A self-assembling nanophotosensitizer and its preparation method and application

Abstract

The present invention discloses a self-assembling nanophotosensitizer, preparation method, and application. The nanophotosensitizer is composed of TPP-Ppa@ATO@CAT@RGD-PEG-BSA, which is primarily self-assembled from the mitochondrial-targeting porphyrin TPP-Ppa, atovaquone (ATO), catalase (CAT), and RGD-PEG-BSA with active cell-targeting function. This photosensitizer can enhance the effect of photodynamic therapy through dual targeting and dual pathways to increase oxygen. It can also induce an immunogenic death effect through photodynamic therapy, and in combination with anti-PD-L1 antibodies, achieve step-by-step enhanced photodynamic immunotherapy for breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a self-assembly nano photosensitizer and a preparation method and application thereof. Background Art

[0002] Cancer remains a public health problem with high morbidity and mortality worldwide. Chemotherapy remains the mainstay, but its side effects and lack of durable responses have prompted the development of novel therapies. In recent years, novel approaches such as photodynamic therapy (PDT) and immunotherapy have garnered significant attention. PDT requires photosensitizers (PSs), a compatible light source, and oxygen molecules to generate cytotoxic reactive oxygen species (ROS), which kill tumor cells. However, due to the rapid proliferation of tumor cells, they rapidly consume the oxygen supply in the blood, creating a hypoxic microenvironment, which limits the therapeutic efficacy of PDT. Immunotherapy has shown great potential in cancer treatment. However, immunotherapy based on immune checkpoint blockade has only a 10-30% response rate in clinical practice, and few patients with solid tumors achieve complete and durable remissions. Studies have shown that PDT can enhance tumor immunotherapy research. Therefore, combining PDT with immunotherapy is of great significance for achieving efficient tumor suppression.

[0003] PDT utilizes photosensitizers, oxygen, and light to generate ROS. However, traditional photosensitizers aggregate in aqueous solutions, leading to quenching and reduced ROS production. Furthermore, they are rapidly metabolized in vivo, hindering their blood circulation and tumor accumulation. Therefore, the preparation of nanoscale photosensitizers is of great significance. Currently, the fourth generation of nanoscale photosensitizers has developed, primarily using porphyrins or phthalocyanines as building blocks to produce nanomaterials. For example, nanomaterials self-assembled from porphyrin or phthalocyanine monomers, metal-organic frameworks, or covalent organic frameworks, address their hydrophobicity and improve their bioavailability. Furthermore, because ROS have a typically short half-life (40 nanoseconds) and are only effective over very short distances (<20 nanometers), it is impossible to effectively control the metastasis of hidden tumors that are not directly exposed to laser irradiation. Studies have found that the combination of PDT and immunotherapy has a significant effect in inhibiting tumor metastasis. Therefore, to overcome the limitations of PDT and immunotherapy, the present inventors have utilized molecular self-assembly methods to construct highly effective nanomedicines for biomedical applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a self-assembling nano photosensitizer and a preparation method thereof, wherein the nano photosensitizer can be used to treat tumors.

[0005] The present invention utilizes TPP-Ppa (porphyrin molecule with mitochondrial targeting function), RGD-PEG-BSA (molecule with active targeting and long blood circulation, namely RGD peptide-polyethylene glycol-bovine serum albumin), ATO (atovaquinone, a molecule that inhibits mitochondrial respiration and reduces oxygen consumption in tumor cells), and CAT (catalase, which can catalyze hydrogen peroxide to produce oxygen) to cleverly design a multifunctional integrated nano-photosensitizer through weak intermolecular forces (hydrophilicity and hydrophobicity, π-π stacking forces, etc.), thereby achieving enhanced photodynamic therapy effect on tumors.

[0006] To achieve the purpose of the present invention, the following embodiments are provided.

[0007] In one embodiment, the present invention provides a self-assembled nanophotosensitizer composed of TPP-Ppa@ATO@CAT@RGD-PEG-BSA, which is mainly self-assembled by TPP-Ppa, atovaquone (ATO), catalase (CAT) and RGD-PEG-BSA. The chemical structure of the TPP-Ppa is as follows:

[0008]

[0009] The mass ratio of TPP-Ppa: ATO: CAT: RGD-PEG-BSA is 1: 0.5: 0.5: 2. In another embodiment, a method for preparing a self-assembled nanophotosensitizer of the present invention, wherein the nanophotosensitizer is composed of TPP-Ppa@ATO@CAT@mPEG-BSA, comprises:

[0010] 1) React RGDfC with Mal-PEG-NHS in solution, then add BSA and stir to react;

[0011] 2) After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain RGD-PEG-BSA;

[0012] 3) ATO and TPP-Ppa were dissolved in an organic solvent to prepare an ATO solution and a TPP-Ppa solution, respectively, and then mixed to form an ATO@TPP-Ppa mixed solution;

[0013] 4) RGD-PEG-BSA and CAT were dissolved in water and then mixed into an aqueous solution. The ATO@TPP-Ppa solution was added dropwise to the mixed solution of CAT and RGD-PEG-BSA under ultrasonic conditions. After the preparation was completed, the nano photosensitizer was dialyzed.

[0014] Wherein, the chemical structure of the TPP-Ppa is as follows:

[0015]

[0016] In the preparation method of the present invention, in step 1), the solution is a PBS solution, and the mass ratio of RGDfC to Mal-PEG-NHS and BSA is 17:147:400.

[0017] In the preparation method of the present invention, the reaction time in step 1) is 12 hours.

[0018] In the above-mentioned preparation method of the present invention, in step 3), the mass ratio of ATO to TPP-Ppa is 1:2.

[0019] In the above preparation method of the present invention, in step 3), the organic solvent is dimethyl sulfoxide.

[0020] In the above preparation method of the present invention, in step 3), the concentration of the ATO solution is 10 mg / mL, and the concentration of the TPP-Ppa solution is 20 mg / mL.

[0021] In the preparation method of the present invention, in step 3), the mass ratio of RGD-PEG-BSA, CAT and ATO is 2:0.5:0.5.

[0022] On the other hand, the present invention also provides a use of the photosensitizer of the present invention and the photosensitizer prepared by the preparation method of the present invention in the manufacture of anti-tumor drugs.

[0023] The above-mentioned application of the present invention further includes combined use with anti-PD-L1 monoclonal antibody, or forming a compound preparation.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising the photosensitizer of the present invention and the photosensitizer prepared by the preparation method of the present invention, and optionally, pharmaceutically acceptable excipients.

[0025] The “@” in the term: “TPP-Ppa@ATO@CAT@RGD-PEG-BSA” indicates the connection of the components of the substance.

[0026] The photosensitizer of the present invention can trigger the immunogenic death (ICD) effect of tumors, stimulate DC maturation, and further combine with anti-PD-L1 antibodies to enhance the immunotherapy effect of breast cancer, thereby achieving step-by-step enhanced photodynamic-immunotherapy for breast cancer.

[0027] The nano photosensitizer of the present invention has the following innovations:

[0028] (1) Improve the loading efficiency of photosensitizers through weak interactions such as hydrophilic and hydrophobic forces and π-π stacking forces between molecules, and solve the problems of hydrophobicity and easy aggregation of photosensitizers;

[0029] (2) RGD-PEG-BSA and TPP-Ppa molecules achieve step-by-step targeting of nanophotosensitizers, targeting not only tumor cells but also mitochondria, thus enhancing the PDT effect;

[0030] (3) Loaded with catalase, it not only decomposes hydrogen peroxide in the tumor to produce oxygen, but also reduces oxygen consumption in the mitochondrial respiratory chain through ATO, increasing oxygen content through a dual pathway of increasing production and reducing consumption, thereby enhancing the PDT effect;

[0031] (4) Under the action of PDT, the primary tumor is killed, the ICD effect of tumor cells is achieved, and the release of tumor-associated antigens (TAA) is promoted, which further stimulates the maturation of DCs, thereby enhancing the immunotherapy effect of Anti-PD-L1 antibodies and inhibiting tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the nuclear magnetic resonance spectrum of TPP-Ppa.

[0033] Figure 2 This is the particle size distribution diagram of nano-photosensitizer TPP-Ppa@ATO@CAT@RGD-PEG-BSA.

[0034] Figure 3 This is the transmission electron microscopy image of the nano-photosensitizer TPP-Ppa@ATO@CAT@RGD-PEG-BSA.

[0035] Figure 4 The diagram shows the endocytosis effect of nano-photosensitizers with and without tumor cell targeting at different time points (2 and 4 hours).

[0036] Figure 5 This is the test result diagram of the evaluation of mitochondrial-targeted endocytosis by nano-photosensitizers.

[0037] Figure 6 This figure shows the therapeutic effects of each group of experiments on tumor cells using CCK-8.

[0038] Figure 7 This is a diagram showing the evaluation of the ability to induce calreticulin (CRT) production after photodynamic therapy of tumor cells and the quantitative evaluation of the fluorescence intensity.

[0039] Figure 8 Figure 2 is the tumor inhibition curve of each experimental group in the bilateral tumor model. DETAILED DESCRIPTION

[0040] The following examples are typical and are used to further understand and illustrate the spirit of the present invention, but are not intended to limit the scope of the present invention in any way.

[0041] Example 1 Preparation of TPP-Ppa

[0042] The synthetic route of TPP-Ppa is as follows:

[0043]

[0044]

[0045] Pyropheophorbide a (140 mg), EDC.HCl (70 mg), HOBt (50 mg), triethylamine (0.05 mL), and BOC-ethylenediamine (200 mg) were mixed in a dichloromethane solution (25 mL) and stirred at 0°C for 1 hour, followed by continued reaction at room temperature for 24 hours. An aqueous solution was added to adjust the pH to acidic, and dichloromethane was added for extraction. The mixture was washed three times with saturated NaCl, twice with water, and dried over sodium sulfate. The obtained substance was concentrated by rotary evaporation and separated by column (3% methanol + CH2Cl2) to obtain compound 3.

[0046] Compound 3 (60 mg) and TFA (5 mL) were added to a solvent of dichloromethane (5 mL) and stirred at 0°C for 5 hours, and then the reaction solution was evaporated to remove the solvent to obtain compound 4. Compound 5 (61 mg, 2 eq), PyBOP (72 mg, 2 eq), and DIPEA (18 mg) were mixed in a dichloromethane solution (10 mL), stirred for 1 hour, and then compound 4 (40 mg) was added and stirred for 24 hours. Dichloromethane was added for extraction, washed three times with saturated NaCl, washed twice with water, dried over sodium sulfate, concentrated by rotary evaporation to obtain a substance, and then separated by column (developing solvent: 3% CH3OH+CH2Cl2 to 10% CH3OH+CH2Cl2 gradient elution) to obtain TPP-Ppa. The magnetic resonance spectrum of the obtained product TPP-Ppa is as follows: Figure 1 .

[0047] Example 2 Preparation of photosensitizer TPP-Ppa@ATO@CAT@RGD-PEG-BSA

[0048] 1) Preparation of polymer RGD-PEG-BSA and mPEG-BSA

[0049] RGDfC (17 mg) was reacted with Mal-PEG-NHS (147 mg) in PBS solution, and then BSA (400 mg) was added to the above solution and stirred for 12 hours. After the reaction, the solution was dialyzed and freeze-dried to obtain RGD-PEG-BSA with cell active targeting function.

[0050] At the same time, mPEG5000-NHS (150 mg) and BSA (400 mg) were stirred and reacted in PBS solution for 12 hours. After the reaction, they were dialyzed and freeze-dried to prepare non-targeted high-molecular-weight mPEG-BSA as a control group.

[0051] 2) Preparation of nano-type photosensitizers

[0052] ATO and TPP-Ppa were dissolved in dimethyl sulfoxide to prepare ATO solution (10 mg / mL) and TPP-Ppa solution (20 mg / mL), 0.05 mL of each was taken and mixed in equal volumes to prepare ATO-@TPP-Ppa mixed solution.

[0053] RGD-PEG-BSA and CAT were dissolved in water to prepare RGD-PEG-BSA solution (10 mg / mL) and CAT solution (1 mg / mL), respectively. RGD-PEG-BSA solution (10 mg / mL, 0.2 mL) was mixed with CAT solution (1 mg / mL, 0.5 mL), and the volume was adjusted to 5 mL to prepare an aqueous solution.

[0054] The ATO-@TPP-Ppa mixed solution was added dropwise to the above aqueous solution under ultrasonic conditions (5%, 5 min, 5 s supersonication and 5 s rest) to prepare TPP-Ppa@ATO@CAT@RGD-PEG-BSA, which was then dialyzed to prepare the nano-photosensitizer.

[0055] The particle size and morphology of the nanophotosensitizer TPP-Ppa@ATO@CAT@RGD-PEG-BSA were characterized. The particle size and morphology of the nanophotosensitizer were investigated using a particle size potentiometer and a transmission electron microscope. Figure 2 and Figure 3 The results showed that the nano-photosensitizer had nano-scale particle size and morphology.

[0056] Example 3 In vitro evaluation of anti-tumor cell activity

[0057] In vitro activity evaluation of the nano-type photosensitizer of Example 2

[0058] (1) Evaluation of cell targeting and PDT effects

[0059] The tumor cell targeting function of the nano-type photosensitizer was demonstrated through endocytosis experiments using flow cytometry and laser confocal imaging; at the cellular level, mitochondria were labeled with fluorescent probes, and the mitochondrial targeting ability of the nano-type photosensitizer was demonstrated through confocal experiments.

[0060] The targeted endocytosis of tumor cells was evaluated using confocal microscopy. Figure 4, showing the endocytic effects of nanophotosensitizers with tumor cell targeting (TPP-Ppa@ATO@CAT@RGD-PEG-BSA, abbreviated as TAC@RPB) and without tumor cell targeting (TPP-Ppa@ATO@CAT@mPEG-BSA, abbreviated as TAC@PB) at different time points (2 and 4 hours).

[0061] And mitochondria-targeted endocytosis evaluation was performed, and the test results are shown in Figure 5 , demonstrating the endocytic effects of nanophotosensitizers with mitochondria targeting (TPP-Ppa@ATO@CAT@RGD-PEG-BSA, abbreviated as TAC@RPB) and without mitochondria targeting (Ppa@ATO@CAT@RGD-PEG-BSA, abbreviated as PAC@RPB).

[0062] The above results demonstrate that the nanophotosensitizer (TAC@RPB) has excellent tumor cell targeting and mitochondrial targeting capabilities.

[0063] (2) Evaluation of the therapeutic effect of nanophotosensitizers on tumor cells and the ability of mitochondria-targeted PDT to induce ICD in tumor cells

[0064] CCK-8 reagent was used to evaluate the therapeutic effect of each group of experiments on tumor cells. Flow cytometry, confocal microscopy imaging, Western Blot and other experimental methods were used to verify the changes in CRT, HMGB1 and ATP after mitochondrial-targeted photodynamic therapy with nanoparticles, thereby characterizing the ability of PDT to induce ICD of tumor cells. Antibodies were further used to detect markers of DC cell maturation to verify the production of tumor-associated antigens after photodynamic therapy of tumor cells, thereby enhancing the immunotherapy of Anti-PD-L1.

[0065] Results: CCK-8 reagent was used to evaluate the therapeutic effect of each group of experiments on tumor cells. The evaluation results are as follows: Figure 6 As shown, compared with the TC@RPB+Laser or TA@RPB+Laser group (TPP-Ppa@CAT@RGD-PEG-BSA, abbreviated as TC@RPB; TPP-Ppa@ATO@RGD-PEG-BSA, abbreviated as TA@RPB), the TAC@RPB+Laser group has excellent synergistic tumor cell killing ability under hypoxic conditions.

[0066] The ability of PDT to kill tumor cells and produce CRT was evaluated by confocal imaging using calreticulin (CRT) antibodies. Figure 7Compared with the ability of each group (Control, TC@RPB, TA@RPB, TAC@RPB) in inducing calreticulin (CRT) production after tumor cell treatment in Figure A, the nanophotosensitizer plus laser irradiation group (TAC@RPB+Laser) has the best ability to induce immunogenic death of tumor cells; Figure B is a quantitative evaluation of the fluorescence intensity generated by each group in Figure A.

[0067] The above results indicate that nanophotosensitizers have excellent tumor cell killing ability and the ability to produce immunogenic death of tumor cells.

[0068] Example 4 Animal Evaluation of Synergistic Anti-tumor Effects of Photodynamic Therapy and Immunotherapy

[0069] Construct a 4T1 bilateral subcutaneous tumor model, i.e., 1×10 6 With 2×10 6 4T1 cells were injected into the subcutaneous tissue of the left and right sides of Balb / c mice, which were respectively called distal tumor and original tumor, thus establishing a bilateral tumor model. When the volume of the original tumor reached about 100mm 3 The mice were divided into 6 groups (5 mice / group) and injected with nano-type photosensitizer (5 mg / kg). According to the results of in vivo fluorescence experiments, the right original tumor site was irradiated with laser (660 nm, 0.5 W / cm 2 , 5min), Anti-PD-L1 antibody was injected intraperitoneally every 2 days (once every 2 days, a total of 3 times, the antibody dosage was 750μg / kg), and the tumor size of the distal tumor and the original tumor was measured with a vernier caliper every two days, and the body weight was weighed at the same time. After 14 days of treatment, the treated mice obtained in vitro tumors for photographing and weighing, the tumor tissue sections were stained with H&E, and the content of various immune cells in the tumor tissue was detected. The spleen and lymph nodes were removed, and the content of various immune cells in the spleen and lymph nodes was detected.

[0070] We conducted an inhibitory evaluation on a bilateral tumor model, irradiating the primary tumor while not irradiating the distal tumor, and monitoring the changes in the tumor volume at both ends over time. The results showed that the photodynamic-immunotherapy implemented by the nanophotosensitizer combined with the antibody can not only inhibit the growth of the primary tumor, but also effectively inhibit the growth of the distal tumor through immunotherapy. The results are as follows Figure 8 shown. Figure 8 Figure A shows the primary tumor inhibition curve for each experimental group, and Figure B shows the distal tumor inhibition curve for each experimental group. The results show that compared to the PBS control group, the PDT plus Anti-PD-L1 combination therapy group not only inhibited the growth of the primary tumor but also achieved immunotherapy to inhibit distal non-irradiated tumors.

Claims

1. A self-assembled nanophotosensitizer, comprising TPP-Ppa@ATO@CAT@RGD-PEG-BSA, which is mainly obtained by self-assembly of TPP-Ppa, atovaquone ATO, catalase CAT and RGD-PEG-BSA, wherein: The chemical structure of the TPP-Ppa is shown below:

2. The nano photosensitizer according to claim 1, wherein the mass ratio of TPP-Ppa:ATO:CAT:RGD-PEG-BSA is 1:0.5:0.5:

2.

3. A method for preparing a self-assembled nanophotosensitizer, wherein the nanophotosensitizer is composed of TPP-Ppa@ATO@CAT@RGD-PEG-BSA, and the preparation method comprises: 1) React RGDfC with Mal-PEG-NHS in solution, then add BSA and stir to react; 2) After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain RGD-PEG-BSA; 3) dissolving atovaquone ATO and TPP-Ppa in an organic solvent to prepare an atovaquone ATO solution and a TPP-Ppa solution, respectively, and then mixing them to form a TPP-Ppa@ATO mixed solution; 4) RGD-PEG-BSA and catalase CAT were dissolved in water and then mixed. The TPP-Ppa@ATO mixed solution was added dropwise to the mixed solution of RGD-PEG-BSA and catalase CAT under ultrasonic conditions to obtain TPP-Ppa@ATO@CAT@RGD-PEG-BSA, which was dialyzed to obtain a nano photosensitizer. Wherein, the chemical structure of the TPP-Ppa is as follows:

4. The preparation method according to claim 3, wherein in step 1), the solution is a PBS solution, the mass ratio of RGDfC to Mal-PEG-NHS to BSA is 17:147:400, and the reaction time is 12 hours.

5. The preparation method according to claim 3, wherein in step 3), the mass ratio of ATO to TPP-Ppa is 1:

2.

6. The preparation method according to claim 3, wherein in step 3), the organic solvent is dimethyl sulfoxide, the concentration of the ATO solution is 10 mg / mL, and the concentration of the TPP-Ppa solution is 20 mg / mL.

7. The preparation method according to claim 3, wherein in step 3), the mass ratio of RGD-PEG-BSA, CAT and ATO is 2:0.5:0.

5.

8. Use of the nanophotosensitizer according to claim 2 or the nanophotosensitizer prepared by the preparation method according to claim 3 in the manufacture of anti-tumor drugs.

9. The use according to claim 8, comprising combining with an anti-PD-L1 monoclonal antibody, or forming a compound preparation.

10. A pharmaceutical composition comprising the nanophotosensitizer according to claim 1 or 2 or the nanophotosensitizer prepared by the preparation method according to claim 3, and optionally, pharmaceutically acceptable excipients.

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