A nano-drug for tumor hypoxia

Abstract

The application discloses a kind of nanomedicine for tumor hypoxia, which is prepared by mPEG-SS-PLGA wrapping drug IR-1048, PFOB and 4-MU.The nanomedicine of the application is constructed by wrapping perfluorooctyl bromide (PFOB), 4-methyl umbelliferone (4-MU) and IR-1048 in tumor microenvironment-responsive amphiphilic polymer mPEG-SS-PLGA, and in response to high GSH in tumor area, 4-MU, PFOB and IR-1048 are released; 4-MU makes the dense tumor extracellular matrix loose, so that tumor vessels are normalized, which is conducive to PFOB carrying oxygen into the tumor interior, long-term solution of tumor hypoxia, remodeling of tumor microenvironment, and reduction of myeloid-derived suppressor cell (MDSC) infiltration; and under the action of 980nm laser in vitro, IR-1048 responds, and the effect of photothermal therapy of tumor is improved.

Description

Technical Field

[0001] This invention relates to a nanomedicine for tumor hypoxia. Background Technology

[0002] Hypoxia is a major pathophysiological feature of the tumor microenvironment, closely related to malignant tumor progression, angiogenesis, tumor cell invasion, metastasis, chemotherapy resistance, and metabolic changes. During proliferation, tumor cells continuously infiltrate surrounding tissues and cells, forming the tumor microenvironment, which becomes the "soil" suitable for tumor cell growth. Although tumor cells have the ability to form microvascular networks during rapid proliferation, their rapid proliferation rate consumes large amounts of oxygen, exceeding the oxygen supply of the microvascular network, resulting in a hypoxic state in the tumor site compared to normal tissues. Under hypoxic conditions, the tumor cell cycle is altered, with most cells remaining in the G1 / S phase, affecting intracellular DNA replication, transcription, and translation, reducing the efficacy of DNA-targeting chemotherapy drugs, such as doxorubicin. Furthermore, it can induce the activation of hypoxia-inducible factors and the expression of P-glycoproteins, leading to strict resistance to chemotherapy drugs. In addition, for photodynamic therapy and sonodynamic therapy, which rely on oxygen to generate reactive oxygen species to kill tumor cells, hypoxia severely limits their efficacy. PFOB is a long-chain, high-density liquid substance among fluorocarbon compounds. It has a high solubility for oxygen and its dissociation curve with oxygen shows a linear relationship. As the partial pressure of oxygen increases or decreases, it dissolves or releases oxygen, making it a promising tissue oxygen carrier for clinical use. PFOB does not participate in biochemical degradation during circulation in the body. It can diffuse into the blood and is eventually excreted through exhalation, exhibiting good in vivo safety and biocompatibility.

[0003] While PFOB application can partially improve tumor hypoxia, the dense extracellular matrix of tumor cells makes it difficult for PFOB to penetrate and thus fails to improve the hypoxic state of the internal region. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies in the existing technology and provide a nanomedicine that can effectively improve tumor hypoxia and enhance the effect of photothermal therapy.

[0005] To achieve the above objectives, the present invention provides a nanomedicine for tumor hypoxia, which is prepared by encapsulating the drug IR-1048, PFOB (perfluorooctyl bromide), and 4-MU (4-methylumbelliferone) in mPEG-SS-PLGA.

[0006] The nanomedicine of this invention is constructed by encapsulating perfluorooctyl bromide (PFOB), 4-methylumbelliferone (4-MU), and IR-1048 in a tumor microenvironment-responsive amphiphilic polymer mPEG-SS-PLGA. It exhibits a high GSH response in the tumor region, releasing 4-MU and PFOB. 4-MU loosens the dense tumor extracellular matrix, normalizing tumor angiogenesis and facilitating the entry of PFOB carrying oxygen into the tumor, thus providing long-term relief from tumor hypoxia, reshaping the tumor microenvironment, and reducing the infiltration of myeloid-derived suppressor cells (MDSCs). Furthermore, under the action of a 980nm laser in vitro, it enhances the photothermal therapy effect on tumors.

[0007] Furthermore, the nanomedicine of the present invention is constructed using a self-assembly technique, with the following specific steps: mPEG-SS-PLGA is dissolved in deionized water to obtain an mPEG-SS-PLGA suspension for later use; IR-1048, PFOB, and 4-MU are dissolved in dichloromethane, and then slowly added to the mPEG-SS-PLGA suspension, and sonicated until the dichloromethane is completely evaporated; the precipitate is collected by repeated centrifugation, and the residue is washed away to obtain the nanomedicine.

[0008] Furthermore, the addition ratio of the above-mentioned mPEG-SS-PLGA, IR-1048, PFOB, and 4-MU is 30mg:(2-4mg):(10-20mg):(2-6mg). The addition amounts of IR-1048, PFOB, and 4-MU have a slight impact on the final encapsulation amount of the nanomedicine, but do not affect the improvement of tumor hypoxia and photothermal therapy effects.

[0009] In some embodiments, as a preferred embodiment, the addition ratio of mPEG-SS-PLGA, IR-1048, PFOB and 4-MU is 30mg:4mg:20mg:6mg.

[0010] This invention also provides the application of the above-mentioned nanomedicine in the preparation of drugs with tumor photothermal therapy effects.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The nanomedicine of this invention (I-4-P@PssP) is constructed by encapsulating perfluorooctyl bromide (PFOB), 4-methylumbelliferone (4-MU), and IR-1048 in an amphiphilic polymer that responds to the tumor microenvironment. I-4-P@PssP responds to high GSH in the tumor region, releasing 4-MU and PFOB; 4-MU loosens the dense tumor extracellular matrix, normalizes tumor angiogenesis, and facilitates PFOB carrying oxygen into the tumor, thus providing long-term relief from tumor hypoxia, reshaping the tumor microenvironment, and reducing the infiltration of myeloid-derived suppressor cells (MDSCs); and under the action of 980nm laser in vitro, it enhances the photothermal therapeutic effect on tumors.

[0013] The nanomedicine of this invention has the effect of long-term relief of tumor microenvironment hypoxia, reduction of tumor MDSC infiltration, and improvement of photothermal therapy efficacy. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the construction of the nanomedicine of the present invention;

[0015] Figure 2 This is a schematic diagram illustrating the mechanism of action of the nanomedicine of the present invention;

[0016] Figure 3 The in vitro release curve of IR-1048 in response to tumor GSH of the nanomedicine of this invention;

[0017] Figure 4 This is the in vitro release curve of the nanomedicine of the present invention in response to tumor GSH with 4-MU;

[0018] Figure 5 This is the oxygen concentration curve of the PFOB oxygen-carrying and maintaining solution for the nanomedicine responding to tumor GSH according to the present invention;

[0019] Figure 6 Immunohistochemical staining images of tumor tissues after different treatments for HABP2;

[0020] Figure 7 Comparison of HA-positive areas in tumor tissues after different treatments;

[0021] Figure 8 Immunohistochemical staining of tumor tissues after different treatments was performed using anti-cd31 antibody and anti-HIF-1α antibody.

[0022] Figure 9 Ultrasound imaging of tumor blood oxygen saturation after different treatments;

[0023] Figure 10 Tumor tissue after different treatments 3 Comparison chart of H-TdR level count monitoring;

[0024] Figure 11 A comparison chart showing the percentage reduction of MDSCs in tumor tissue after different treatments;

[0025] Figure 12 Comparison images of tumor sites after different treatments;

[0026] Figure 12 In the image, a shows a comparison of images of the tumor site, b shows a comparison of H&E staining, and c shows a comparison of the weight of the tumor site. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings.

[0028] Example 1

[0029] Construction of the nanomedicine (I-4-P@PssP) of this invention

[0030] The construction of I-4-P@PssP employs a self-assembly technique, such as... Figure 1 As shown, specifically: dissolve 30 mg of mPEG-SS-PLGA in 30 mL of deionized water. Dissolve IR-1048 (2-4 mg), PFOB (10-20 mg), and 4-MU (2-6 mg) in 3 mL of dichloromethane, then slowly add this solution to the mPEG-SS-PLGA suspension. Sonicate until the dichloromethane is completely evaporated. Centrifuge repeatedly (10 minutes, 4000 g), collect the precipitate, and wash three times to remove residues. This yields the nanomedicine of this patent, abbreviated as I-4-P@PssP in the following examples.

[0031] This embodiment uses the optimal solution of IR-1048 (4mg), PFOB (20mg), and 4-MU (6mg).

[0032] Among them, mPEG-SS-PLGA was purchased from Xi'an Ruixi Biotechnology Co., Ltd., IR-1048 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., PFOB was purchased from Beijing Bailingwei Technology Co., Ltd., and 4-MU was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0033] The nanomedicine I-4-P@PssP of this invention is constructed by encapsulating perfluorooctyl bromide (PFOB), 4-methylumbelliferone (4-MU), and IR-1048 in a tumor microenvironment-responsive amphiphilic polymer (mPEG-SS-PLGA). After intravenous injection, I-4-P@PssP releases its contents at the tumor site (high GSH level), such as... Figure 2 As shown, 4-MU loosens the dense extracellular matrix of tumor cells, which is conducive to PFOB carrying oxygen into the tumor, effectively solving tumor hypoxia in the long term, and improving the efficacy of tumor photothermal therapy under the action of 980nm laser in vitro.

[0034] The preparation of the drugs used for comparing the effects is as follows:

[0035] The preparation process of I@PssP is basically similar to that of I-4-P@PssP. The difference is that only IR-1048 (4 mg) is dissolved in 3 mL of dichloromethane and then slowly added to the mPEG-SS-PLGA suspension. The nanomedicine prepared contains only IR-1048.

[0036] The preparation process of I-4@PssP is basically similar to that of I-4-P@PssP. The difference is that IR-1048 (4 mg) and 4-MU (6 mg) are dissolved in 3 mL of dichloromethane and then slowly added to mPEG-SS-PLGA suspension. The nanomedicine prepared contains only IR-1048 and 4-MU.

[0037] The preparation process of IP@PssP is basically similar to that of I-4-P@PssP. The difference is that IR-1048 (4 mg) and PFOB (20 mg) are dissolved in 3 mL of dichloromethane and then slowly added to the mPEG-SS-PLGA suspension. The nanomedicine prepared contains only IR-1048 and PFOB.

[0038] Example 2: Tumor Microenvironment Response Release

[0039] To investigate the responsive release of I-4-P@PssP nanomedicine, we simulated a high GSH environment in tumors in vitro: 2 mL of I-4-P@PssP (50 M PBS) prepared in Example 1 was transferred to a dialysis bag (molecular weight cutoff 3000 Da), and the dialysis bag was then immersed in 10 mL of GSH-containing PBS solution (0.1, 0.5, and 1 mM) and continuously shaken (100 rpm) at 37°C. 200 μL of release medium was extracted at different time points (0, 12, 24, 36, 48, and 60 h), with the same volume of fresh buffer added to maintain volume. The amounts of IR-1048 and 4-MU in the extracted release medium were determined by UV-Vis-NIR absorption spectroscopy. Based on the measurement data, release curves of IR-1048 and 4-MU were plotted, as shown below. Figure 3 , 4 As shown.

[0040] In addition, the PFOB oxygen release capacity of I-4-P@PssP was measured. 2 mL of I-4-P@PssP (50 M PBS) was added to a vial and further covered with mineral oil to isolate it from air. The oxygen concentration in the solution was measured in real time using a portable dissolved oxygen meter (JPBJ-608, INESA). Figure 5 As shown.

[0041] As shown in the figure, the nanomedicine of the present invention can respond to high GSH in the tumor region and release IR-1408, PFOB and 4-MU in a long-term and stable manner, effectively solving the problem of tumor hypoxia.

[0042] The CT26 tumor-bearing mice used in the following examples were modeled using the following methods:

[0043] To evaluate the in vivo therapeutic effect, female BALB / c mice (16-20g, 4-6 weeks old) were purchased from the Model Animal Research Center of Nanjing University and subcutaneously injected with 100 μL LCT26 cells (5 × 10⁻⁶). 7 CT26 cells / mL PBS). When the tumor volume reaches 80–150 mmHg. 3 At that time, it was considered that the CT26 tumor-bearing mouse model had been successfully established.

[0044] Example 3

[0045] I-4-P@PssP nanomedicine addresses the loose and dense tumor extracellular matrix and resolves hypoxia.

[0046] CT26 tumor-bearing mice were randomly divided into groups and intravenously injected with 0.1 mL PBS, 0.1 mL I@PssP, 0.1 mL I-4@PssP, 0.1 mL IP@PssP, and 0.1 mL I-4-P@PssP. Three days later, the mice were euthanized, and tumor tissue was obtained. Immunohistochemistry (IHC) was used to stain the obtained tumor tissue for HABP2, anti-cd31 antibody, and anti-HIF-1α antibody. Figure 6-8 As shown.

[0047] As can be seen, the I-4-P@PssP nanomedicine of this invention loosens the dense extracellular matrix of tumor cells. Immunohistochemistry (IHC) was used to stain hyaluronic acid in tumor tissue with hyaluronic acid-binding protein 2 (HABP2). Brown indicates HA-positive areas.

[0048] The results show that the I-4-P@PssP nanomedicine can release its contents at the tumor site in response. The released 4-MU can play a good role in loosening the extracellular matrix of tumor cells, which is conducive to drug penetration.

[0049] Simultaneously, after treatment with the I-4-P@PssP nanomedicine of this invention, vascular normalization was significantly improved and hypoxia was reduced, indicating that the nanomedicine of this invention can effectively promote vascular normalization (CD31) and resolve tumor hypoxia (HIF-1α).

[0050] Example 4

[0051] Photoacoustic imaging

[0052] Photoacoustic imaging technology can characterize the blood oxygen saturation within tumors, indirectly demonstrating the effectiveness of nanomedicines in addressing hypoxia in the loose and dense extracellular matrix of tumor cells. Specifically, CT26 tumor-bearing mice were treated with PBS, I@PssP, I-4@PssP, IP@PssP, and I-4-P@PssP (all administered intravenously at 0.1 mL) for 24 hours. In vivo photoacoustic imaging (PA) of intratumoral vascular saturation oxygen in the CT26 tumor-bearing mice was performed using a photoacoustic imaging system (Endra Nexus 128, USA). Oxyhemoglobin (HbO2) at an excitation wavelength of 850 nm and deoxyhemoglobin (Hb) at an excitation wavelength of 700 nm were measured, respectively.

[0053] like Figure 9 As shown, the I-4-P@PssP nanomedicine of the present invention can significantly improve intratumoral blood oxygen saturation, while IP@PssP cannot improve internal blood oxygen saturation and its oxygen-carrying capacity is limited to the periphery of the tumor.

[0054] Example 5

[0055] MDSCs inhibition experiment

[0056] CT26 tumor-bearing mice received different treatments (PBS, I@PssP, I-4@PssP, IP@PssP, and I-4-P@PssP, all administered intravenously at 0.1 mL) and were humanely sacrificed 14 days later. Tumor tissue was harvested and a single-cell suspension was prepared. MDSCs were further isolated and purified using an MDSCs isolation kit. T cells were obtained from the mouse spleen. Subsequently, the prepared MDSCs and T cells were co-cultured at ratios of 1:1 / 1:5 / 1:10 for 3 days. Anti-CD3 / CD28 antibody was added to the culture medium as a T cell stimulant, and the cells were treated with 0.5 μCi / well 3H-thymidine (3H-TdR). Finally, the results were monitored using an LS 6500 multifunction scintillation counter (Beckman Coulter, USA). 3 H-TdR levels were used to assess cell proliferation, such as Figure 10 As shown.

[0057] like Figure 11 As shown, compared with other treatment groups, the percentage of MDSCs (Gr-1+ and CD11b+) in tumor tissue was significantly reduced after treatment with the nanomedicine I-4-P@PssP of the present invention, indicating that it can significantly inhibit the infiltration of MDSCs.

[0058] Example 6

[0059] I-4-P@PssP Anti-tumor photothermal therapy effect

[0060] The established CT26 tumor-bearing mouse model was divided into 5 groups (7 mice per group), and each group was intravenously injected with PBS, I@PssP, I-4@PssP, IP@PssP, and I-4-P@PssP (2 mg / kg), respectively. One hour later, the mice were treated with a 980 nm laser (1 W / cm²). 2 Irradiate the tumor site for 3 minutes. Record the tumor weight for each group. Use H&E staining to evaluate the efficacy of different treatment methods.

[0061] like Figure 12 As shown, compared with other treatment groups, the nanomedicine I-4-P@PssP of the present invention can significantly reduce the volume and weight of the tumor site after treatment, and has a better therapeutic effect.

Claims

1. A nanomedicine for tumor hypoxia, characterized in that, The nanomedicine was prepared by encapsulating the drugs IR-1048, PFOB, and 4-MU in mPEG-SS-PLGA.

2. The nanomedicine according to claim 1, characterized in that, The nanomedicine was prepared by the following method: mPEG-SS-PLGA was dissolved in deionized water to obtain an mPEG-SS-PLGA suspension for later use; IR-1048, PFOB and 4-MU were dissolved in dichloromethane and then slowly added to the mPEG-SS-PLGA suspension, and the mixture was sonicated until the dichloromethane was completely evaporated; the precipitate was collected by repeated centrifugation, and the residue was washed to remove the residue to obtain the nanomedicine.

3. The nanomedicine according to claim 2, characterized in that, The addition ratio of mPEG-SS-PLGA, IR-1048, PFOB and 4-MU is 30mg:(2-4mg):(10-20mg):(2-6mg).

4. The nanomedicine according to claim 3, characterized in that, The ratio of mPEG-SS-PLGA, IR-1048, PFOB and 4-MU added is 30mg:4mg:20mg:6mg.

5. The use of the nanomedicine according to any one of claims 1 to 4 in the preparation of a drug with tumor photothermal therapeutic effect.

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