An acidic tumor microenvironment-activating nanoprobe, its synthesis method, and its application.

Abstract

This invention discloses an acidic tumor microenvironment-activated nanoprobe, its synthesis method, and its application, belonging to the field of nanomaterial preparation technology. Addressing the shortcomings of existing photosensitizers in photodynamic therapy (PDT), such as low treatment efficiency and lack of tumor targeting due to tumor microenvironment hypoxia, this invention utilizes metallic Fe... 2+ A low-pH-responsive smart nanoprobe was prepared by self-assembling the anti-angiogenic drug thalidomide and the FDA-approved photosensitizer hematoporphyrin monomethyl ether. Before degradation at the tumor site, the probe exhibits photodynamic therapy activity while its magnetic resonance imaging (MRI) signal is deactivated. As the material accumulates in the tumor tissue, under the low-pH stimulation of the tumor microenvironment, the MRI signal gradually activates, releasing thalidomide. This amplifies oxidative stress by increasing ROS / consuming GSH and inducing ferroptosis in tumor cells, ultimately resulting in a synergistic effect of chemokinetics / chemotherapy / photodynamic therapy on an excellent anti-tumor response.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology, specifically relating to an acidic tumor microenvironment activating nanoprobe, its synthesis method, and its application. Background Technology

[0002] Malignant tumors have always been one of the most serious threats to human health. Currently, the main clinical treatments for cancer include surgical resection, chemotherapy, and radiotherapy. However, these strategies have certain limitations in terms of significant damage to normal tissues and the hypoxic tumor microenvironment (TME) and high systemic toxicity.

[0003] In recent years, photodynamic therapy (PDT) has emerged as a leading treatment option for cancer due to its high success rate, non-invasive nature, and minimal side effects, effectively addressing the challenges faced by traditional cancer treatments. The principle of PDT is that a photosensitizer (PS) absorbs near-infrared light of a specific wavelength, activating the PS from its ground state (S0) to a single excited state (S1), followed by an intersystem transition to a triple excited state (T1), generating cytotoxic reactive oxygen species (ROS) that ultimately lead to tumor cell death. Despite its rapid development, PDT has not yet been widely accepted as a first-line cancer treatment due to its low penetration depth in tissues and significant dark toxicity. More importantly, the complexity of the tumor microenvironment (TME), including its abnormal physiological characteristics such as acidity, hypoxia, nutrient deprivation, metabolite accumulation, and redox imbalance, prevents PDT from completely eradicating tumors. Therefore, there is an urgent need to explore cascade-amplified anti-tumor strategies to optimize and enhance PDT and improve cancer treatment efficacy. Although nanomaterials possess high permeability and retention (EPR) in solid tumor regions, nanomedicine delivery driven by a single EPR effect suffers from insufficient accumulation and tumor retention. To address these issues, developing tumor microenvironment-activated nanoprobes with on / off modes is crucial. These probes leverage the low pH and unbalanced redox state of the tumor microenvironment to activate imaging therapy at the tumor site while remaining "silent" in non-tumor lesions, significantly contributing to precise tumor diagnosis and treatment. Therefore, the rational design of stimulus-responsive nanomedicines is essential for enhancing effective uptake by tumor cells and achieving superior anti-tumor performance. Summary of the Invention

[0004] To address the shortcomings of existing photosensitizers in photodynamic therapy (PDT), such as low treatment efficiency and lack of tumor targeting due to the limitation of tumor microenvironment hypoxia, this invention provides an acidic tumor microenvironment-activated nanoprobe, its synthesis method, and its application.

[0005] Therefore, given the acidic nature of the tumor microenvironment, it is necessary to explore Fe, which possesses reliable biosafety and highly effective PDT tumor therapy. 2+ Starting with supramolecular nanoassembly, we selected the FDA-approved photosensitizer hematoporphyrin monomethyl ether (HMME), the anti-angiogenic drug thalidomide (Thal), and Fe... 2+ Using [material name] as raw material, a tumor microenvironment-responsive activated nanoprobe was prepared. This probe achieved a low-pH-driven cascade amplification of tumor therapeutic effects. The HMME in this nanoprobe exhibited good aggregation-induced singlet oxygen ([…]) through J-aggregation. 1 The production capacity of O2. As materials continue to accumulate in tumor tissue, the H2 in the tumor microenvironment... + Disruption of the J-aggregate structure leads to disordered HMME accumulation, resulting in PDT shutdown. PDT therapy is driven by near-infrared (NIR) radiation before probe degradation, and Thal and Fe are released after degradation. 2+ This probe, used separately for chemotherapy and chemokinetic therapy (CDT), addresses the issue of poor efficacy with single-treatment PDT. Furthermore, the Fe released during probe degradation... 2+ MRI signals were activated to indicate the optimal timing for PDT laser irradiation. A cascaded, expanded tumor treatment strategy was achieved through a two-stage treatment approach (first: PDT, second: CDT / chemotherapy) before and after probe degradation. This work proposes a self-assembled responsive nanoplatform constructed from endogenous components, integrating MRI / PDT / CDT / chemotherapy functions. It features safety, low toxicity, and easy metabolism after degradation, addressing the issue of poor efficacy with single PDT therapy and providing a strategy for maximizing the tumor therapeutic efficacy of materials.

[0006] This material exhibits quenched MRI signals and good PDT effect under neutral conditions, while opening MRI signals in the acidic environment of tumors and combining chemotherapy and CDT performance. Through ROS-mediated oxidative stress, GSH consumption and GPX4 downregulation cascade amplification of photodynamic therapy, it induces ferroptosis and apoptosis in tumor cells, realizing the integration of tumor diagnosis and treatment.

[0007] To achieve the above objectives, the present invention employs the following technical solutions:

[0008] An acidic tumor microenvironment-activating nanoprobe, HMME-Fe-Thal (HFT), is a nanoprobe self-assembled from iron ions, the photosensitizer hematoporphyrin monomethyl ether (HMME), and thalidomide (Thal).

[0009] Furthermore, iron ions coordinate with oxygen atoms in HMME and Thal to form a square bipyramidal geometry, and adjacent secondary building units are stacked into three-dimensional supramolecular nanoparticles through hydrogen bonds and π–π interactions.

[0010] Furthermore, the iron ions are Fe. 2+ .

[0011] Furthermore, the particle size of the nanoprobe is 55~65 nm.

[0012] Furthermore, the loading efficiency of the chemotherapy drug thalidomide (Thal) was approximately 68%.

[0013] A method for synthesizing an acidic tumor microenvironment-activated nanoprobe includes the following steps:

[0014] Step 1: Dissolve thalidomide (Thal) and hematoporphyrin monomethyl ether (HMME) in N,N-dimethylformamide (DMF), mix and vortex to obtain a mixed solution;

[0015] Step 2: Slowly add ferrous chloride tetrahydrate aqueous solution (FeCl2·4H2O) to the mixed solution obtained in Step 1, and stir vigorously at room temperature to react.

[0016] Step 3: The product obtained in Step 2 is subjected to ultrafiltration centrifugation and washing several times to obtain HMME-Fe-Thal nanoparticles.

[0017] Furthermore, the mass ratio of thalidomide, hematoporphyrin monomethyl ether, and ferrous chloride tetrahydrate is 2:1:10.

[0018] Furthermore, the volume ratio of the solvents used in steps 1 and 2 is 1:2.

[0019] Furthermore, in step 2, the stirring speed is 600 rpm / min to 800 rpm / min, and the time is 2 min.

[0020] Furthermore, in step 3, the molecular weight cutoff for ultrafiltration centrifugation is 30 kDa, the centrifugation speed is 2500 rpm, the time is 10 min, and the centrifugation is performed 3 times.

[0021] Application of an acidic tumor microenvironment-activated nanoprobe in magnetic resonance imaging.

[0022] Application of an acidic tumor microenvironment-activated nanoprobe in photodynamic / chemokinetic / chemotherapy drug cascade amplification for tumor treatment.

[0023] Application of an acidic tumor microenvironment-activated nanoprobe in inducing ferroptosis in tumor cells.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] This invention utilizes self-assembled Fe 2+A smart nanoprobe responsive to the acidic tumor microenvironment was prepared using the anti-angiogenic drug thalidomide and the photosensitizer HMME. Before degradation at the tumor site, the material exhibits a closed MRI signal and photosensitizing phototherapy (PDT) efficacy. As the material accumulates in the tumor tissue, under the low pH stimulation of the tumor microenvironment, the MRI signal gradually turns on, releasing thalidomide. This not only distinguishes cascade therapy processes but also enhances PDT efficacy and amplifies oxidative stress-induced ferroptosis in tumor cells through the synergistic effect of CDT / chemotherapy / glutathione (GSH) consumption. This preparation method is simple and efficient, and the obtained nanomaterials are safe, have low toxicity, and are easily metabolized after degradation, showing great promise for clinical applications in oncology. Attached Figure Description

[0026] Figure 1 This is a transmission electron microscope image of the activated self-assembled nanoprobe synthesized in Example 1 of the present invention.

[0027] Figure 2 The image shows the ultraviolet spectra of the activated self-assembled nanoprobe synthesized in Example 1 of this invention.

[0028] Figure 3 This is a TEM image of the activated self-assembled nanoprobe synthesized in Example 1 of the present invention after incubation under acidic conditions for 12 h.

[0029] Figure 4 The T1 relaxation signal of the activated self-assembled nanoprobe synthesized in Example 1 of this invention under different acidic pH conditions.

[0030] Figure 5 The cumulative release curves of Thal under different acidic pH conditions are shown for the activated self-assembled nanoprobe synthesized in Example 1 of this invention.

[0031] Figure 6 This study investigates the change in reactive oxygen species generated over time after the activated self-assembled nanoprobe synthesized in Example 1 of this invention is co-incubated with hydrogen peroxide at pH 6.5.

[0032] Figure 7 The study on the generation of singlet oxygen by the activated self-assembled nanoprobe synthesized in Example 1 of this invention under near-infrared (NIR) laser irradiation: Figure (a) pH 7.4; Figure (b) pH 6.5.

[0033] Figure 8 This study investigates the time-dependent consumption of the activated self-assembled nanoprobe synthesized in Example 1 of this invention.

[0034] Figure 9This study investigates the cytotoxicity of the activated self-assembled nanoprobe synthesized in Example 1 of this invention against CCK-8 under different treatment conditions.

[0035] Figure 10 These are trypan blue staining images of the activated self-assembled nanoprobe synthesized in Example 1 of this invention under different treatment conditions after co-incubation with tumor cells.

[0036] Figure 11 This is a protein immunoblotting analysis of the activated self-assembled nanoprobes synthesized in Example 1 of the present invention, which induced ferroptosis in tumor cells under different treatment conditions. Figure (a) shows the immunoblotting analysis of GPX4 under different treatment conditions, Figure (b) shows the change in the GSH / GSSG ratio under different treatment conditions, and Figure (c) shows the content analysis of malondialdehyde (MDA) under different treatment conditions.

[0037] Figure 12 This study investigates the in vivo antitumor effect of the activated self-assembled nanoprobe synthesized in Example 1 of this invention. Figure (a) shows the change in relative tumor volume in mice during treatment, Figure (b) shows the change in body weight of mice in each group after treatment, and Figure (c) shows a photograph of mouse tumors collected on day 14. Detailed Implementation

[0038] To gain a deeper understanding of this invention, we will provide a comprehensive and detailed description. However, this invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a full understanding of the disclosure of this invention.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Thalidomide was purchased from Maclean Biotechnology Co., Ltd. (Shanghai, China), catalog number T911502-25 mg; hematoporphyrin monomethyl ether was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S61821-100 mg; ferrous chloride tetrahydrate and N,N-dimethylformamide were both purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China).

[0041] Example 1: Synthesis of HFT NPs

[0042] Weigh 1 mg of thalidomide (Thal) and 0.5 mg of hematoporphyrin monomethyl ether (HMME), add 500 μL of N,N-dimethylformamide (DMF), and vortex for 1 min to obtain a mixed solution;

[0043] Add 1 mL of a 10 mg / mL aqueous solution of ferrous chloride tetrahydrate (FeCl2·4H2O) to the mixed solution, and stir vigorously for 1 min.

[0044] After ultrafiltration and centrifugation (2500 rpm, 10 min), the nanoparticles were washed three times with deionized water to obtain HMME-Fe-Thal nanoparticles (HFT NPs), which were then stored at 4°C for further use.

[0045] Example 2: Characterization of HFT NPs

[0046] The sample was dropped onto a 400-mesh copper grid covered with a carbon film, allowed to dry naturally, and then observed under a transmission electron microscope. Figure 1 The image shown is a transmission electron microscope image of the self-assembled nanoprobe synthesized in Example 1 of the present invention. As can be seen from the image, the synthesized nanoparticles have a uniform, monodisperse spherical morphology, a smooth surface, and a diameter of 55~65 nm.

[0047] Thalidomide (Thal), hematoporphyrin monomethyl ether (HMME), and HFT NPs samples were dissolved in aqueous solution, and the absorbance in the wavelength range of 200–800 nm was measured using a UV spectrophotometer. Figure 2 This is the ultraviolet spectrophotometer of a self-assembled nanoprobe for tumor cascade amplification therapy synthesized in Example 1 of this invention, which is an acidic tumor microenvironment (TME) activated nanoprobe. As shown in the figure, the synthesized HFT NPs exhibit two characteristic peaks at 397 nm and 302 nm, respectively, indicating the successful synthesis of the nanoprobe.

[0048] Example 3: Performance Study of HFT NPs

[0049] HFT NPs were incubated with phosphate buffer at pH 6.5 for 12 h. Samples were then dropped onto a 400-mesh copper grid covered with a carbon film. After natural drying, the morphological changes of the samples were observed under an electron microscope. Figure 3 The image shown is a TEM image of a self-assembled nanoprobe synthesized in Example 1 of this invention, designed for amplified tumor cascade therapy, and incubated for 12 hours under acidic conditions at pH 6.5. Figure 3 As can be seen, the spherical HFT NPs are gradually destroyed and deformed into a gel-like structure, which indicates that the synthesized HFT NPs can disintegrate in response to acidic conditions.

[0050] Different concentrations of HFT NPs were incubated with phosphate buffer at pH 6.5 and pH 5.5 for 1 h, respectively, and then the magnetic resonance of the nanoprobes was detected. T 1 Relaxation rate. For example... Figure 4As shown, the self-assembled nanoprobe synthesized in Example 1 of this invention, which is an acidic tumor microenvironment (TME) activated type for tumor cascade amplification therapy, exhibits magnetic resonance imaging under different acidic pH conditions. T 1 Relaxation rate. Figure 4 As can be seen, the r1 value of HFT NPs increases significantly as pH decreases, indicating that HFT NPs have a low-pH activated MRI switch function.

[0051] The release behavior of HFT NPs in two acidic buffer solutions (pH 6.5 and pH 5.5) was studied using dialysis bags. The cumulative release of Thal was calculated by measuring its absorbance at 302 nm, and a cumulative release curve of Thal was plotted. Figure 5 The figure shows the cumulative release curve of Thal, a self-assembled nanoprobe synthesized in Example 1 of this invention for tumor cascade amplification therapy, under acidic pH conditions. As can be seen from the figure, after incubation at pH 6.5 for 12 h, the release of Thal from HFT NPs reached 49%, while at pH 5.4, the release of Thal reached 68%. This indicates that HFT NPs have good pH-dependent degradation behavior, enabling them to degrade in the acidic environment of the tumor and release chemotherapeutic drugs to exert antitumor effects.

[0052] Methylene blue (MB) was added to a solution containing HFT and hydrogen peroxide (pH 6.5), and then stirred in the dark for 10 min. The formation of ·OH was assessed by monitoring the absorbance intensity of MB at 664 nm. Figure 6 The figure shows a study on the change in reactive oxygen species (ROS) generated over time after co-incubation of a self-assembled nanoprobe for tumor cascade amplification therapy synthesized in Example 1 of this invention with hydrogen peroxide at pH 6.5. As can be seen from the figure, the characteristic peak of MB gradually decreases under acidic conditions, indicating that HFT NPs generate ROS due to low pH-driven Fe. 2+ It is released and reacts with hydrogen peroxide in a Fenton reaction to produce ·OH.

[0053] Using DPBF as 1 O2 chemical probe to detect HFT NPs induced by 808 nm laser irradiation. 1 The generation of O2. For example... Figure 7The figure shows a study on the generation of singlet oxygen by a self-assembled nanoprobe synthesized in Example 1 of this invention, which is an acidic tumor microenvironment (TME) activated nanoprobe for tumor cascade amplification therapy, under near-infrared laser irradiation. As can be seen from the figure, under neutral conditions with a laser irradiation duration of 20 min, the absorbance of the characteristic absorption peak of DPBF at 410 nm gradually decreased from 1.8 to 1.1 with increasing irradiation time; while under acidic conditions, the characteristic absorption peak of DPBF only decreased to 1.5, indicating that... 1 O2 production was significantly suppressed because HMME in HFT NPs could be generated under laser irradiation via J-aggregation. 1 O2, however, under acidic conditions, H + The ions disrupted the J-aggregate structure of HMME in HFT NPs, leading to the closure of PDT.

[0054] Different concentrations of HFT NPs were co-incubated with GSH solution and hydrogen peroxide in a pH 6.5 buffer solution. A certain amount of the mixture was taken at different time points, and the glutathione consumption capacity of the HFT NPs was detected using 5,5'-dithiomethyl (2-nitrobenzoic acid) (DTNB). Figure 8 The figure shows a study on the time-dependent GSH consumption of a self-assembled nanoprobe synthesized in Example 1 of this invention, which is an acidic tumor microenvironment (TME) activated nanoprobe for tumor cascade amplification therapy. As can be seen from the figure, under acidic conditions, the absorbance of DTNB at 412 nm decreases with increasing nanoparticle concentration, indicating a gradual decrease in GSH concentration. This demonstrates that HFT NPs have the ability to consume GSH.

[0055] Example 4: In vitro antitumor activity of HFT NPs

[0056] Figure 9 This study investigates the cytotoxicity of a self-assembled, acidic tumor microenvironment (TME)-activated nanoprobe synthesized in Example 1 of this invention for tumor cascade amplification therapy under different treatment conditions using CCK-8 assays. The HFT NPs concentration reached 200 μg / mL. -1 At that time, the cell viability of the HFT group, HFT + 808 nm Laser and HFT + pH 5.5 were 82.73%, 44.09% and 27.92%, respectively. After treatment with HFT + pH 5.5 + H2O2, the cell viability decreased to 22.34%, indicating that HFT has the potential to be an effective therapeutic drug for killing cancer cells.

[0057] Figure 10 shows trypan blue staining images of tumor cells under different treatment conditions after co-incubation of a self-assembled nanoprobe for tumor cascade amplification therapy synthesized in Example 1 of this invention with tumor cells. Cells were treated according to different grouping conditions, and dead cells were observed using trypan blue staining. Compared with the control group, some cells in the HFT + 808 nm Laser and HFT + pH 5.5 groups were stained blue. However, after HFT + pH 5.5 + H2O2 treatment, a large number of dead cells (blue cells) were visible, further demonstrating the excellent in vitro antitumor effect of HFT NPs.

[0058] Studies on the induction of ferroptosis in tumor cells by nanoprobes under different treatment conditions

[0059] The expression level of GPX4 under different treatment conditions was detected by Western blotting, such as... Figure 11 The diagram shown illustrates the results of research on the induction of ferroptosis in tumor cells under different treatment conditions by a self-assembled nanoprobe synthesized in Example 1 of this invention, which is an acidic tumor microenvironment (TME) activated nanoprobe for tumor cascade amplification therapy. Figure 11 As shown in (a), GPX4 expression was significantly downregulated in the pH 5.5 + hydrogen peroxide group, while it showed a slight decreasing trend in the pH 6.5 + hydrogen peroxide group; in addition, the levels of GSH and GSSG were measured using a GSH colorimetric assay kit, and the GSG / GSSH ratio was calculated. Figure 11 (b) The expression of GSG / GSSH was significantly downregulated in the pH 5.5 + hydrogen peroxide group, indicating that HFT NPs have a significant GSH-consuming capacity, leading to a decrease in intracellular GPX4 and inducing ferroptosis in tumor cells. Lipid peroxide accumulation is one of the basic characteristics of ferroptosis. The lipid peroxide content after different treatment conditions was detected using malondialdehyde (MDA), such as... Figure 11 As shown in (c), the MDA content in the pH 5.5 + hydrogen peroxide group was significantly increased, indicating that HFT NPs induced ferroptosis in tumor cells under these conditions.

[0060] Example 5: In vivo antitumor activity of HFT NPs

[0061] 4T1 cells were subcutaneously injected into the right thigh of nude mice to establish a 4T1 tumor-bearing mouse model. The tumor volume reached 100 mm². 3 Mice were randomly divided into four groups: (1) PBS group (as control); (2) HFT + V c(Vitamin C) group (chemotherapy); (3) HFT group (CDT / chemotherapy); (4) HFT + L group (PDT / CDT / chemotherapy), were given the corresponding treatments, and the weight and tumor volume of the mice were recorded every other day. After 14 days, all tumor-bearing mice were sacrificed, and the tumor tissue was isolated and photographed. Figure 12 The image shows a study of the in vivo antitumor effect of a self-assembled acidic tumor microenvironment (TME)-activated nanoprobe synthesized in Example 1 of this invention for tumor cascade amplification therapy. Figure 12 As can be seen, the tumors in the PBS group grew rapidly to 18.1 times their initial volume, while the tumors in the HFT + Vc group and the HFT group grew to 7.2 times and 7.6 times their initial volume, respectively. In contrast, the tumors in the HFT + L group (PDT / CDT / chemotherapy) mice grew to only 2.9 times their initial volume, demonstrating a strong tumor-suppressive effect. There were no significant changes in mouse body weight among the groups, confirming the good biocompatibility of HFT NPs.

[0062] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. An acidic tumor microenvironment-activated nanoprobe, characterized in that: The nanoprobe HMME-Fe-Thal is self-assembled by iron ions, a photosensitizer hemin monomethyl ether (HMME) and thalidomide (Thal). 2.The acid tumor microenvironment-activated nano probe according to claim 1, characterized in that: The square bipyramidal geometry is formed by the coordination of iron ions with oxygen atoms in HMME and Thal, and adjacent secondary building units are stacked into a three-dimensional supramolecular structure of nanoparticles through hydrogen bonds and π-π interactions. 3.The acid tumor microenvironment-activated nanoprobe according to any one of claims 1 or 2, characterized in that: The iron ion is Fe 2+ . 4.The acid tumor microenvironment-activated nano probe according to any one of claims 1 or 2, characterized in that: The nanoprobe has a particle size of 55-65 nm.

5. A method for synthesizing an acid tumor microenvironment-activated nanoprobe, characterized in that, The method comprises the following steps: Step 1, dissolving thalidomide and hemin monomethyl ether in N,N-dimethylformamide, mixing and vortexing to obtain a mixed solution; Step 2, slowly adding an aqueous solution of ferrous chloride tetrahydrate to the mixed solution obtained in step 1, and stirring vigorously at room temperature; Step 3, performing ultrafiltration centrifugation and washing the product obtained in step 2 several times to obtain HMME-Fe-Thal nanoparticles.

6. The method for synthesizing an acidic tumor microenvironment-activating nanoprobe according to claim 5, characterized in that: The mass ratio of thalidomide, hemin monomethyl ether and ferrous chloride tetrahydrate is 2:1:10, and the volume ratio of the solvents used in steps 1 and 2 is 1:

2.

7. The method for synthesizing an acidic tumor microenvironment-activating nanoprobe according to claim 5, characterized in that: The stirring speed in step 2 is 600 rpm / min-800 rpm / min, and the stirring time is 2 min; the molecular weight cut-off of the ultrafiltration centrifugation in step 3 is 30 kDa, the centrifugation speed is 2500 rpm, the centrifugation time is 10 min, and the centrifugation is performed for 3 times.

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