Light-promoted release supramolecular diagnosis and treatment prodrug as well as preparation method and application thereof
By using supramolecular diagnostic and treatment prodrug composed of cuproaromatic prodrugs and photosensitizers, the problem of unsatisfactory efficacy of diagnostic and treatment prodrugs in breast cancer hypoxia microenvironment is solved, and high-selective drug release and real-time imaging in hypoxia environment is achieved Tracer simplifies the preparation process and improves the efficacy.
Patent Information
- Application Number
- CN202411640458.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-02-18
AI Technical Summary
The existing diagnostic and treatment prodrugs for breast cancer hypoxia microenvironment are not ideal due to uneven hypoxia levels, and it is difficult to achieve real-time trace of drug release and efficacy. The synthesis process is complex, which limits its wide application.
The supramolecular diagnostic and therapeutic prodrug consisting of a cuvron aromatic prodrug and a photosensitive agent included in the cuvron aromatic prodrug, and a stable supramolecular complex is formed through non-covalent interactions between the host and the guest, and the coupons of the cuvron aromatic prodrug are used. The nitrogen bond fracture characteristics release anti-tumor drugs in an hypoxia environment, and light-promoted drug release and near-infrared fluorescence imaging traceability are achieved through the photodynamic characteristics of photosensitizers.
It has achieved a high selective response to the hypoxic microenvironment of breast cancer, real-time near-infrared fluorescence imaging and tracing, simplified the preparation process, improved the efficacy, and met the accurate and controllable diagnosis and treatment needs of breast cancer.
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Figure CN119455014A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a light-promoted release supramolecular diagnostic and therapeutic prodrug, a preparation method thereof, and an application thereof in the diagnosis and treatment of breast cancer. Background Art
[0002] For women, breast cancer is the most common type of cancer. Hypoxia is a prominent feature of the breast cancer microenvironment. Hypoxia in breast cancer is caused by the distance between cancer cells and blood vessels exceeding the limit of oxygen diffusion. In the occurrence and development of breast cancer, hypoxia can stimulate angiogenesis, leading to malignant proliferation of cancer cells, inhibition of cell apoptosis, immune evasion, etc. On the other hand, the hypoxic microenvironment also brings opportunities for the diagnosis and treatment of breast cancer. So far, various hypoxia-activated prodrugs, imaging probes or drug delivery carriers have been developed and show good prospects in the diagnosis and treatment of breast cancer. Among them, diagnostic and therapeutic prodrugs, as an effective product combining analytical chemistry with precision cancer treatment, combine tumor diagnosis and targeted treatment, realize probes for characteristic metabolites or proteins in the tumor site and targeted treatment of related tumors, and show good application prospects in the diagnosis and treatment of breast cancer.
[0003] However, one of the biggest challenges facing therapeutic prodrugs targeting the hypoxic microenvironment of breast cancer is the uneven distribution of hypoxia levels in breast cancer tissue, which leads to unsatisfactory efficacy of these prodrugs. Therefore, accurate analysis and control of hypoxia levels are particularly important for the development of hypoxia-targeted prodrugs for breast cancer. In recent years, prodrugs that combine chemotherapeutic drugs and photosensitizers have attracted much attention. Through this strategy, the photodynamic properties of photosensitizers can lead to local hypoxia in the tumor microenvironment, which can achieve light-promoted drug release ( Yuan, J.; Zhou, QH; Xu, S.; et al., Angew. Chem. Int. Ed. 2022, 61, e202206169 ). However, these types of prodrugs can rarely achieve real-time tracking of drug release and efficacy; in addition, most of these prodrugs are bound by covalent bonds, and the synthesis, purification and separation processes are complicated, which limits their further widespread application. Summary of the invention
[0004] In view of the above technical problems, the present invention provides a supramolecular diagnostic and therapeutic prodrug with light-promoted release, and its preparation method and application. The supramolecular diagnostic and therapeutic prodrug has the advantages of simple preparation method, targeted treatment of breast cancer hypoxia response, real-time near-infrared fluorescence imaging tracing and light-promoted drug release.
[0005] The present invention adopts the following technical scheme: 1. Supramolecular theranostic prodrugs with light-promoted release The light-promoted release supramolecular diagnostic and therapeutic prodrug of the present invention is composed of a calixarene prodrug and a photosensitizer included in the calixarene prodrug. The chemical structure of the calixarene prodrug is as follows: The chemical structural formula of the photosensitizer is as follows: Wherein, R is methyl, ethyl, or n-propyl; The light-promoted release supramolecular diagnostic and therapeutic prodrug provided by the present invention is a stable supramolecular complex formed by a calixarene prodrug and a photosensitizer through non-covalent interactions between the host and the guest, and utilizes the azo bond breaking property of the calixarene prodrug in the hypoxic microenvironment of breast cancer to release active anti-tumor drugs for treatment, while simultaneously releasing the photosensitizer to produce an obvious fluorescence emission signal for imaging analysis and detection, and utilizes the photodynamic property of the photosensitizer to cause local hypoxia in the tumor microenvironment to achieve light-promoted drug release (such as Figure 1 as shown).
[0006] 2. Preparation of supramolecular theranostic prodrugs with light-promoted release The calixarene prodrug and the photosensitizer are fully mixed in proportion and dissolved in a buffer to obtain a supramolecular diagnostic and therapeutic prodrug solution. The molar ratio of the calixarene prodrug and the photosensitizer group is 1:1 to 1:3 (preferably 1:1). The buffer is a PBS buffer with a pH of 5.0 to 8.0 (preferably pH 7.4).
[0007] The preparation method of the supramolecular diagnostic and therapeutic prodrug provided by the present invention has simple steps, and only requires mixing the calixarene prodrug and the photosensitizer in proportion and then dissolving them in a buffer solution. The two raw materials can also be pre-dissolved in a solvent such as DMSO in proportion and then added to the buffer solution to be configured to a target concentration, which is easy for industrial production.
[0008] 3. Application of supramolecular theranostic prodrugs with light-promoted release The light-promoted release supramolecular diagnostic and therapeutic prodrug provided by the present invention can highly selectively respond to the hypoxic microenvironment of breast cancer to release anti-tumor drugs for the treatment of breast cancer, and make the near-infrared fluorescence signal light up for imaging analysis, and can also use the photodynamic properties of the photosensitizer to cause local hypoxia in the tumor microenvironment to achieve light-promoted drug release. The following uses the prepared supramolecular diagnostic and therapeutic prodrug A as an example to illustrate the application of the supramolecular diagnostic and therapeutic prodrug of the present invention in fluorescence imaging, breast cancer treatment and light-promoted drug release. The remaining supramolecular diagnostic and therapeutic prodrugs in the claims can produce the same effect as supramolecular diagnostic and therapeutic prodrug A.
[0009] First, human breast cancer MCF-7 cells were cultured in a cell culture incubator at 37°C, hypoxia (5% oxygen), and 5% carbon dioxide for 24 hours. The experimental group was added with the supramolecular diagnostic and therapeutic prodrug A solution prepared in Example 1 of the present invention; the control group was only added with the same amount of PBS solution. The above two groups of cells were cultured in a cell culture incubator at 37°C, hypoxia (5% oxygen), and 5% carbon dioxide for 4 hours, and then the culture medium in the imaging dish was washed away with PBS buffer, and fluorescence imaging was performed using a laser scanning confocal microscope. Figure 4 It can be seen that there is no obvious fluorescence signal in the control group cells, while strong fluorescence appears in the experimental group cells. This shows that the probe can enter breast cancer cells and specifically respond to the hypoxic microenvironment of cells, produce a stronger fluorescence signal, and realize the imaging analysis of hypoxic breast cancer cells.
[0010] 4T1 breast cancer tumor-bearing mice were used as the research subjects and divided into a drug-treated group and a blank control group. The mice in the drug-treated group were injected intratumorally with 0.1 mL of supramolecular diagnostic and therapeutic prodrug A; the mice in the blank control group were injected intratumorally with 0.1 mL of PBS solution. Fluorescence imaging was performed using a small animal in vivo fluorescence imager before and 8 hours after administration. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that the fluorescence signal at the tumor site of the mice in the drug-treated group was significantly higher than that in the blank control group. This is because the interaction between the supramolecular diagnostic and therapeutic prodrug A and the hypoxic microenvironment of mouse breast cancer causes the near-infrared fluorescence signal to light up, thus realizing in vivo fluorescence imaging analysis of breast cancer.
[0011] First, human breast cancer MCF-7 cells were cultured in a cell culture incubator at 37°C, under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 24 hours. They were then randomly divided into a non-illuminated group and an illuminated group. The non-illuminated group was added with different concentrations of supramolecular diagnostic and therapeutic prodrug A and then cultured in a cell culture incubator under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 24 hours; the illuminated group was added with different concentrations of supramolecular diagnostic and therapeutic prodrug A and then cultured in a cell culture incubator under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 4 hours, then irradiated with an 808 nm laser for 10 minutes, and then cultured in a cell culture incubator under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 20 hours. The MTT method was then used to test the survival rate of human breast cancer MCF-7 cells in different groups. Figure 6 It can be seen that the cell survival rate of the non-illumination group and the illumination group was higher when the supramolecular diagnostic prodrug A was not added. The survival rate of the cells in the non-illumination group and the illumination group was lower than that of the cells in the control group, indicating that the supramolecular diagnostic prodrug A can release anti-tumor drugs under hypoxic conditions for tumor treatment. In addition, the survival rate of the cells in the illumination group was lower than that in the non-illumination group, indicating that light can promote the release of supramolecular diagnostic prodrug A and improve the efficacy.
[0012] In summary, the beneficial effects of this application are: 1. The supramolecular diagnostic and therapeutic prodrug with light-promoted release provided in this application can efficiently respond to the hypoxic microenvironment of breast cancer to release anti-tumor drugs for treatment, and at the same time release photosensitizers to produce obvious fluorescence emission signals for imaging analysis and detection, and use the photodynamic properties of photosensitizers to cause local hypoxia in the tumor microenvironment to achieve light-promoted drug release. It has the advantages of novel structure, good breast cancer targeting, light-promoted drug release and good imaging performance, and can meet the needs of accurate and controllable diagnosis and treatment of breast cancer.
[0013] 2. The method for preparing a light-promoted release supramolecular therapeutic prodrug provided in the present application has a simple and effective process, which can quickly and efficiently obtain the therapeutic prodrug, avoiding complex processes such as synthesis, purification and separation.
[0014] 3. In view of the good imaging performance and anti-breast cancer activity, the light-promoted release supramolecular diagnostic and therapeutic prodrug provided in this application is of great significance for the diagnosis and treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a schematic diagram of the application of light-promoted release supramolecular diagnostic and therapeutic prodrugs in the diagnosis and treatment of breast cancer; Figure 2 (a) is the fluorescence spectrum of the non-covalent bonding between the calixarene prodrug and the photosensitizer host and guest; Figure 2 (b) is the titration curve of the bonding between the calixarene prodrug and the photosensitizer in Example 1; Figure 3 is the stability diagram of supramolecular theranostic prodrug A; Figure 4 This is the imaging image of supramolecular theranostic prodrug A in hypoxic breast cancer cells; Figure 5 This is the imaging image of supramolecular diagnostic and therapeutic prodrug A in 4T1 breast cancer tumor-bearing mice; Figure 6 This is a graph of cell survival rate of supramolecular theranostic prodrug A in the treatment of breast cancer. DETAILED DESCRIPTION
[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0017] Example 1: Preparation of supramolecular diagnostic and therapeutic prodrugs The present embodiment provides a supramolecular diagnostic and therapeutic prodrug, and the preparation method thereof is as follows: 0.5 μmol of calixarene prodrug and 0.5 μmol of photosensitizer (according to a molar ratio of 1:1) are mixed and dissolved in 10 μL of DMSO, and then added to 100 mL of PBS buffer (10 mM, pH = 7.4) to obtain supramolecular diagnostic and therapeutic prodrug A.
[0018] The chemical formula of calixarene prodrug is: The chemical structure of the photosensitizer is as follows: Fluorometric titration was used to determine the binding constants of calixarene prodrug and photosensitizer. Specifically, different concentrations of calixarene prodrug were added to the photosensitizer (5 μmol / L) solution at room temperature, and the test solution was heated at λ ex = Fluorescence spectrum changes at 690 nm and at λ ex / em =690 / 760 nm fluorescence intensity changes. The combination of calixarene prodrug and photosensitizer will produce a quenching effect on the fluorescence of the photosensitizer, and a set of data on the change of fluorescence intensity with the concentration of the added calixarene prodrug is obtained by titration. The obtained bonding titration curve is fitted using the host-guest 1:1 binding model. Figure 2 (a) and Figure 2 (b) It can be seen that the calixarene prodrug and the photosensitizer can specifically produce host-guest interactions, and the bonding constant between the calixarene prodrug and the photosensitizer is K a = (6.3± 0.7) × 10 6 M -1 , the bonding constant is large and can be effectively combined at low concentrations.
[0019] Example 2: Stability of supramolecular diagnostic prodrugs A supramolecular diagnostic and therapeutic prodrug provided in the present application has good in vitro stability. This example studies the in vitro stability of the supramolecular diagnostic and therapeutic prodrug A prepared in Example 1. The supramolecular diagnostic and therapeutic prodrug prepared in Example 1 is stored at room temperature, and the stability of the supramolecular diagnostic and therapeutic prodrug is tested using ultraviolet absorption spectroscopy. The test uses a quartz cuvette as a sample cell, and the supramolecular diagnostic and therapeutic prodrug is placed at room temperature for 12 hours. The changes in the ultraviolet absorption spectrum of the sample solution after 0, 0.5, 1.0, 4.0, 6.0, 8.0 and 12 hours are tested respectively. The results are as follows. Figure 3 As shown, from Figure 3 It can be seen that the ultraviolet absorption spectrum of the supramolecular therapeutic prodrug has no obvious change within 12 hours, proving that the supramolecular therapeutic prodrug has good stability.
[0020] Example 3: Application of supramolecular theranostic prodrugs in breast cancer cell imaging The supramolecular diagnostic and therapeutic prodrug provided in the present application can highly selectively respond to the hypoxic microenvironment of breast cancer to release anti-tumor drugs and light up near-infrared fluorescence signals. Therefore, this example studies the application of supramolecular diagnostic and therapeutic prodrug A prepared in Example 1 in fluorescence imaging of hypoxic cells in breast cancer.
[0021] First, human breast cancer MCF-7 cells were cultured in a cell culture incubator at 37°C, hypoxia (5% oxygen), and 5% carbon dioxide for 24 hours. The experimental group was added with the supramolecular diagnostic and therapeutic prodrug A solution prepared in Example 1 of the present invention; the control group was only added with the same amount of PBS solution. The above two groups of cells were cultured in a cell culture incubator at 37°C, hypoxia (5% oxygen), and 5% carbon dioxide for 4 hours, and then the culture medium in the imaging dish was washed away with PBS buffer, and fluorescence imaging was performed using a laser scanning confocal microscope. Figure 4 It can be seen that there is no obvious fluorescence signal in the control group cells, while strong fluorescence appears in the experimental group cells. This shows that the probe can enter breast cancer cells and specifically respond to the hypoxic microenvironment of breast cancer cells, produce a strong fluorescence signal, and realize the imaging analysis of breast cancer cells.
[0022] Example 4: Application of supramolecular diagnostic and therapeutic prodrugs in in vivo imaging of breast cancer The supramolecular diagnostic and therapeutic prodrug provided in the present application can be used for imaging analysis of hypoxic microenvironment of breast cancer in vivo. Therefore, this example studies the application of supramolecular diagnostic and therapeutic prodrug A prepared in Example 1 in in vivo fluorescence imaging analysis of breast cancer.
[0023] 4T1 breast cancer tumor-bearing mice were randomly divided into a treatment group and a blank control group. The mice in the treatment group were injected with 0.1 mL of supramolecular diagnostic and therapeutic prodrug A intratumorally; the mice in the blank control group were injected with 0.1 mL of normal saline intratumorally. Fluorescence imaging was performed using a small animal in vivo fluorescence imager before and 8 hours after administration. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that the fluorescence signal at the tumor site of the mice in the drug-treated group was significantly higher than that in the blank control group. This is because the interaction between the supramolecular diagnostic and therapeutic prodrug A and the hypoxic microenvironment of mouse breast cancer causes the near-infrared fluorescence signal to light up, thus realizing in vivo fluorescence imaging analysis of breast cancer.
[0024] Example 5: Application of supramolecular theranostic prodrugs in breast cancer treatment and light-promoted drug release The present application provides a supramolecular diagnostic and therapeutic prodrug that can highly selectively respond to the hypoxic microenvironment of breast cancer and release anti-tumor drugs for the treatment of breast cancer, and utilizes the photodynamic properties of the photosensitizer to cause local hypoxia in the tumor microenvironment to achieve light-promoted drug release. Therefore, this example studies the application of the supramolecular diagnostic and therapeutic prodrug A prepared in Example 1 in the treatment of breast cancer and light-promoted drug release.
[0025] First, human breast cancer MCF-7 cells were cultured in a cell culture incubator at 37°C, under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 24 hours. They were then randomly divided into a non-illuminated group and an illuminated group. The non-illuminated group was added with different concentrations of supramolecular diagnostic and therapeutic prodrug A and then cultured in a cell culture incubator under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 24 hours; the illuminated group was added with different concentrations of supramolecular diagnostic and therapeutic prodrug A and then cultured in a cell culture incubator under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 4 hours, then irradiated with an 808 nm laser for 10 minutes, and then cultured in a cell culture incubator under hypoxia (5% oxygen) conditions and a 5% carbon dioxide environment for 20 hours. The MTT method was then used to test the survival rate of human breast cancer MCF-7 cells in different groups. Figure 6 It can be seen that the cell survival rate of the non-illumination group and the illumination group was higher when the supramolecular diagnostic prodrug A was not added. The survival rate of the cells in the non-illumination group and the illumination group was lower than that of the cells in the control group, indicating that the supramolecular diagnostic prodrug A can release anti-tumor drugs under hypoxic conditions for tumor treatment. In addition, the survival rate of the cells in the illumination group was lower than that in the non-illumination group, indicating that light can promote the release of supramolecular diagnostic prodrug A and improve the efficacy.
Claims
1. A supramolecular diagnostic and therapeutic prodrug with light-promoted release, characterized in that: It is composed of a calixarene prodrug and a photosensitizer contained inside the calixarene prodrug. The chemical structure of the calixarene prodrug is as follows: 。 2. The supramolecular diagnostic and therapeutic prodrug with light-promoted release according to claim 1, characterized in that: The chemical structural formula of the photosensitizer is as follows: Among them, R is methyl, ethyl, or n-propyl.
3. The supramolecular diagnostic and therapeutic prodrug with light-promoted release according to claim 1, characterized in that: The calixarene prodrug and the photosensitizer form a stable supramolecular complex through non-covalent interaction between the host and the guest.
4. A method for preparing the light-promoted release supramolecular diagnostic and therapeutic prodrug as claimed in claim 1, characterized in that: The calixarene prodrug and the photosensitizer are fully mixed and dissolved in a buffer solution to obtain a supramolecular diagnostic and therapeutic prodrug solution.
5. The method for preparing the light-promoted release supramolecular diagnostic and therapeutic prodrug according to claim 4, characterized in that: The molar ratio of the calixarene prodrug to the photosensitizer group is 1:1 to 1:
3.
6. The method for preparing the light-promoted release supramolecular diagnostic and therapeutic prodrug according to claim 4, characterized in that: The buffer solution is a PBS buffer solution with a pH value of 5.0 to 8.
0.
7. Use of the light-promoted release supramolecular diagnostic and therapeutic prodrug as claimed in claim 1 in the preparation of a fluorescent imaging detection reagent for breast cancer.
8. Use of the light-promoted release supramolecular diagnostic and therapeutic prodrug as claimed in claim 1 in the preparation of drugs for treating breast cancer.