Preparation method of pH-sensitive fluorine-containing oxygen-carrying polymer nano delivery platform and application thereof in tumor treatment

Abstract

This invention belongs to the field of biomedical technology and discloses a method for preparing a pH-sensitive fluorinated oxygen-carrying polymer nanodelivery platform and its application in tumor treatment. This invention uses an oxygen-carrying, pH-responsive amphiphilic block copolymer as a carrier and employs a self-assembly method to encapsulate small-molecule antitumor drugs, achieving co-delivery of oxygen and the small-molecule antitumor drugs. The protonable pH-sensitive monomer enables the nanoparticles to respond to the acidic tumor microenvironment and release the drug, while the perfluorinated carbon monomer achieves efficient oxygen carrying, reversing the hypoxic tumor microenvironment. The small-molecule antitumor drugs encapsulated in the nanodelivery platform and the carried oxygen exert a synergistic effect in tumor tissue, relieving the inhibitory effect of the hypoxic microenvironment on tumor treatment, improving efficacy, and providing an effective strategy for tumor treatment. Furthermore, this platform has high safety, strong applicability, and can encapsulate a variety of drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanodelivery platform based on protonable perfluorinated carbon copolymer, used to simultaneously deliver oxygen and small molecule tumor therapeutic drugs to overcome the hypoxic tumor microenvironment and enhance the effect of immunotherapy. Background Technology

[0002] Hypoxia is a key characteristic of the tumor microenvironment, primarily caused by rapid tumor growth, abnormal vascular structure, and insufficient blood perfusion. Hypoxia not only promotes malignant tumor progression but also significantly inhibits the effectiveness of various treatments. Hypoxia significantly weakens the efficacy of tumor treatments through multiple mechanisms: First, hypoxia directly reduces radiosensitivity because radiation-induced DNA damage depends on the formation of oxygen free radicals; second, hypoxia activates the HIF-1α signaling pathway, promoting abnormal angiogenesis and epithelial-mesenchymal transition, enhancing tumor invasiveness and forming a physical barrier to hinder drug penetration; third, it induces the enrichment of tumor stem cells and overexpression of P-glycoprotein, leading to chemotherapy resistance; fourth, it suppresses effector T cell function while simultaneously expanding immunosuppressive cells, recruiting regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) to form an immunosuppressive microenvironment. This dynamically evolving hypoxic ecosystem, through metabolic reprogramming and epigenetic regulation, ultimately enables tumors to acquire multi-modal treatment resistance. Therefore, intervention strategies targeting hypoxia need to integrate multimodal therapies to overcome the bottlenecks of existing tumor treatments.

[0003] Current nanomedicine delivery systems for hypoxia still have significant drawbacks: ① Traditional oxygen-carrying materials (such as perfluorocarbon and hemoglobin carriers) lack intelligent responsiveness, prematurely releasing oxygen during circulation (in vitro experiments show that over 60% of oxygen leaks within 6 hours), resulting in insufficient improvement in local tumor oxygen partial pressure (preclinical models show only a 1.5-2 fold increase); ② Passive targeting (EPR effect) is inefficient (<5% ID / g) and lacks a specific activation mechanism for the hypoxic microenvironment; ③ Co-delivery with small molecule therapeutic drugs lacks spatiotemporal matching, making it difficult to achieve therapeutic synergy. Therefore, there is an urgent need for a nanoplatform capable of precisely regulating oxygen release and synergistically enhancing tumor treatment. Summary of the Invention

[0004] This invention provides a nanodelivery platform with high oxygen-carrying capacity and capable of encapsulating various types of antitumor drugs. It is responsive to the acidic tumor microenvironment, enabling precise delivery and release of oxygen and drugs. It comprises a fluorinated, oxygen-carrying, pH-responsive amphiphilic block copolymer, micelles formed by the self-assembly of this copolymer, micelles formed by the copolymer and antitumor drugs, its preparation method, and its pharmaceutical applications.

[0005] The fluorinated, oxygen-carrying, pH-responsive amphiphilic block copolymer and a small molecule drug (e.g., the STING agonist SR717) described in this invention are encapsulated in micelles formed by the polymer through hydrophobic interactions. Under normal physiological conditions, the drug molecules can be stably present in the micelle core through polymer self-assembly, reducing toxic side effects on normal tissues and minimizing off-target effects. Through enhanced permeability and retention effects, the drug passively accumulates at the tumor site via systemic blood circulation and specifically dissipates within the tumor tissue. Simultaneously, it precisely delivers oxygen and the small molecule drug, overcoming hypoxia to amplify the anti-tumor drug's effect and improve tumor treatment efficacy, providing a safer and more effective strategy for targeted tumor therapy. Furthermore, this amphiphilic block copolymer can also be used in combination with photothermal therapy, chemotherapy, photodynamic therapy, sonodynamic therapy, and immunotherapy to exert its therapeutic effect.

[0006] The amphiphilic block copolymer described in this invention is composed of two parts: hydrophilic segments and hydrophobic segments.

[0007] The hydrophilic block is selected from one or more of polyethylene glycol, polyethylene oxide, polycarboxylate betaine, poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, and polymethyl methacrylate phosphatidylcholine.

[0008] Hydrophobic blocks are copolymerized from one or more protonable tertiary amine monomers and oxygen-carrying perfluorinated carbon monomers via atom transfer radical polymerization or reversible addition-chain transfer polymerization.

[0009] The polymer of the present invention has hydrophobic blocks having a structure as shown in Formula 1:

[0010]

[0011] The protonable tertiary amine monomers (R1, R2) are selected from one or more of 2-(diisopropylamino)ethyl methacrylate (iDPA), 2-(dipropylamino)ethyl methacrylate (DPA), diethylaminoethyl methacrylate (DEA), ethylpropylaminoethyl methacrylate (EPA), dibutylaminoethyl methacrylate (DBA), cyclopentylaminomethacrylate (C5A), cyclohexylaminomethacrylate (C6A), and cycloheptylaminomethacrylate (C7A). R1 ​​and R2 are selected from the following structures:

[0012]

[0013] m and n are integers from 0 to 500; where the sum of m and n is an integer from 20 to 5000.

[0014] The oxygen-carrying perfluorocarbon monomer (R3) is selected from one or more of 2-(perfluoropropyl)ethyl methacrylate (2-(F3MA), 2-(perfluorohexyl)ethyl methacrylate (2-(HFMA), and 2-(perfluorooctyl)ethyl methacrylate (2-(F8MA),) and can efficiently load oxygen through hydrophobic interactions. R3 is selected from the following structures:

[0015]

[0016] z is an integer from 0 to 100, and the three units m, n, and z can be arranged in any order.

[0017] The amphiphilic block copolymers described in this invention can form stable nanomicelles in solution through self-assembly under ultrasonication, serving as carriers for nanodrug delivery and effectively encapsulating small molecule drugs (in this invention, PDPA-encapsulated SR717 is referred to as the SP group, and PDPA-HFMA-encapsulated SR717 is referred to as the SPF group).

[0018] Among them, small molecule drugs can be different types of drugs related to tumor immunotherapy, which can be encapsulated in the core of nanoparticles through self-assembly. These drugs are selected from chemotherapy drugs such as cisplatin, paclitaxel, and doxorubicin; immune enhancers such as interleukin-2, interferon, and thymosin α1; and STING agonists such as 2,3'-cyclic guanosine monophosphate-adenosine monophosphate (2,3'-cGAMP), 5,6-dimethylxanthoxanone-4-acetic acid (DMXAA), 4,5-difluoro-2-(((6-(1H-imidazol-1-yl)-3-pyridazinyl)carbonyl)amino)-benzoate lithium salt (SR-717), and benzimidazole derivatives (GSK). 37417), macrocyclic bridged dinucleotide analogs (E7766), diaminobenzimidazole (diABZI), acridinone acetate, 4-((4-(2-(tert-butyl)-5,5-dimethyl-1,3-dioxane-2-yl)benzyl)amino-3-methoxybenzoic acid (NVS-STG2), MSA-2, MK-1454, TAK-676; TLR agonists such as imiquimod, lipopolysaccharide, CpG oligonucleotides; photosensitizers such as 5-aminolevulinic acid, porphyrins, phthalocyanines, and sonosensitive agents such as methylene blue, indigo green, and nanomaterials, one or more of these drugs.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention provides a nanodelivery platform with high oxygen-carrying capacity and capable of encapsulating various types of small molecule anti-tumor drugs. It can respond efficiently to the acidic tumor microenvironment and simultaneously achieve targeted tumor delivery and release of oxygen and drugs, effectively overcoming the limitations of hypoxia on the therapeutic effect of small molecule drugs and improving the therapeutic effect.

[0021] The polymer micelles provided by this invention can remain stable under blood circulation and normal physiological conditions, avoiding drug release and effectively reducing the toxic side effects of drugs on normal tissues; while in tumor tissues, they respond rapidly to the acidic microenvironment, and the micelles disintegrate to achieve intelligent response release of drugs, further improving the effectiveness and safety of tumor treatment.

[0022] The preparation method of this invention is simple, reliable, and highly practical, and can be combined with various treatment strategies to achieve synergistic anti-tumor effects. For example, it can be combined with chemotherapy and immunotherapy, or with chemotherapy and photothermal therapy. The synthesized amphiphilic block copolymer can serve as a nanodelivery platform for various small-molecule anti-tumor drugs, demonstrating versatility. Attached Figure Description

[0023] 1. Figure 1 This is a synthetic route diagram for synthesizing amphiphilic block copolymers using atom transfer radical polymerization (ATRP).

[0024] 2. Figure 2 Synthesized pH-sensitive polymer PEG 113 -bP(DPA 67 -HFMA 16 The proton NMR spectrum of 10 ...

[0025] 3. Figure 3 For the synthesis of pH-sensitive polymer PEG 113 -bP(DPA 67 -HFMA 16 The nuclear magnetic resonance fluorine spectrum of ).

[0026] 4. Figure 4 This is a pathway diagram for the synthesis of SPF nanoparticles.

[0027] 5. Figure 5 This is a particle size distribution diagram of SPF.

[0028] 6. Figure 6 This is a zeta potential diagram of SPF.

[0029] 7. Figure 7 This is a transmission electron microscope image of SPF.

[0030] 8. Figure 8 The oxygen concentration diagram is shown when saturated oxygen PF nanoparticles are added to oxygen-free water.

[0031] 9. Figure 9 The oxygen release curves of PF at different pH values ​​when it is filled to saturation with oxygen.

[0032] 10. Figure 10 The graph shows the release curves of SR717 from SPF nanoparticles at different pH values.

[0033] 11. Figure 11 A quantitative graph showing the activation of IFN-I in THP-1 reporter cells (THP1-Lucia™ STING Reporter Cells) by PBS, PF, SR717, and SPF.

[0034] 12. Figure 12 The Western blot plot was used to verify the ability of PF to alleviate hypoxia.

[0035] 13. Figure 13 The Western blot plot verifies the activation effect of SPF on the STING pathway under hypoxic conditions.

[0036] 14. Figure 14 The Western blot plots demonstrate the superior activation effect of SPF on the STING pathway compared to SP under hypoxic conditions.

[0037] 15. Figure 15 The tumor growth curves of CT26 tumor-bearing BALB / c mice under the treatment of PBS, SR717, PF, and SPF are shown. The tumor inhibition rate of the SPF group reached 88.9%. Detailed Implementation

[0038] The following embodiments are intended to illustrate the present invention and not to further limit it. The present invention is further described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments and the preparation methods used. Moreover, those skilled in the art can make equivalent substitutions, combinations, improvements, or modifications to the present invention based on the description, but all such substitutions and modifications will be included within the scope of the present invention.

[0039] Example 1: Synthesis of polyethylene glycol-block-poly(diisopropylaminoethyl methacrylate-perfluorohexylethyl methacrylate) (PEG-bP(DPA-HFMA)) copolymer

[0040] This invention utilizes atom transfer radical polymerization (ATRP) technology to successfully synthesize a polyethylene glycol-block-poly(diisopropylaminoethyl methacrylate-perfluorohexylethyl methacrylate) (PEG-bP(DPA-HFMA)) copolymer.

[0041] The specific synthesis steps are as follows: First, mPEG-Br (250 mg, 0.05 mmol), DPA (700 mg, 3.28 mmol), HFMA (400 mg, 0.93 mmol), and PMDETA (13 μL, 0.06 mmol) were dissolved in a mixed solvent of 1 mL N,N-dimethylformamide (DMF) and 1 mL isopropanol (IPA). Then, after three freeze-thaw cycles, CuBr (8.5 mg, 0.06 mmol) was added to the above solution, and the reaction was carried out overnight at 42 °C. After the reaction was complete, the reaction product was treated with a neutral Al₂O₃ column to remove residual catalyst CuBr; then, the organic solvent and excess reactant impurities were removed by dialysis. The synthesis steps are as follows: Figure 1 As shown, in addition, the present invention also performs precise calculations of the degree of polymerization, and uses 1H-NMR and 19 F-NMR technology clearly characterized and confirmed the chemical structure of the copolymer, such as Figure 2 , Figure 3 As shown.

[0042] Example 2: Preparation of SPF nanoparticles

[0043] The PEG-bP(DPA-HFMA) (5 mg) and the non-nucleotide STING agonist SR717 (0.5 mg) prepared in Example 1 were dissolved in 200 μL of methanol (CH3OH). The organic solvent was then uniformly dispersed in 4 mL of deionized water under ultrasonic conditions at 70 W to form nanoparticles. To remove residual methanol and unencapsulated SR717, the nanoparticle solution was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, ultimately obtaining a pure SPF nanoparticle dispersion. SR717 was not added in the preparation steps of the SPF nanoparticle dispersion; the remaining preparation methods were the same as for SPF. The nanoparticle preparation steps are as follows: Figure 4 As shown.

[0044] Example 3: Characterization of SPF nanoparticles

[0045] The particle size, PDI, and Zeta potential of SPF nanoparticles were determined using dynamic light scattering and a Malvern laser particle size analyzer. Figure 5 , Figure 6 As shown, the hydrated particle size of the obtained SPF is 43.8 nm, and the Zeta potential is positive. The SPF achieves an encapsulation efficiency of 98% for SR717, with a drug loading capacity as high as 5.9%. Furthermore, the excellent drug loading capacity of SPF makes it suitable for encapsulating other STING agonists, as well as chemotherapeutic drugs, immune enhancers, TLR agonists, photosensitizers, sonosensitive agents, and nanomaterials.

[0046] Example 4: Characterization of pH responsiveness of oxygen and antitumor molecules

[0047] Figure 7 Transmission electron microscopy images showed that SPF was a spherical nanoparticle with clear boundaries at pH 7.4, and disintegrated at pH 5.5, demonstrating the pH-responsiveness of SPF. Figure 8 As shown, after PF nanoparticles were filled with oxygen and then injected into deoxygenated water, the oxygen content did not drop within 2 minutes, indicating their good oxygen-carrying capacity. Furthermore, the oxygen release rates of PF and SR717 at different pH values ​​were investigated. Figure 9 and Figure 10 The release of both oxygen and SR717 was shown to be pH-dependent.

[0048] Example 5: Activation effect of SPF nanodelivery platform on STING pathway

[0049] STING reporter cells THP1-Lucia TM STING Reporter Cells were co-incubated with PBS, SR717, PF, and SPF medium, and samples were taken at 12h, 24h, and 36h to detect luciferase secretion.

[0050] like Figure 11 As shown, PF-induced luciferase induction was negligible over time, while SR717 treatment exhibited moderate STING activation. In contrast, SPF treatment resulted in the highest level of STING activation, peaking at 24 hours. Importantly, SPF was also shown to have sustained activation capabilities, producing potent IFN-I induction at 24 hours, 2.9-fold and 12.5-fold higher than that of SR717 and PF, respectively.

[0051] SPF can maximally activate STING by overcoming hypoxia-induced inhibition, thus a significant decrease in HIF-1α protein can be clearly observed, such as... Figure 12 As shown. Furthermore, we investigated the activation effect of SPF on bone marrow-derived dendritic cells under hypoxic conditions. SPF successfully activated the STING pathway under hypoxic conditions, leading to increased phosphorylation levels of STING, TBK1, and NF-κB, consistent with the results observed in reporter cells. SPF-induced p-STING protein levels were significantly higher, 2.0-fold and 6.0-fold higher in SR717 and PBS under hypoxic conditions, respectively. Figure 13 As shown. Meanwhile, under hypoxic conditions, SP failed to elicit a response in the same STING / TBK1 / NF-κB pathway as SPF, and the upregulation of p-STING protein was 57.6% of that in the SPF group, indicating the crucial role of restoring oxygen in STING phosphorylation, as... Figure 14 As shown.

[0052] Example 6: Evaluation of SPF Treatment Efficacy

[0053] Given SPF's significant effect in alleviating tumor hypoxia and activating STING, we further evaluated its antitumor efficacy. Tumors with a volume of approximately 50 mm were selected. 3 Mice were treated with PBS, SR717, PF, or SPF every 3 days. Results showed that SR717 and PF treatments only moderately reduced tumor volume, indicating limited therapeutic activity. In contrast, the SPF group demonstrated superior therapeutic efficacy, achieving a tumor inhibition rate of 88.9%, and 33% of the mice achieved complete tumor regression. Figure 15 As shown.

Claims

1. A pH-sensitive fluorinated oxygen-carrying polymer nanodelivery platform, characterized in that: The platform comprises a copolymer of a protonable tertiary amine and an oxygen-carrying perfluorinated carbon, and an encapsulated small molecule antitumor drug.

2. The nanodelivery platform according to claim 1, characterized in that, It is composed of both hydrophilic and hydrophobic segments. The hydrophilic segments are selected from one or more of polyethylene glycol, polyethylene oxide, polycarboxylate betaine, poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, and polymethyl acrylate phosphatidylcholine.

3. The nanodelivery platform according to claim 1 or 2, characterized in that, The hydrophobic segment has the structure shown in Formula 1: Among them, the protonable tertiary amine monomers R1 and R2 are independently selected from one or more of 2-(diisopropylamino)ethyl methacrylate, 2-(dipropylamino)ethyl methacrylate, diethylaminoethyl methacrylate, ethylpropylaminoethyl methacrylate, dibutylaminoethyl methacrylate, cyclopentylaminomethacrylate, cyclohexylaminomethacrylate, and cycloheptylaminomethacrylate. m and n are integers from 0 to 500, and their sum is an integer from 20 to 500; z is an integer from 0 to 100, and the three units m, n, and z can be arranged in any order; The perfluorocarbon monomer R3 accounts for 5%-40% of the total monomers in the polymer and is selected from one or more of 2-(perfluoropropyl)ethyl methacrylate, 2-(perfluorohexyl)ethyl methacrylate (2-) and 2-(perfluorooctyl)ethyl methacrylate (2-), and is used for efficient oxygen carrying.

4. A method for synthesizing the nanodelivery platform according to any one of claims 1-3, characterized in that, include: pH-sensitive fluorinated oxygen-carrying polymers are synthesized from monomers via atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer polymerization (RAFT).

5. The polymer nanomedicine delivery platform according to claim 1, characterized in that, The small molecule antitumor drugs include chemotherapy drugs such as cisplatin, paclitaxel, and doxorubicin; immune enhancers such as interleukin-2, interferon, and thymosin α1; and STING agonists such as 2,3'-cyclic guanosine monophosphate-adenosine monophosphate (2,3'-cGAMP), 5,6-dimethylxanthoxanone-4-acetic acid (DMXAA), lithium 4,5-difluoro-2-(((6-(1H-imidazol-1-yl)-3-pyridazinyl)carbonyl)amino)-benzoate (SR-717), benzimidazole derivatives (GSK37417), and macrocyclic bridged dinucleotide analogs (E776). 6) Dimeric aminobenzimidazole (diABZI), acridinone acetic acid, 4-((4-(2-(tert-butyl)-5,5-dimethyl-1,3-dioxane-2-yl)benzyl)amino-3-methoxybenzoic acid (NVS-STG2), MSA-2, MK-1454, TAK-676; TLR agonists such as imiquimod, lipopolysaccharide, CpG oligonucleotides; photosensitizers such as 5-aminolevulinic acid, porphyrin compounds, phthalocyanine compounds, and sonosensitive agents such as methylene blue, indigo green, and nanomaterials, one or more of these drugs form micelles through self-assembly.

6. The use of the nanodelivery platform according to any one of claims 1-5 in the preparation of antitumor drugs.

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