ag 3d3/2), which correlate with the characteristic peaks for Ag (0).
Fig. 5 shows the photo-thermal properties of different concentrations of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in embodiment 1 of the present invention. As can be seen from the figure, the diagnosis and treatment integrated nanoprobes with different concentrations are irradiated in the near infrared (1064nm, 1W/cm)2) The lower temperature rise curve shows that the higher the concentration the faster the temperature rise, the higher the temperature increase with irradiation time.
Fig. 6 is a fluorescence spectrum of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. As can be seen, BSA can significantly enhance the fluorescence property of CQ4T, and the product probe can quench the fluorescence of BSA-CQ 4T.
Fig. 7 shows a cytotoxicity CCK-8 study of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in example 1 of the present invention. As can be seen from the figure, the photo-thermal combined ion therapy nanoprobes with different concentrations and guided by bimodal imaging have no obvious influence on the proliferation inhibition rate of cells (normal cell HUVEC and tumor cell 4T1), and are low in toxicity.
Fig. 8 is a fluorescence imaging diagram of the tumor microenvironment-responsive diagnosis and treatment integrated nanoprobe synthesized in 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 sequentially arranged from left to right; as can be seen, compared with the pure probe set, the fluorescent lighting effect of the NIR-II region is obvious after incubation with FAP.
Fig. 9 is a cell photoacoustic imaging diagram of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in embodiment 1 of the present invention. As can be seen, the PA signal at 660nm (PA660) shows a continuous decay and eventually disappears, but the PA intensity at 1050nm (PA1050) remains almost unchanged.
Fig. 10 is a diagram illustrating the fluorescence imaging effect of the nude mouse living body of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in embodiment 1 of the present invention. As shown in the figure, after the synthesized probe is injected into the tail vein of the tumor-bearing nude mouse and the healthy nude mouse, the nude mouse is photographed at different times by using a near-infrared two-zone fluorescence camera. Compared with a healthy nude mouse group, the nude mouse group with tumor can see obvious NIR-II area fluorescence at the tumor part, and the fluorescence lighting effect is good.
Fig. 11 is a graph showing the photoacoustic imaging effect of the nude mouse living body of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe synthesized in embodiment 1 of the present invention. As shown in the figure, the synthesized probe was injected into the tail vein of tumor-bearing nude mice, and dynamic changes of PA signals of the tumor-bearing mice were recorded using MOST photoacoustic imaging system and displayed in red and green, respectively. At 660nm, a significant PA signal (PA660) was clearly observed in the tumor area within 2 hours after probe injection, with the PA660 intensity showing a slight decrease of some extent 4 hours after injection, until the PA660 intensity started to gradually increase again 6 hours after dosing. And as time goes on, PA1050 gradually increases until it reaches a plateau at 8 hours. This data effectively demonstrates H in living TME2O2Activation of the synthesized nanoprobe.
Fig. 12 is a diagram illustrating the photothermal treatment effect of the nude mouse living body 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 irradiation is given 8h and 12h after the nano probe is injected into the tail vein of the nude mouse, the treatment temperature of the tumor part is obviously increased and is far higher than the irradiation given 4h and 48h after the PBS and the probe are injected, and the photo-thermal treatment effect is good.
Although only the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art, and all changes are included in the scope of the present invention.
Example 2:
50mg of Pd (acac)2The powder, 30mg of PVP (Mw ═ 29000) powder, and 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, filled with argon gas, and stirred in a water bath at 37 ℃ for 30 minutes to dissolve the material sufficiently, so that a bright orange-yellow transparent solution was obtained. Introducing CO gas into the obtained solution at 4 deg.C for 1min to remove other gases, filling CO, placing into 80 deg.C oil bath, keeping CO continuously introduced and removed, and heating at 80 deg.C under stirring for 3 hr. 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 near-black solution. Adding 12mL of acetone, centrifuging at 10000rpm for 8min, and removing the supernatant to obtain a blue-black precipitate. This step was repeated 2 times. Using ethanol: the volume ratio of acetone is 1:8, washing the mixed solution, centrifuging the solution at 12000rpm for 10min, discarding the supernatant to obtain a blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was mixed with bovine serum albumin at a ratio of 1:5 in phosphate buffer, and stirring at 70 ℃ for 5 min. The resulting solution was BSA-CQ4T, stored at 4 ℃ in the dark.
And (3) dissolving 10mg of the obtained blue-black solid in 20mL of phosphate buffer, adding 5mg of the customized amino acid short chain, slowly stirring for 24h at 4 ℃ in a CO atmosphere, centrifuging by using an ultrafiltration centrifugal tube, and freeze-drying to obtain the deep blue near-black solid, namely the Pd-pep.
Taking 2mg of Pd-pep solid, dissolving the Pd-pep solid in a phosphate buffer solution, slowly stirring the Pd-pep solid in ice bath, sequentially adding 0.13mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.8mg of N-hydroxysuccinimide (NHS), slowly stirring the mixture for 24 hours in a CO atmosphere, centrifuging the mixture by using an ultrafiltration centrifugal tube, taking the supernatant, adding 100 mu L of BSA-CQ4T solution, and slowly stirring the mixture for 24 hours at a dark temperature of 4 ℃.
After 24h, aqueous silver nitrate solution (2.5 x 10) was added in sequence under slow stirring in the dark-3M)0.5mL, L-ascorbic acid aqueous solution (0.1M)0.7mL, and sodium hydroxide aqueous solution (0.1M)0.3mL, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nanoprobe.
Example 3:
50mg of Pd (acac)2The powder, 30mg of PVP (Mw 29000) powder, and 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, filled with argon gas, and stirred in a water bath at 37 ℃ for 30min to fully dissolve the materials, thereby obtaining a bright orange-yellow transparent solution. And (3) rapidly introducing CO gas into the obtained solution at 4 ℃ for 1min to completely discharge other gases in the container, rapidly placing the solution into an oil bath kettle at 80 ℃ after the CO is full, keeping the CO continuously introduced and discharged, and heating and stirring the solution at 80 ℃ for 3 h. And after 3h, closing the gas inlet and the gas outlet, rapidly cooling the solution to room temperature in an ice-water bath, and taking out the solution to obtain a dark blue near-black solution. Adding 12mL of acetone, centrifuging at 10000rpm for 8min, and discarding the supernatant to obtain a blue-black precipitate. This step was repeated 2 times. Using ethanol: the volume ratio of acetone is 1:8, washing the mixed solution, centrifuging the solution at the speed of 10000rpm for 10min, discarding supernatant to obtain blue-black precipitate, and repeating the steps for 3 times. The obtained precipitate was dried in a vacuum drying oven and stored.
CQ4T was mixed with bovine serum albumin at a ratio of 1:8 in phosphate buffer, and stirring at 70 ℃ for 5 min. The resulting solution, BSA-CQ4T, was stored at 4 ℃ in the dark.
Dissolving 1.5mg of the obtained bluish-black solid in 4mL of phosphate buffer, adding 3.0mg of custom amino acid short chain, slowly stirring for 24h at 4 ℃ in a CO atmosphere, centrifuging by using an ultrafiltration centrifuge tube, taking the supernatant, slowly stirring in an ice bath, sequentially adding 0.69mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.42mg of N-hydroxysuccinimide (NHS), slowly stirring for 24h in a CO atmosphere, centrifuging by using an ultrafiltration centrifuge tube, taking the supernatant, adding 100 mu L of BSA-CQ4T solution, and slowly stirring for 24h at 4 ℃ in a dark place.
After 24h, aqueous silver nitrate solution (2.5 x 10) was added in sequence under slow stirring in the dark-3M)1.0mL, L-ascorbic acid aqueous solution (0.1M)1.4mL and sodium hydroxide aqueous solution (0.1M)0.6mL, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
Example 4:
50mg of Pd (acac)2The powder and 30mg of PVP (Mw 29000) powder were dissolved in 8mL of N, N-dimethylformamide. 52.2mg of NaBr powder was taken and dissolved in 2mL of deionized water. The 2mL aqueous sodium bromide solution was slowly added dropwise to 8mL dimethylformamide with low stirring to give a bright yellow-orange clear solution. Introducing CO gas into the obtained solution at 4 deg.C for 1min to remove other gases, filling CO, placing into 80 deg.C oil bath, keeping CO continuously introduced and removed, and heating at 80 deg.C under stirring for 3 hr. 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 near-black solution. Adding 12mL of acetone, centrifuging at 10000rpm for 8min, and discarding the supernatant to obtain a blue-black precipitate. This step was repeated 2 times. The reaction solution is prepared by using ethanol: the volume ratio of acetone is 1:8, centrifuging at 12000rpm for 10min, discarding the supernatant to obtain a blue-black precipitate, and repeating the step for 3 times. The obtained precipitate was dried in a vacuum oven and stored.
CQ4T was mixed with bovine serum albumin at a ratio of 1: 9 in phosphate buffer, and stirring at 70 ℃ for 5 min. The resulting solution was BSA-CQ4T, stored at 4 ℃ in the dark.
And (3) dissolving 1mg of the obtained blue-black solid in 20mL of phosphate buffer, adding 5mg of the customized amino acid short chain, slowly stirring for 24 hours at 4 ℃ in a CO atmosphere, centrifuging by using an ultrafiltration centrifugal tube, and freeze-drying to obtain the deep blue and near-black solid, namely the Pd-pep.
2mg of Pd-pep solid is taken and dissolved in a phosphate buffer solution, slowly stirred in an ice bath, 0.39mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.24mg of N-hydroxysuccinimide (NHS) are sequentially added, slowly stirred for 24 hours in a CO atmosphere, centrifuged by an ultrafiltration centrifugal tube, the supernatant is taken, 100 mu L of BSA-CQ4T solution is added, and slowly stirred for 24 hours at a temperature of 4 ℃ in a dark place.
After 24h, aqueous silver nitrate solution (2.5 x 10) was added in sequence under slow stirring in the dark-3M)0.5mL, L-ascorbic acid aqueous solution (0.1M)0.7mL, and sodium hydroxide aqueous solution (0.1M)0.3mL, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nanoprobe.
Example 5:
50mg of Pd (acac)2The powder, 30mg of PVP (Mw ═ 29000) powder, and 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, filled with argon gas, and stirred in a water bath at 37 ℃ for 30 minutes to dissolve the material sufficiently, so that a bright orange-yellow transparent solution was obtained. And (3) rapidly introducing CO gas into the obtained solution at 4 ℃ for 1min to completely discharge other gases in the container, rapidly placing the solution into an oil bath kettle at 80 ℃ after the CO is full, keeping the CO continuously introduced and discharged, and heating and stirring the solution at 80 ℃ for 3 h. 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 near-black solution. Adding 12mL of acetone, centrifuging at 10000rpm for 8min, and removing the supernatant to obtain a blue-black precipitate. This step was repeated 2 times. Using ethanol: the volume ratio of acetone is 1:8, centrifuging at 12000rpm for 10min, discarding the supernatant to obtain a blue-black precipitate, and repeating the step for 3 times. The obtained precipitate was dried in a vacuum drying oven and stored.
CQ4T was mixed with bovine serum albumin at a ratio of 1: 6 in phosphate buffer, and stirring at 70 ℃ for 5 min. The resulting solution was BSA-CQ4T, stored at 4 ℃ in the dark.
And (3) dissolving 1mg of the obtained blue-black solid in 20mL of phosphate buffer, adding 3mg of customized amino acid short chain, slowly stirring for 24 hours at 4 ℃ in a CO atmosphere, centrifuging by using an ultrafiltration centrifugal tube, and freeze-drying to obtain the deep blue and near-black solid, namely the Pd-pep.
2mg of Pd-pep solid is taken and dissolved in a phosphate buffer solution, slowly stirred in an ice bath, 0.35mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.21mg of N-hydroxysuccinimide (NHS) are sequentially added, slowly stirred for 24 hours in a CO atmosphere, centrifuged by an ultrafiltration centrifugal tube, the supernatant is taken, 200 mu L of BSA-CQ4T solution is added, and slowly stirred for 24 hours at a temperature of 4 ℃ in a dark place.
After 24h, aqueous silver nitrate solution (2.5 x 10) was added in sequence under slow stirring in the dark-3M)0.5mL, L-ascorbic acid aqueous solution (0.1M)0.7mL, sodium hydroxide aqueous solution (0.1M)0.3mL, stirring for 20min, ultrafiltering and centrifuging to obtain Pd-CQ4T-Ag nanoprobe.
Although only the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art, and all changes are included in the scope of the present invention.
Claims (10)
1. A tumor microenvironment response type diagnosis and treatment integrated nanoprobe is characterized in that: the nano probe comprises a palladium nano sheet, nano silver, fluorescent organic micromolecule BSA-CQ4T and an amino acid short chain; the palladium nanosheet accounts for 43.4 wt% of the nano probe, the nano silver accounts for 1.6 wt% of the nano probe, the fluorescent organic micromolecule BSA-CQ4T accounts for 40 wt% of the nano probe, and the amino acid short chain accounts for 15 wt% of the nano probe.
2. The tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 1, wherein: the short chain of amino acids can be recognized and cut by fibroblast activation protein; the fluorescent organic micromolecule BSA-CQ4T is modified by bovine serum albumin; the nano silver is reductive nano silver; the nano probe takes a palladium nano sheet as a main body; the short chain of amino acid is connected with fluorescent organic micromolecule BSA-CQ4T in an amidation mode.
3. The tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 1, wherein: the edge length of the nano probe is 20-40 nm.
4. A preparation method of a tumor microenvironment response type diagnosis and treatment integrated nanoprobe is characterized by comprising the following steps:
s1 general formula Pd (acac)2Mixing the powder, polyvinylpyrrolidone powder and sodium bromide powder, dissolving the mixture into a mixed solution of deionized water and N, N-dimethylformamide, putting the mixture into a glass pressure container, and stirring the mixture in water bath at 30 ℃ until the mixture is dissolved to obtain a bright light yellow transparent solution;
s2, introducing CO gas into the container in which the solution obtained in the S1 is positioned at 4 ℃, and exhausting air;
s3, placing the S2 container in an oil bath kettle at the temperature of 80 ℃, keeping CO continuously introduced and discharged, and stirring for 3 hours;
s4, moving the container S3 out of the oil bath, closing the gas inlet and outlet of the 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 a blue-black precipitate;
s6, precipitation of S5 with ethanol: washing with a mixed solution with the volume ratio of acetone of 1:8, and centrifuging at a high speed to obtain a blue-black precipitate;
s7, carrying out vacuum drying on the precipitate obtained in the step S6, and storing at room temperature to obtain solid palladium nanosheet Pd NSs;
s8, dissolving fluorescent organic micromolecules BSA-CQ4T and albumin in phosphate buffer solution according to the mass ratio of 1:5-1:10, stirring at 70 ℃ to obtain BSA-CQ4T solution, and storing at 4 ℃ in a dark place;
s9, mixing the raw materials in a mass ratio of (2: 1-1): 5, mixing the solid palladium nanosheet Pd NSs obtained in the step S7 with a phosphate buffer solution with a short chain of amino acid, slowly stirring in a CO atmosphere at 4 ℃, performing ultrafiltration centrifugation, and freeze-drying to obtain a dark blue and nearly black solid Pd-pep;
s10, activating the solid Pd-pep obtained in the S9 in a phosphate buffer solution by using N-hydroxysuccinimide/1-3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride NHS/EDC, performing ultrafiltration and centrifugation after activation, taking the supernatant, adding a BSA-CQ4T solution in S8, keeping out of the sun in a CO atmosphere, and slowly stirring in an ice water bath to obtain a liquid Pd-pep-BSA-CQ 4T;
and S11, adding a silver nitrate solution and a reducing agent into the liquid Pd-pep-BSA-CQ4T obtained in the S10 in sequence under the conditions of keeping out of the sun and slowly stirring, stirring for 20min, and performing ultrafiltration and centrifugation to obtain the Pd-CQ4T-Ag nano probe.
5. The preparation method of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 4, which is characterized in that: pd (acac) in step S12The 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 is 29000.
6. The preparation method of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 4, which is characterized by comprising the following steps: step S5 and step S6, the centrifugal speed is 10000rpm, and the centrifugal time is 8 min; step S5 washing, centrifuging and repeating for 2 times; step S6 washing and centrifugation were repeated 3 times.
7. The preparation method of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 4, which is characterized in that: in step S8, the albumin is bovine serum albumin.
8. The preparation method of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 4, which is characterized by comprising the following steps: the amino acid short chain in step S9 has the amino acid sequence Ac-Asp-Ala-Gly-Pro-Asn-Gln-Cys-NH2。
9. The preparation method of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe according to claim 4, which is characterized by comprising the following steps: the reducing agent in step S11 is 0.1M L-ascorbic acid, 0.1M NaOH; the mass concentration of the silver nitrate solution is 2.5 x 10-3M。
10. The application of the tumor microenvironment responsive diagnosis and treatment integrated nanoprobe is characterized in that: use of a nanoprobe according to any of claims 1 to 4 or prepared according to any of claims 5 to 9 for fluorescence switching on in the near infrared two-domain for FAP-responsive applications.
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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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