CN114010803B - Multifunctional targeting molecular probe for detecting Treg cells in living tumor microenvironment and application thereof - Google Patents
Multifunctional targeting molecular probe for detecting Treg cells in living tumor microenvironment and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of immune medicine research, in particular to a multifunctional targeted molecular probe for detecting Treg cells in a living tumor microenvironment based on a photoacoustic imaging technology, and further discloses application of the multifunctional targeted molecular probe in preparing an anti-tumor drug. The multifunctional targeted molecular probe for detecting the Treg cells in the living body tumor microenvironment forms the nano probe capable of responding to the tumor microenvironment in a mode of coating and processing the CD-25 fluorescent antibody, the chemotherapeutic drug and the immune checkpoint inhibitor IDO together, the nano probe can be accumulated at the tumor part under the action of EPR, and the CD-25 fluorescent antibody, the chemotherapeutic drug and the immune checkpoint inhibitor IDO coated inside are released in the presence of glutathione, so that the Treg cells in the tumor microenvironment can be traced through photoacoustic imaging and fluorescence imaging, the number of the Treg cells in the tumor microenvironment can be quantitatively detected in vivo, the growth in the tumor can be inhibited, and the survival rate of a patient can be improved.
Description
Technical Field
The invention belongs to the technical field of tumor diagnosis research, and particularly relates to a multifunctional targeting molecular probe for detecting Treg cells in a living tumor microenvironment based on a photoacoustic imaging technology, and further discloses application of the multifunctional targeting molecular probe in preparation of antitumor drugs.
Background
Gliomas are a diffusely infiltratively growing, heterogeneous, fatal tumor, and even with the greatest degree of surgery and drug therapy, the treatment of glioblastoma remains a serious intractable problem. Median survival of brain gliomas is reported to be less than 2 years, with few patients surviving for long periods. Moreover, because the boundary of the brain glioma is not obvious and has strong erosiveness, and the tumor boundary is fuzzy and inaccurate, incomplete surgical resection is easy to cause, and the recurrence rate of patients is high. It has been studied that the factors responsible for the poor prognosis of GBM are mainly the strong immunosuppressive tumor environment, whereas the induced maintenance of immunosuppression in GBM is due in part to infiltration and accumulation of highly immunosuppressive regulatory T cells (tregs).
Studies have shown that tregs express high levels of glucocorticoid-induced tumor necrosis factor receptor-associated protein, inhibiting the function of Antigen Presenting Cells (APCs) by immunosuppressing the expression of the cytokine interleukin 10, secretion of transforming growth factor-beta, and suppression of Antigen Presenting Cell (APC) function from thymus-derived or induced progenitor cells. However, the current monitoring of Treg cells is confirmed by pathological section, but pathological section is not only much harmful to patients, but also suitable for long-term monitoring. Therefore, the development of the nano-targeted drug which can monitor the Treg cells in the tumor prognosis for a long time in a non-invasive manner, especially is beneficial to anti-tumor treatment, and has positive significance for the treatment of the glioma.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a multifunctional targeting molecular probe for detecting Treg cells in a living tumor microenvironment, which not only can realize noninvasive detection of the Treg cells, but also can further improve the survival rate of patients through targeted release of antitumor drugs;
the invention also aims to provide the application of the multifunctional targeted molecular probe for detecting the Treg cells in the living tumor microenvironment in preparing anti-tumor medicines, in particular anti-glioma medicines.
In order to achieve the aim, the invention provides a preparation method of a multifunctional targeted molecular probe for detecting Treg cells in a living tumor microenvironment, which comprises the following steps:
(1) Respectively dissolving the coated copolymer material, the fluorescent antibody material and the anti-tumor drug in an organic solvent, and fully dispersing to obtain a mixed solution for later use;
(2) Adding deionized water into the mixed solution, stirring thoroughly and violently, and removing residual organic solvent in the solution to obtain the required nanoparticles;
(3) And (3) dialyzing the obtained nano particles to remove non-encapsulated substances, and freeze-drying to obtain the required nano probe.
Specifically, in the step (1), the fluorescent antibody material includes CD25-Cy7.
Specifically, in the step (1), the coating copolymer material comprises DSPE-SS-PEG 2000 。
Specifically, in the step (1), the antitumor drug comprises temozolomide.
Specifically, the step (1) further comprises a step of adding an immune checkpoint inhibitor for common coating.
In particular, the immune checkpoint inhibitor comprises an IDO inhibitor.
Specifically, the dosage ratio of the coating copolymer material, the fluorescent antibody material, the antitumor drug and the immune checkpoint inhibitor is 10:0.1:2:0.1.
specifically, the organic solvent includes THF.
The invention also discloses a multifunctional targeting molecular probe for detecting Treg cells in a living tumor microenvironment, which is prepared by the method.
The invention also discloses application of the multifunctional targeting molecular probe for detecting Treg cells in a living tumor microenvironment in preparation of antitumor drugs.
Specifically, the anti-tumor drug is a drug for treating brain glioma.
The multifunctional targeted molecular probe for detecting the Treg cells in the living body tumor microenvironment forms a nano molecular probe capable of responding to the tumor microenvironment in a mode of coating and processing the CD-25 fluorescent antibody, the chemotherapeutic drug and the immune checkpoint inhibitor IDO together, the molecular probe can be accumulated at the tumor part under the action of EPR, and the probe can be cracked in the presence of glutathione to release the CD-25 fluorescent antibody, the chemotherapeutic drug and the immune checkpoint inhibitor IDO which are coated in the probe. The released chemotherapeutic drug can be well accumulated to a tumor part, kill tumor cells, inhibit the growth of tumors and improve the survival rate of patients; in addition, the released CD-25 fluorescent antibody can be combined with Treg cells, so that the Treg cells in a tumor microenvironment can be traced through photoacoustic and fluorescence imaging, the number of the Treg cells in the tumor microenvironment can be quantitatively detected in vivo, and the relationship between the number of Treg cell infiltration and the survival rate of a patient can be favorably detected; and the released immune checkpoint inhibitor IDO is further combined, so that the infiltration of Treg cells in the tumor can be effectively reduced, and the survival rate of patients is further improved.
The multifunctional targeted molecular probe for detecting the Treg cells in the microenvironment of the living body tumor monitors the immune microenvironment of the brain glioma through the photoacoustic imaging living body, provides real-time evaluation and monitoring, can realize visual monitoring of the important biological event of Treg cell infiltration of the tumor microenvironment, and provides a new scheme for treatment and prognosis of the brain glioma. According to the targeting molecular probe, by researching a targeting action mechanism and influencing factors, researching the detection capability of the targeting probe on tumor treatment and tumor microenvironment immune cells and combining the imaging effect of the existing imaging method, the problems of complex heterogeneity and unclear boundary blurring of brain glioma are solved, the problem of poor diagnosis and treatment caused by erosive growth and fuzzy boundary of brain glioma is effectively solved, a slice diagnosis mode with complicated steps in the traditional monitoring method can be overcome, and the diagnosis accuracy is effectively improved; in addition, the molecular probe is combined with an anti-tumor drug and an immune checkpoint inhibitor, so that the treatment effect is improved, the survival rate of patients is improved, and the molecular probe has a good clinical application prospect.
According to the multifunctional targeting molecular probe for detecting the Treg cells in the living tumor microenvironment, the PEG wraps the temozolomide, so that the biocompatibility of the temozolomide is effectively improved, the treatment effect of the temozolomide is obviously improved, and the problem that the application effect of the temozolomide in vivo is influenced due to poor water solubility of the temozolomide is effectively solved.
Drawings
FIG. 1 is a schematic diagram of the synthesis and release process of the targeting molecular probe of the present invention;
FIG. 2 is a hydrated particle size of a targeted molecular probe of the present invention, showing that the probe diameter is about 50nm;
FIG. 3 is the UV-VIS absorption spectrum of the targeting molecular probe of the present invention, showing 275nm and 750nm absorption peaks from the spectrum, which are characteristic absorption peaks of the probe;
FIG. 4 shows the results of drug release testing of the targeted molecular probe of the present invention;
FIG. 5 shows the results of the cellular uptake assay of the targeted molecular probes of the present invention;
FIG. 6 shows the result of the killing effect of the targeting molecular probe of the present invention on tumor cells;
FIG. 7 shows the effect of targeting the brain glioma using the targeting molecular probe of the present invention.
Detailed Description
Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
As shown in fig. 1, the molecular probe capable of responding to a tumor microenvironment can accumulate at a tumor site under the action of EPR, and can be cracked in the presence of glutathione to release a CD-25 fluorescent antibody, a chemotherapeutic drug temozolomide and an immune checkpoint inhibitor IDO wrapped inside, so that the chemotherapeutic drug can be well accumulated at the tumor site, kill tumor cells, inhibit growth in the tumor and improve the survival rate of a patient; in addition, the released CD-25 fluorescent antibody can be combined with Treg cells, so that the Treg cells in a tumor microenvironment can be traced through photoacoustic and fluorescence imaging, and the quantity of the Treg cells in the tumor microenvironment can be quantitatively detected in vivo; by combining with an immune checkpoint inhibitor IDO, the infiltration of Treg cells in tumors can be effectively reduced, and the survival rate of patients is further improved.
EXAMPLE 1 preparation of nanoparticles
Mixing 10mg DSPE-SS-PEG 2000 Dissolving 10 mu L of CD25-Cy7, 2mg of temozolomide and 10 mu L of indoleamine 2, 3-dioxygenase inhibitor (IDO-inhibitor) in 1mL of THF (commercially available material), adding the THF into 9mL of deionized water which is vigorously stirred for 15min, and then stirring overnight to remove residual THF in the solution to obtain target nanoparticles; and dialyzing the target nano particles for 3 times by using a dialysis membrane of 2KDa, removing substances which are not coated, and freeze-drying to obtain the target molecular probe.
Example 2 characterization of nanoparticles
The morphology and size of the nanoparticles were examined with a field emission high resolution 2100F Transmission Electron Microscope (TEM) at an accelerating voltage of 200 kV. As shown in FIG. 2, the nanoprobe has a uniform spherical shape and a diameter of about 50nm.
The size distribution of the nanoparticles was measured by a Malvern Zetasizer Nano ZS90 (Malvern, UK) instrument and the results showed agreement with the TEM results shown in fig. 2.
The infrared spectrum of the sample from 4000 to 500 was measured using a Nicolet 759 type Fourier transform infrared spectrometer (FT-IR) and a KBr crystal, and the result shown in FIG. 3 showed characteristic peaks of the respective substances.
The optical properties of CD25-Cy7 and IR780 were measured by UV-visible absorption (Lambda-35 UV-visible spectrophotometer, perkinelmer, mass., USA) and showed characteristic peaks for each material.
The photoacoustic effect of Cy7 and IR780 was detected by a tunable pulsed laser as the light source (Nd: YAG Surelight-II-20), and the results showed that a strong photoacoustic signal was displayed at 750 nm.
Example 3 cell culture
GL261 cells were incubated in DMEM medium containing 10% FBS and 1% antibiotic (penicillin-streptomycin, 10000U/mL) in a humidified atmosphere of 5% CO2 at 37%. Treg cells isolated in mice were cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotic (penicillin-streptomycin, 10000U/mL) in a 5% humidified environment at 37 ℃ with carbon dioxide incubated under standard medium and conditions.
Cell count assays (CCK-8, dojindo laboratories, kumamoto, japan) are used to measure cell viability.
Example 4 drug Release test
The dispersion was placed in PBS buffer (0 mM glutathione, 10mM glutathione) at pH 7.4 and transferred to a dialysis tube (molecular weight cut-off: 2000 Da) immersed in the same buffer medium. After changing the time interval, an equal amount of external medium was removed and replaced with the same volume of fresh buffer. The results are shown in FIG. 4, in which the amount of the released nanoprobe was 50% in 2 hours under the condition of 0mM glutathione and 98% in 2 hours under the condition of 10mM glutathione.
Example 5 cellular uptake
EXAMPLE 6 killing of tumor cells by nanoparticles
The killing effect of the chemotherapeutic drug on cells is verified through a cell layer. We performed cell activity assays on cells from different treatment groups using CCK8 kit to determine the reasonable nanoparticle concentrations we used for tumor therapy later.
GL261 cells were first seeded into 96-well plates and cultured for 12h to moderate density. The seeded cells were then divided into 24 groups and treated as follows: control, PBS, various concentrations of TMZ. After 4h incubation, 10 microliters of CCK-8 reagent was added to all the well plates of cultured cells, incubated for 1h, and absorbance was measured in each well with a microplate reader at a wavelength of 450 nm.
The average relative activity of the cells was calculated as follows: cell average relative activity = (treatment group absorbance/control group absorbance) × 100%.
The results of the experiment are shown in FIG. 6, the cell killing effect of the chemotherapeutic drug is increased along with the increase of the concentration, and 80 percent of the killing effect can be achieved at 100 mu g.
Then, to evaluate the change in the morphology of apoptotic nuclei caused by chemotherapy, GL261 cells were cultured in 35 mm glass-bottom plates with Control, PBS, and TMZ at various concentrations, respectively. After incubation at 37 ℃ for 12 h. These samples were co-stained with calcein-acetylmethoxylate (calcein-AM) and PI at 37 ℃ for 30 minutes and washed twice with PBS. Images were acquired using a confocal laser scanning microscope (zeiss LSM 510META, germany). calcein-AM and PI are available from Sigma-Aldrich Corporation (Mo. ). The experimental results show that the results are consistent with the effect of CCK 8.
Example 7 in situ monitoring of Treg cells from brain gliomas by optical imaging
The brain of the mouse is imaged through photoacoustic microscopy imaging, and the tumor growth condition and the tumor microvascular distribution condition of the mouse are accurately observed. Under the action of a tumor microenvironment, the shell of the nano probe is decomposed to release the temozolomide which is a chemotherapeutic drug in the encapsulated nucleus, and the treatment condition of the tumor is monitored by a photoacoustic microscope. After 21 days of treatment, the tumor is taken out, the tumor is ground into single cells, the number of Tregs in a tumor microenvironment is detected by a parallel flow cytometer, and the immunosuppressive effect of the brain tumor is verified, and the result is shown in figure 7, so that the nano probe can be gathered at the tumor part, and the gathering effect of other control groups is very poor.
EXAMPLE 8 Effect of nanoparticles on Living tissues
After treatment of all treatment groups was completed, tumors and major organs (including heart, liver, spleen, lung, kidney) were collected and fixed in 4% (w/v) paraformaldehyde solution, and tissues were embedded in paraffin overnight. The paraffin embedded tissue was then cut to about 5.0 μm. Then stained with hematoxylin and eosin (H & E). And finally, observing the H & E staining result by using a microscope, and performing biochemical histological analysis on different treatment groups. The experimental result shows that the nano probe has no damage to each organ in the mouse body, and the safety of the nano probe is proved.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (4)
1. A preparation method of a multifunctional targeting molecular probe for detecting Treg cells in a living tumor microenvironment is characterized by comprising the following steps:
(1) Respectively taking DSPE-SS-PEG 2000 Dissolving a fluorescent antibody CD25-Cy7, temozolomide and indoleamine 2, 3-dioxygenase inhibitor in an organic solvent, and fully dispersing to obtain a mixed solution for later use;
(2) Adding deionized water into the mixed solution, stirring thoroughly and violently, and removing residual organic solvent in the solution to obtain the required nanoparticles;
(3) And (3) dialyzing the obtained nanoparticles to remove unencapsulated substances, and freeze-drying to obtain the target molecular probe.
2. The method for preparing the multifunctional targeting molecular probe for detecting the Treg cells in the microenvironment of the living tumor according to claim 1, wherein the DSPE-SS-PEG is used for detecting the Treg cells in the microenvironment of the living tumor 2000 The mass ratio of the fluorescent antibody CD25-Cy7 to the temozolomide to the indoleamine 2, 3-dioxygenase inhibitor is 10:0.1:2:0.1.
3. a multifunctional targeting molecular probe for the detection of Treg cells in a living tumor microenvironment, prepared by the method of any one of claims 1-2.
4. The use of the multifunctional targeted molecular probe for detecting Treg cells in a microenvironment of a living tumor as claimed in claim 3 for preparing an anti-tumor medicament for treating brain glioma.
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