CN117731664B - Preparation and application of tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring - Google Patents
Preparation and application of tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring Download PDFInfo
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Abstract
The invention discloses preparation and application of a tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring, and belongs to the technical field of biological medicines. Adding polyacrylic acid, thioacetamide and triethanolamine into an ethylene glycol solution containing manganese acetylacetonate and trisodium citrate to obtain a manganese sulfide-polyacrylic acid nano material; and adding a proton pump inhibitor to obtain the manganese sulfide-polyacrylic acid-proton pump inhibitor nano material, namely the tumor MRI diagnosis and treatment integrated nano probe. The tumor MRI diagnosis and treatment integrated nano probe prepared by the invention can be used for integration of acidosis monitoring tumor MRI diagnosis and treatment; the synthesis method is simple and convenient, and the operability is strong; the synthesized product is stable and has repeatability; the magnetic resonance imaging system has good nuclear magnetic imaging capability, and can monitor the degree of tumor acidosis in the tumor cell chemokinetics treatment process; has excellent effect in inducing acidosis and chemical kinetics combined treatment of tumor cells, and is beneficial to clinical application.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to preparation and application of a tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring.
Background
Acidosis is a phenomenon in which pH decreases due to accumulation of acidic substances produced by cells in tissues and blood in the body. The fast-proliferating tumor cells meet the requirement of large energy supply, adapt to low-oxygen environment, accelerate metabolism, up-regulate glycolysis reaction level, generate large amount of acidic metabolites such as lactic acid, carbonic acid and the like, form tumor microenvironment with stronger acidity, and induce tumor acidosis. Because the protein capable of regulating the intracellular and extracellular hydrogen ion level exists on the surface of the tumor cell, the protein has stronger capability of regulating the intracellular and extracellular acid-base balance, so that the tumor cell is well adapted to the acidic environment, acidosis in the cell can be avoided, and the meta-acid environment can promote invasion and metastasis of tumors. A proton pump is a hydrogen ion transporter that has the ability to regulate intracellular and extracellular hydrogen ion levels. The proton pump inhibitor can inhibit the high-expression V-type proton pump on the surface of a tumor cell membrane, reduce the activity of the V-type proton pump, weaken the capability of the tumor cell to regulate the acid-base balance inside and outside the cell, and prevent the discharge of acid metabolites, so that the acid metabolites are accumulated in the cytoplasm of the tumor cell, and the acidity inside the cell is enhanced to cause acidosis of the tumor cell. The role and mechanism of vacuole ATPase in tumor (Chinese biochemistry and molecular biology report of garden beam, etc.) reviews the multiple functions of vacuole ATPase in the process of tumorigenesis by regulating the pH of the intracellular and extracellular environments, such as inhibiting tumor apoptosis, participating in autophagy of tumor cells, promoting invasion, migration and proliferation of tumor, participating in the generation of tumor drug resistance, etc. Current acidosis research remains to inhibit metabolic activity in tumor cells and reduce tumor cell viability, thus a need exists for a therapeutic approach that can link acidosis to tumor therapy.
Magnetic Resonance Imaging (MRI) is widely used in medical diagnostics as a means of imaging body tissue using the principles of nuclear magnetic resonance. Among them, manganese-based contrast agents are hot spots of nuclear magnetic contrast agents in recent years, and have attracted much attention. Manganese ions prove to have good magnetic resonance imaging capability, and can strengthen the signal intensity of the T 1 weighted nuclear magnetic resonance image, so that the contrast enhancement and imaging of tumor parts are more obvious, and the accurate diagnosis of tumors is facilitated. In addition, the manganese ion has chemical kinetics treatment characteristics, and can convert hydrogen peroxide in tumor cells into hydroxyl free radicals (OH) by catalyzing Fenton-like reaction, so that tumor cell necrosis or apoptosis is initiated. The combination of proton pump inhibitors (Proton Pump Inhibitors, PPI) with manganese-based nanomaterials for the preparation of nanoprobes for monitoring the degree of acidosis of tumor cells and for carrying out treatments has not been reported.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide preparation and application of an integral tumor MRI diagnosis and treatment nano probe for acidosis monitoring. The proton pump inhibitor and the manganese-based contrast agent are combined, and the synthesized tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring has the characteristics of inducing acidosis and chemical kinetics treatment of tumor cells, has good nuclear magnetic resonance imaging capability, and can monitor the acidosis degree of the tumor cells in real time in the tumor treatment process, thereby providing real-time diagnosis for the treatment of diseases and being beneficial to clinical application. The synthesis method of the tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring is simple and convenient and has strong operability; the synthesized product has stability and repeatability.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect of the present invention, a tumor MRI diagnosis and treatment integrated nanoprobe for acidosis monitoring is provided, and the tumor MRI diagnosis and treatment integrated nanoprobe is prepared by the following method:
(1) Adding polyacrylic acid into glycol solution containing manganese acetylacetonate and trisodium citrate, stirring for the first time, adding glycol solution of thioacetamide, stirring for the second time, adding triethanolamine, stirring for the third time, heating, centrifuging, and collecting precipitate to obtain manganese sulfide-polyacrylic acid nanomaterial;
(2) Mixing the manganese sulfide-polyacrylic acid nanomaterial prepared in the step (1) with a proton pump inhibitor solution, stirring in an ice bath, and centrifugally collecting precipitate to obtain the manganese sulfide-polyacrylic acid-proton pump inhibitor nanomaterial, namely the tumor MRI diagnosis and treatment integrated nano probe.
Further, in the step (1), the adding proportion of the manganese acetylacetonate, the trisodium citrate, the polyacrylic acid and the thioacetamide is (1-500) to (1-100) to (1-200) to (1-100).
In the step (1), the adding ratio of the glycol to the triethanolamine is (1-100) to (0.1-10).
Further, in the step (1), the first stirring speed is 100-5000 rpm, and the time is 10-500 min; the second stirring and the third stirring have the speed of 100-5000 rpm and the time of 1-100 min.
Further, in the step (1), the heating temperature is 25-500 ℃ and the heating time is 1-200 min.
Further, in the step (1), the particle diameter of the manganese sulfide-polyacrylic acid nano material is 10-300 nm.
In the step (2), the mass ratio of the manganese sulfide-polyacrylic acid to the proton pump inhibitor is (0.1-10) to 1, wherein the proton pump inhibitor is pantoprazole sodium.
Further, the composition also comprises pharmaceutically acceptable carriers or auxiliary materials.
Furthermore, the application method of the tumor MRI diagnosis and treatment integrated nano probe is injection.
In a second aspect, the invention provides an application of the tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring in any one of the following 1) to 3):
1) Preparing an integrated diagnosis and treatment preparation;
2) Preparing a preparation for monitoring acidosis degree;
3) Preparing a tumor therapeutic preparation.
The invention has the beneficial effects that:
(1) The synthesis method of the tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring is simple and convenient, and has strong operability; the synthesized product is stable and has repeatability.
(2) The tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring prepared by the invention has good nuclear magnetic imaging capability, and can monitor the degree of tumor acidosis in the tumor cell chemical kinetics treatment process.
(3) The tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring prepared by the invention has excellent effect in inducing tumor cell acidosis and chemical kinetics combined treatment, and is beneficial to clinical application.
(4) The invention uses PPI, the PPI has the capability of inhibiting the proton pump over-expressed on the surface of tumor cells, the addition of manganese sulfide can provide a carrier for the PPI, the effect of the PPI can increase intracellular acidity, the degradation degree of the manganese sulfide is increased, and the imaging of T 1 is enhanced, so that the visualization of acidosis is achieved. The proton pump inhibitor and the manganese-based contrast agent are combined, and the internal functions of the proton pump inhibitor and the manganese-based contrast agent are exerted, so that the nano probe for monitoring the acidosis degree of tumor cells and implementing treatment can be prepared, and the proton pump inhibitor and the manganese-based contrast agent can be used for integrating tumor MRI diagnosis and treatment for acidosis monitoring so as to monitor and induce acidosis of tumor parts with different degrees.
Drawings
FIG. 1 is a transmission electron microscope picture of manganese sulfide-polyacrylic acid (MnS-PAA, MP) nanomaterial prepared in the examples;
FIG. 2 is a hydrated particle size of MP nanomaterial;
FIG. 3 is a graph of MP 3.0T magnetic resonance gel imaging at different concentrations;
Fig. 4 shows the inhibition effect of western blot (westem blot, WB) to protein expression by MPP nanomaterial with different drug loading rates: a in fig. 4 is WB developing strip; FIG. 4b is a graph showing the quantification of GLS protein expression; FIG. 4 c is a graph showing the quantification of V-ATPase protein expression;
FIG. 5 is a graph showing the cell killing effect of manganese sulfide-polyacrylic acid-proton pump inhibitor nanomaterials (MPP nanomaterials) at different concentrations and different drug loading rates on mouse non-small cell lung cancer (LLC);
fig. 6 shows the effect of 9.4T magnetic resonance imaging after tail vein injection with different drug loads: fig. 6 a is a magnetic resonance image of a mouse; fig. 6 b is a signal-to-noise ratio extremum quantization chart.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, the current study of acidosis remains in inhibiting the metabolic activity in tumor cells and reducing the viability of tumor cells, and the combination of intracellular acidosis and tumor diagnosis and treatment cannot be realized.
Based on the above, the invention aims to provide a tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring and a preparation method and application thereof. According to the invention, manganese acetylacetonate and polyacrylic acid are used as raw materials, a precursor solution is obtained through simple stirring reaction, then solvothermal reaction is carried out to obtain manganese sulfide-polyacrylic acid (MnS-PAA, MP) nano-materials, and finally the manganese sulfide-polyacrylic acid-proton pump inhibitor nano-materials (tumor MRI diagnosis and treatment integrated nano-probe for acidosis monitoring, MPP) are obtained after mixing and stirring with pantoprazole sodium solution serving as a proton pump inhibitor. The tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring prepared by the invention has the advantages of simple synthesis method, strong operability and good nuclear magnetic imaging capability, and can induce acidosis of tumor cells in the process of chemodynamics treatment of the tumor cells and monitor the acidosis degree of the tumor cells.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available. The polyacrylic acid used in the present invention was purchased from ala Ding Gongsi, CAS:9003-01-4. Mouse non-small cell lung cancer cells (LLC) were purchased from a national laboratory cell resource sharing platform. Mice used in this experiment were purchased from the experimental animal breeding limited company, jinan Pengyue, following the rule of welfare of experimental animals.
Example 1
(1) Preparation of manganese sulfide-polyacrylic acid nano material
213.174Mg of manganese acetylacetonate and 58.82mg of trisodium citrate are dissolved in 20mL of ethylene glycol, after complete dissolution, 120mg of polyacrylic acid is added, and the mixture is reacted for 120min under magnetic stirring at 800rpm under normal temperature. Then 56.3475mg of thioacetamide is dissolved in15 mL of ethylene glycol, added into the solution, reacted for 5min under the magnetic stirring of 800rpm at normal temperature, 0.5mL of triethanolamine is added dropwise, reacted for 5min under the magnetic stirring of 800rpm at normal temperature, after the reaction is finished, all the liquid is transferred into a microwave synthesizer, and heated for 60min at 200 ℃. And finally, centrifuging at 14000rpm for 10min to collect precipitate, re-dissolving the precipitate with absolute ethyl alcohol, and repeatedly centrifuging for 3 times to clarify supernatant to obtain the product manganese sulfide-polyacrylic acid (MnS-PAA, MP) nanomaterial. Dispersing the product in ethanol to obtain ethanol solution of manganese sulfide-polyacrylic acid, taking appropriate amount of product, quantifying with inductively coupled plasma emission spectrometer (ICP-OES), and storing at 4deg.C or-20deg.C.
(2) Preparation of manganese sulfide-polyacrylic acid-proton pump inhibitor nanomaterial (MPP)
Taking ethanol solution of manganese sulfide-polyacrylic acid prepared according to the method in the step (1), centrifuging at 14000rpm for 10min, discarding supernatant, collecting precipitate, re-suspending the precipitate with pure water, uniformly mixing by ultrasonic, and repeating the centrifugal re-suspending step for 3 times to obtain 1mg/mL manganese sulfide-polyacrylic acid aqueous solution. 3mg of pantoprazole sodium serving as a proton pump inhibitor is weighed and dissolved in 3mL of pure water to obtain 1mg/mL of pantoprazole sodium serving as the proton pump inhibitor. Manganese sulfide-polyacrylic acid aqueous solution and proton pump inhibitor pantoprazole sodium aqueous solution are mixed according to solid mass, and the ratio is 4: 1. 2: 1. 1:1, adding pure water to the mixture to a constant volume of 2mL, and the specific adding amount is shown in Table 1.
Table 1: manganese sulfide-polyacrylic acid and proton pump inhibitor pantoprazole sodium mixing proportion
Stirring the solution after constant volume in ice bath for 30min, centrifuging at 14000rpm for 10min at 4deg.C, and collecting precipitate and supernatant respectively. Washing the precipitate with pure water for 1-2 times to obtain products of manganese sulfide-polyacrylic acid-proton pump inhibitor nanomaterial MPP-1 (4:1), MPP-2 (2:1) and MPP-3 (1:1), respectively for the above three nanomaterials, mixing the supernatant obtained by first centrifugation after ice bath stirring with the supernatant collected by subsequent water washing. The absorbance of the 292nm supernatant was measured using an ultraviolet spectrophotometer. Calculating the load rate of the proton pump inhibitor in each group of materials according to the standard curve of the proton pump inhibitor; the manganese ion content of each set of products was then determined using an inductively coupled plasma emission spectrometer (ICP-OES), the results are shown in table 2:
Proton pump inhibitor load rate calculation formula: LE (%) = [ (m a-mb)/(mc+(ma-mb)) ] ×100%
M a: total mass of PPI added
M b: PPI unloaded mass measured by UV and calculated by PPI labelling
M c: added MnS-PAA mass
Table 2: manganese ion content in each group of products and loading rate of proton pump inhibitor
Test example: analysis of Material Properties
Transmission electron microscope observation
Taking 100 mu g of MP ethanol solution prepared according to the method in the step (1), centrifuging to remove supernatant, re-suspending 100 mu L of pure water to prepare 1mg/mL solution, taking 10 mu L of solution by using a 10 mu L pipette, dripping 30-40 mu L of solution on a copper mesh, drying to prepare a transmission electron microscope detection sample, and observing by using a transmission electron microscope.
As shown in FIG. 1, the MP nanomaterial is spherical and has a particle size of 90-150nm, and the MP loading PPI does not change the particle size of the nanomaterial because the PPI is a small molecule drug.
Dynamic light scattering analyzer (DLS) test:
30. Mu.g of the MP ethanol solution prepared in the method of step (1) was centrifuged to remove the supernatant, and then resuspended in 1mL of pure water to prepare a 30. Mu.g/mL solution, and the hydrated particle size of MP was measured using a dynamic light scattering analyzer (DLS).
The results are shown in fig. 2, which shows that the hydration particle size of the MP nanomaterial is about 170nm, and that the MP loading the PPI does not change the particle size of the nanomaterial because the PPI is a small molecule drug.
Relaxation performance detection:
MP nanomaterial dispersions at concentrations 320, 160, 80, 40, 20, 10, and 0 μg/mL were formulated with PBS at pH 7.4, 6.5, 6, 5.5, 5, 4.5, respectively. 1mL of each concentration dispersion was taken in a 2mL EP tube, 1mL of 1% agarose gel (1% agarose gel preparation: 1g agarose gel was weighed and added to 99mL of 1 xTAE buffer, and heated with a microwave oven for about 2 minutes with high fire to obtain clear agarose gel), and the clear agarose gel was mixed uniformly to achieve the final concentration of the gel to 160, 80, 40, 20, 10, 5 and 0. Mu.g/mL, relaxation properties were detected using 3.0T nuclear magnetism, and then the images were analyzed.
As shown in FIG. 3, the stronger the manganese ions are released under the condition of stronger acidity, the stronger the imaging capability is, the capability also has concentration dependence, the simulated acidosis state provides basis for the subsequent experiment, and the result shows that the MP nanomaterial can respond to different pH environments, present different nuclear magnetic signal intensities and has the capability of presenting acidosis degree.
Western Blot (Western Blot) experiment:
Mouse non-small cell lung cancer cells (LLC) were plated in six well plates, with five groups of treatment cells: (1) NC (Mn concentration of 0. Mu.g/mL, no PPI loading); (2) MP (Mn at a concentration of 20. Mu.g/mL, without PPI loading); (3) MPP-1 (Mn concentration 20. Mu.g/mL, MP, PPI feed ratio 4:1); (4) MPP-2 (Mn concentration 20. Mu.g/mL, MP, PPI feed ratio 2:1); (5) MPP-3 (Mn concentration 20. Mu.g/mL, MP, PPI feed ratio 1:1). After 12h incubation, each group of cells was collected, total cell proteins were extracted by adding protein lysate, and protein quantification was performed according to BCA reagent instructions. After electrophoresis, the separated proteins were transferred to a PVDF membrane. After transfer of the membrane, it was blocked with skimmed milk powder for 1h, with GAPDH as an internal control, diluted anti-GLS primary antibody (abcam) or anti-ATP 6V1A antibody (abeam) solution (diluted according to the recommended concentration for each antibody specification) was added and incubated for 2h at 37 ℃. After PBST washing, a secondary antibody shaking table was added for 1h, PBST was washed, developed, and photographed, and the WB experimental results were analyzed.
As shown in fig. 4, the results demonstrate that PPI-loaded MPs are able to down-regulate the expression of GLS and V-atpase receptors, key transporters of Gln, in LLC cells.
Cytotoxicity experiment:
Preparing 96-well plates, adding LLC cells of 1X 10 4 into each well, culturing for 24 hours in a constant temperature incubator at 37 ℃ containing 5% CO 2, incubating MP, MPP-1, MPP-2 and MPP-3 prepared in the step (2) with the cells for 12 hours at different concentrations of 0, 5, 10, 20 and 30 mug/mL respectively, sucking off culture solution, adding MTT-containing culture medium into each well for culturing for 3-3.5 hours, sucking off MTT culture medium, adding 150 mug of DMSO into each well, oscillating a shaking table for 10-15 minutes to completely dissolve purple solids, reading absorbance value (OD value) at 490nm by using an enzyme-labeled instrument, judging the number of living cells according to the measured OD value, and making a result graph.
The results are shown in fig. 5, and the LLC cell viability gradually decreases with increasing concentration of the formulation and drug loading, indicating that the MPP formulation has significant cytotoxic effects on tumor cells, and increases cell killing with increasing concentration and loading.
Magnetic resonance imaging test:
The 6-week-old healthy C57 mice are taken, male and female mice are respectively half-suspended in PBS, MP, MPP-1, MPP-2 and MPP-3 are respectively injected into the bodies of the mice through tail veins according to the drug concentration of 2mg/kg, and the images are quantitatively analyzed after scanning is carried out by a 9.4T small animal nuclear magnetic instrument before injection, after injection for 0.5h, after injection for 1h, after injection for 1.5h, after injection for 2h and after injection for 2.5 h.
As shown in fig. 6, after the nano preparation is injected, the image signal to noise ratio is obviously increased compared with that before injection, and the imaging effect is enhanced along with the increase of the PPI load, which indicates that the nano preparation prepared in the step (2) has stronger magnetic resonance imaging capability, and the effect of monitoring the acidosis of tumor cells can be achieved through different PPI loads.
Claims (7)
1. The tumor MRI diagnosis and treatment integrated nano-probe is characterized by being prepared by the following method:
(1) Adding polyacrylic acid into glycol solution containing manganese acetylacetonate and trisodium citrate, stirring for the first time, adding glycol solution of thioacetamide, stirring for the second time, adding triethanolamine, stirring for the third time, heating, centrifuging, and collecting precipitate to obtain manganese sulfide-polyacrylic acid nanomaterial;
(2) Mixing the manganese sulfide-polyacrylic acid nanomaterial prepared in the step (1) with a proton pump inhibitor solution, stirring in an ice bath, and centrifugally collecting precipitate to obtain the manganese sulfide-polyacrylic acid-proton pump inhibitor nanomaterial, namely the tumor MRI diagnosis and treatment integrated nano probe;
in the step (1), the adding ratio of the manganese acetylacetonate, the trisodium citrate, the polyacrylic acid and the thioacetamide is (1-500): (1-100): (1-200): (1-100); the adding ratio of the ethylene glycol to the triethanolamine is (1-100): (0.1-10);
In the step (2), the mass ratio of the manganese sulfide-polyacrylic acid to the proton pump inhibitor is (0.1-10) 1, wherein the proton pump inhibitor is pantoprazole sodium.
2. The tumor MRI diagnosis and treatment integrated nano-probe for acidosis monitoring according to claim 1, wherein in the step (1), the first stirring speed is 100-5000 rpm, and the time is 10-500 min; the second stirring speed and the third stirring speed are 100-5000 rpm, and the time is 1-100 min.
3. The tumor MRI diagnosis and treatment integrated nano-probe for acidosis monitoring according to claim 1, wherein in the step (1), the heating temperature is 25-500 ℃ and the heating time is 1-200 min.
4. The tumor MRI diagnosis and treatment integrated nano-probe needle for acidosis monitoring according to claim 1, wherein in the step (1), the particle diameter of the manganese sulfide-polyacrylic acid nano-material is 10-300 nm.
5. The tumor MRI diagnosis and treatment integrated nanoprobe for acidosis monitoring according to any one of claims 1 to 4, further comprising a pharmaceutically acceptable carrier or adjuvant.
6. The tumor MRI diagnosis and treatment integrated nano-probe for acidosis monitoring according to claim 5, wherein the application method of the tumor MRI diagnosis and treatment integrated nano-probe is injection.
7. The use of the tumor MRI diagnosis and treatment integrated nanoprobe for acidosis monitoring according to claim 5 in any one of the following 1) to 3):
1) Preparing an integrated diagnosis and treatment preparation;
2) Preparing a preparation for monitoring acidosis degree;
3) Preparing a tumor therapeutic preparation.
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