Oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy, preparation method and application

Abstract

The present invention provides oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy. The oxygen nanobubbles are lipid-coated and contain an anti-HER2 scFv-nCytc (Pro), a photosensitizer (PS), and a lipid. The preparation method includes: first, using a gas-liquid mixing pump to prepare NBs-O2 water, then dissolving the phospholipid mixture and the photosensitizer in an organic solvent, removing the organic solvent by rotary evaporation, adding NBs-O2 water for hydration, and finally adding the anti-HER2 scFv-nCytc and incubating in the dark to obtain Pro@Lipo-PS-NBs-O2. The Pro@Lipo-PS-NBs-O2 prepared by the present invention can simultaneously deliver oxygen and drugs, improve the hypoxia of the tumor microenvironment, and enhance the efficiency of PDT. At the same time, the combination of anti-HER2 scFv-nCytc and PDT can produce a synergistic anti-tumor effect and enhance the therapeutic effect. In addition, the Pro@Lipo-PS-NBs-O2 prepared in the present invention induced breast cancer cell apoptosis through a non-inflammatory process without pro-inflammatory effects, and showed good safety both in vitro and in vivo.

Description

Technical field:

[0001] The present invention belongs to the field of medicine, and in particular relates to oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy, a preparation method and an application thereof. Background technology:

[0002] The tumor microenvironment (TME) is characterized by hypoxia in a subset of nearly every tumor type. Hypoxia leads to complex biological responses, including genomic instability, epigenetic reprogramming, angiogenesis and vasculogenesis, epithelial-mesenchymal transition, invasion and metastasis, metabolic reprogramming, immunosuppression, resistance to apoptosis, and resistance to drugs and radiation. Consequently, hypoxia significantly impacts many cancer treatments, including chemotherapy, radiotherapy, photodynamic therapy (PDT), sonodynamic therapy, and immunotherapy. Consequently, hypoxia limits therapeutic options and compromises tumor therapeutic efficacy. PDT typically requires three main components: light irradiation, a photosensitizer (PS), and oxygen. In PDT, the PS converts light energy into oxygen, generating high concentrations of reactive oxygen species (ROS), which cause irreversible oxidative damage to biomacromolecules and lead to cell death. Notably, the PDT process depletes oxygen from the TME, thereby exacerbating local hypoxia and further compromising therapeutic outcomes. A hypoxic TME severely limits the efficacy of PDT and is considered a major obstacle to cancer treatment. Numerous oxygen-supplementing materials have been investigated to alleviate hypoxia. For example, red blood cells and hemoglobin have been used as carriers, perfluorocarbons have been used to load oxygen, and strategies have been developed to generate oxygen through in situ reactions. However, the degradability of most oxygen carrier components remains an issue, and the safety and efficacy of treatments must be carefully considered. For example, several inorganic nanoparticles have been reported to release proinflammatory cytokines. Therefore, a safer treatment approach is urgently needed.

[0003] Nanobubble water is a "solution" formed by dispersing gas in water as free bubbles of nanometer diameter using specialized equipment. The resulting system contains a large number of bulk nanobubbles (BNBs), which can remain stable in the aqueous phase for days, weeks, or even months without any surfactants. BNBs are also attracting considerable attention due to their high specific surface area, high internal pressure, negative surface charge, good biocompatibility, and mechanical, thermal, optical, and acoustic responses.

[0004] Although the mechanisms underlying BNB longevity remain controversial, BNBs have been widely used in industry. For example, RNS60, a physically modified saline solution that is safe for enteral or parenteral administration, has been approved by the US Food and Drug Administration (FDA) for use in a Phase II clinical trial for the treatment of amyotrophic lateral sclerosis (ALS) (NCT02988297). RNS60 is a stable oxygen-carrying BNB composed solely of water, sodium chloride, and 55 ± 5 ppm oxygen. The resulting gas nanobubble solution has the potential to increase gas solubility in the aqueous phase, expand the gas diffusion interface area, and reduce the potential for gas embolism in the blood. These advantages of BNB have attracted attention for its promising application in oxygen delivery. However, few reports have described novel drug delivery systems using aqueous BNB as a dispersion medium. Further exploration is needed to determine whether the use of BNB as a dispersion medium encapsulated by encapsulating materials can enhance the long-term presence of oxygen and provide more space for drug loading.

[0005] Anti-HER2 scFv-nCytc is a fusion protein constructed from a single-chain antibody against human epidermal growth factor receptor 2 (anti-HER2 scFv) and tandem repeats of cytochrome c (nCytc) (Patent No.: 202010678663.4). Anti-HER2 scFv is designed to bind to HER2. + Cytc is a key endogenous inducer of apoptosis. + The results showed good targeting and efficacy in breast tumor growth. Similarly, the fusion protein modified oxygen carrier can enhance the targeting ability.

[0006] This paper studies the effect of membrane excipients on the oxygen stability of nanobubbles (NB)-O2 in water, evaluates the effect of oxygen in nanobubbles on PDT efficiency, and ultimately develops an anti-HER2 scFv-nCytc-modified, photosensitizer-carrying lipid-encapsulated oxygen nanobubbles Pro@Lipo-PS-NBs-O2. This is a drug delivery system that simultaneously carries PS, oxygen, and fusion protein. It can induce breast cancer cell apoptosis in a non-inflammatory manner through a combined treatment strategy of antibodies and PDT, showing an anti-cancer effect. Summary of the invention:

[0007] The purpose of the present invention is to address the deficiencies of the existing technology and provide an oxygen nanobubble for inducing cell apoptosis and improving photodynamic therapy, a preparation method and an application thereof.

[0008] The present invention adopts the following technical solutions:

[0009] (1) The present invention provides an oxygen nanobubble for inducing cell apoptosis and improving photodynamic therapy, wherein the oxygen nanobubble is modified with anti-HER2 scFv-nCytc, carries a photosensitizer, and is lipid-encapsulated.

[0010] (II) The present invention also provides a method for preparing oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy, comprising the following steps:

[0011] S1. Preparation of NBs-O2 water: oxygen and water are introduced into a gas-liquid mixing pump to form a supersaturated gas solution, which is then introduced into a water storage tank through a pipeline. The supersaturated gas solution is decompressed under atmospheric pressure to generate bubble nucleation and generate NBs-O2, thereby obtaining NBs-O2 water;

[0012] S2. Preparation of Pro@Lipo-PS-NBs-O2: The phospholipid mixture and photosensitizer were dissolved in an organic solvent, and then the organic solvent was removed by rotary evaporation to produce a thin film at the bottom of the bottle. NBs-O2 water was added for hydration. The mixture was sealed and incubated in the dark for 8 hours. The whole particles were filtered to make the particle size uniform. The filtrate was taken, anti-HER2 scFv-nCytc was added, and the mixture was incubated in the dark for another 8 hours to obtain Pro@Lipo-PS-NBs-O2.

[0013] Furthermore, in S1, the pressure of the gas-liquid mixing pump is 0.3-0.4 MPa.

[0014] Furthermore, in the S1, the oxygen content in the NBs-O2 water is higher than 15 mg / L at room temperature.

[0015] Furthermore, the filtering operation is specifically: filtering through a 0.45 μm filter membrane, or filtering through a 0.22 μm filter membrane, or filtering through a 0.45 μm filter membrane and a 0.22 μm filter membrane in sequence.

[0016] Furthermore, in S2, the phospholipid mixture includes DPPG, egg yolk lecithin and DSPE-MPEG2000, wherein the weight ratio of DPPG, egg yolk lecithin and DSPE-MPEG2000 is 2:20:1.

[0017] Furthermore, in S2, the usage ratio of the phospholipid mixture, photosensitizer, NBs-O2 water and anti-HER2scFv-nCytc is 128 mg:0.25 mg:25 ml:265 μg.

[0018] Furthermore, in S2, the photosensitizer is a fat-soluble photosensitizer used for PDT, specifically copper phthalocyanine, zinc phthalocyanine, Ce6 or IR780.

[0019] (3) The present invention also provides the use of the above-mentioned oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy in the preparation of drugs for treating breast cancer.

[0020] Beneficial effects of the present invention:

[0021] (1) The Pro@Lipo-PS-NBs-O2 prepared in the present invention has strong stability in aqueous solution and can simultaneously deliver oxygen and drugs (photosensitizer and fusion protein). The local release of oxygen and PS is mutually beneficial. PDT can promote the simultaneous release of oxygen and loaded drugs. Local oxygen release is also beneficial to reverse the hypoxia of the tumor microenvironment.

[0022] (2) In the Pro@Lipo-PS-NBs-O2 prepared by the present invention, the presence of anti-HER2 scFv-nCytc can improve the endocytosis efficiency. The fusion protein allows more oxygen to enter the cells for intracellular PDT. Oxygen is transported through the nanocarrier and enters the cells synchronously with PS, which can improve the PDT efficiency and enhance the therapeutic effect.

[0023] (3) The Pro@Lipo-PS-NBs-O2 prepared by the present invention can induce apoptosis of tumor cells by generating ROS during PDT. Cytc in the anti-HER2 scFv-nCytc is an endogenous apoptosis inducer that is released from the fusion protein when cleaved by caspase-3. Therefore, the combination of anti-HER2 scFv-nCytc and PDT can produce a synergistic anti-tumor effect.

[0024] (4) The Pro@Lipo-PS-NBs-O2 prepared by the present invention exhibits an anti-cancer effect by inducing apoptosis in breast cancer cells in a non-inflammatory manner. This effect is the opposite of necrosis or pyroptosis, which can cause inflammation and may promote tumor metastasis. This is because Cytc itself or its apoptotic metabolites have the effect of inhibiting inflammation, while cell death through necrosis or pyroptosis can promote immune response and lead to immunosuppression. Therefore, compared with the treatment strategy of inflammatory activation of tumor immunity, the Pro@Lipo-PS-NBs-O2 of the present invention has almost no pro-inflammatory effect and exhibits good safety both in vitro and in vivo. Description of the drawings:

[0025] Figure 1 Schematic diagrams of the present invention, where (A) shows the preparation of NBs-O2 water using a gas-liquid mixing pump; (B) shows the preparation of Pro@Lipo-PS-NBs-O2; and (C) shows the treatment of Pro@Lipo-PS-NBs-O2 combined with laser (NIR) irradiation.

[0026] Figure 2Characterization diagram of NBs-O2 of the present invention, wherein (A) schematic diagram of oxygen release of oxygen nanobubble water (NBs-O2), ordinary double distilled water (DD-water), and oxygen-saturated water at atmospheric pressure (O2-water) for one week; (B) Tyndall effect diagram of NBs-O2 from fresh preparation to storage for one week, with arrows indicating the light path; (C) average particle size and PDI value of NBs-O2 after preparation and after storage for one week; (D) according to total reflection infrared spectroscopy, DD-water as background, after preparation and storage Characterization diagram of hydrogen bonds between NBs-O2 and NBs-SF6 after 1 week; (E) Schematic diagram of the change in oxygen content of NBs-O2 diluted with degassed water within 1 week; (F) Schematic diagram of the change in average particle size of NBs-O2 within 1 week when diluted with degassed water; (G) Schematic diagram of the oxygen content of NBs-O2 under different pH conditions over 1 week; (H) Schematic diagram of the change in oxygen content of NBs-O2 solutions with different concentrations of PEI added within 1 week; (I) Schematic diagram of the change in oxygen content after adding different concentrations of DPPG to NBs-O2 within 1 week;

[0027] Figure 3 : Characterization diagram of Pro@Lipo-PS-NBs-O2 of the present invention, wherein, (A) average hydrodynamic size diagram of different drug delivery systems; (B) zeta potential diagram of different drug delivery systems; (C) oxygen release curve of Lipo and Lipo-NBs-O2 within 3 days; (D) ROS detection based on UV-visible absorption of DPBF during NIR irradiation, schematic diagram of absorption of DPBF in DD-water, NBs-O2+PS and DD-water+PS systems after 0.5, 1, 2, 3, 5 and 7 minutes of NIR irradiation; (E) zeta potential change diagram with the concentration of fusion protein added; (F) super-resolution fluorescence image of liposome-bound fusion protein; (G) intermolecular interaction between Lipo-NBs-O2 and Anti-HER2 scFv-Cytc was determined by micro-thermophoresis, and the temperature-related intensity change of anti-HER2 scFv-Cytc-dependent change was plotted as Fnorm value in dose-response curve and ligand (anti-HER2 The relationship between the concentration of scFv-Cytc and the fluorescence intensity of the laser is shown in Table 1. FORM = F1 / F0, where F1 corresponds to the fluorescence value under heating, and F0 is the fluorescence value measured before the laser is turned on. K d The value is 364 nM; (H) TEM image of the micromorphology of Pro@Lipo-PS-NBs-O2 (120 kV), scale bar is 1 μm; (I) Image of the micromorphological changes of Pro@Lipo-PS-NBs-O2 after near-infrared irradiation. The red circle in the left image indicates the lipid residue after irradiation (scale bar = 500 nm), and the upper and lower right images show the aggregation of PS after irradiation;

[0028] Figure 4Pro@Lipo-PS-NBs-O2 and HER2 + Breast cancer cell targeting and receptor-mediated endocytosis, including: (A) Confocal microscopy images of MDA-MB-231, MCF-7, SK-BR-3, and T47D cells treated with Pro@Lipo-PS-NBs-O2 (blue: DAPI, cell nuclei; green: fusion protein labeled with FITC fluorescent secondary antibody), and the right image is a digital quantification of the left image; (B) Confocal microscopy images of Lipo-PS-NBs-O2, CDDP@Lipo-PS-NBs-O2, and Pro@ Confocal microscopy images of SK-BR-3 cells treated with Lipo-NBs-O2 (blue: cell nucleus; red: RhoB-labeled liposomes); (C) Confocal microscopy images of SK-BR-3 cells treated with rhodamine-labeled Lipo-PS-NBs-O2, CDDP@Lipo-PS-NBs-O2, and Pro@Lipo-NBs-O2 combined with near-infrared irradiation; (D) Confocal microscopy images of the endocytic pathway of Pro@Lipo-NBs-O2 in SK-BR-3 cells.

[0029] Figure 5 To determine the cell death mechanism and inflammatory factors in SK-BR-3 cells after treatment, the following are shown: (A) Fluorescence images of SK-BR-3 cells co-stained with Calcein AM / PI after treatment with different agents, with red representing dead cells and green representing live cells; (B) Fluorescence images of SK-BR-3 cells co-stained with AO / EB after treatment with different agents, with green representing live or apoptotic cells and red representing necrotic cells; (C) Schematic diagram of flow cytometry for detecting cell apoptosis induced by different drugs; (D) Levels of IL-1β, IL-6, and TNF-α in SK-BR-3 cells after treatment; (E) Fluorescence images of SK-BR-3 cells stained with JC-1 to observe mitochondrial membrane potential, with green representing JC-1 monomers and red representing JC-1 aggregates;

[0030] Figure 6 Cellular pharmacodynamics after combined treatment with Pro@Lipo-PS-NBs-O2 and PDT, including: (A) Fluorescence images of DCFH-DA in SK-BR-3 cells with or without NIR irradiation; (B) Schematic diagram of cell viability of SK-BR-3 cells treated with 0, 10, 50, 100, and 150 nM fusion protein; (C) Schematic diagram of cell viability of SK-BR-3 cells treated with Lipo-PS-NBs-O2 and then irradiated for 0, 1, 3, 5, and 10 min; (D) HSA synergistic score of the combination of fusion protein and PDT calculated by SynergyFinder (<10, antagonism; 10 to 10, additive effect; >10, synergistic effect);

[0031] Figure 7 Schematic diagram of the distribution of Pro@Lipo-PS-NBs-O2 in tumor-bearing mice, and in vitro images of major tissues (heart, liver, spleen, lung, kidney, and tumor) 24 hours after injection;

[0032] Figure 8 Figures show the results of combined treatment with Pro@Lipo-PS-NBs-O2 and PDT, including: (A) Diagram of the tumor treatment process in mice; (B) Photos of tumors after treatment with different preparations (Group 1: normal saline; Group 2: Lipo-PS; Group 3: Lipo-PS-NBs-O2; Group 4: free fusion protein; Group 5: CDDP@Lipo-PS-NBs-O2; Group 6: Pro@Lipo-PS-NBs-O2); (C) Tumor growth curves after treatment with different preparations; (D) Schematic diagram of average tumor weight at the end of treatment with different preparations; (E) Graph of weight changes of mice treated with different preparations; (F) TUNEL staining of tumor slides after treatment with different preparations; (G) Results of detecting tumor hypoxia using the pimozole hydrochloride hypoxia probe, where the right figure is a digital quantification of the left figure;

[0033] Figure 9 Schematic diagram of the safety and inflammatory factor levels of Pro@Lipo-PS-NBs-O2 combined with PDT, including: (A) Schematic diagram of the levels of IL-1β, IL-6, and TNF-α in tumor tissues after treatment with different preparations; (B) Schematic diagram of the levels of serum biochemical indicators (ALP, ALT, AST, Cre, BUN) in tumor tissues after treatment with different preparations. Specific implementation method:

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0035] 1. Implementation

[0036] The present invention provides an oxygen nanobubble for inducing cell apoptosis and improving photodynamic therapy. The oxygen nanobubble is modified with anti-HER2 scFv-nCytc and is lipid-encapsulated and carries a photosensitizer. The materials and preparation method are as follows:

[0037] (1) Materials:

[0038] DPPG and DSPE-MPEG2000 were obtained from Jiangsu Southeast Nanomaterials Co., Ltd., China; egg yolk lecithin (ePC, PC-98T) was from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., China; anti-HER2 scFv-nCytc was prepared by the method described in the invention patent No. CN202010678663.4.

[0039] Gas-liquid mixing pump: 20QY-1DS, China Southern Pump Industry.

[0040] (2) Preparation of NBs-O2 water:

[0041] Oxygen is passed through a gas-liquid mixing pump (0.3-0.4 MPa) to form a supersaturated gas solution, which is then input into a water storage tank through a nozzle at the outlet of the pipeline. The supersaturated gas solution is decompressed under atmospheric pressure, causing bubble nucleation and generation of NBs-O2 to obtain NBs-O2 water; the oxygen content in NBs-O2 at room temperature is 25 mg / L.

[0042] (III) Preparation of Pro@Lipo-PS-NBs-O2:

[0043] A thin film hydration method was used, specifically: a phospholipid mixture (DPPG, egg yolk lecithin and DSPE-MPEG2000 weight ratio of 2:20:1) and copper phthalocyanine were dissolved in chloroform and placed in a distillation flask, and then the chloroform was removed by rotary evaporation to produce a thin film at the bottom of the flask, NBs-O2 water was added for hydration, the flask was sealed and incubated in the dark for 8 hours, and then the whole particles were filtered through a 0.45μm filter membrane to make the particles uniform in size. Anti-HER2 scFv-nCytc was then added to the filtrate and incubated in the dark for another 8 hours. The fusion protein was fixed on the particles through ionic bonds to obtain Pro@Lipo-PS-NBs-O2.

[0044] The dosage of phospholipid mixture, copper phthalocyanine, NBs-O2 water and anti-HER2 scFv-nCytc is 128 mg: 0.25 mg: 25 ml: 265 μg.

[0045] 2. Characterization of the obtained preparation

[0046] 1. Characterization of NBs-O2:

[0047] Oxygen content determination:

[0048] The oxygen content of the NBs-O2 water system, double-distilled water (DD-water), and water saturated with oxygen at atmospheric pressure (O2-water) was determined using an oxygen electrode method on a dissolved oxygen analyzer (JPB-607A, Shanghai Yidian Scientific Instruments). The hydrodynamic size and zeta potential of the resulting particles were measured at room temperature using a Zetasizer (NanoZS90, Malvern, UK).

[0049] The results are as follows Figure 2 As shown in Figure A, within 7 days of preparation, the oxygen content of NBs-O2 water was significantly higher than that of DD-water and O2-water (p < 0.01), indicating that an oxygen-saturated water system was successfully obtained. NBs-O2 water remained stable as a colloidal dispersion for at least one week, and the Tyndall phenomenon ( Figure 2 B). However, the average hydrodynamic particle size decreased from 843.4 to 679.3 nm, and the polydispersity index (PDI) increased from 0.289 to 0.318 ( Figure 2 C). After standing for one week with DD-water as the background, the study of hydrogen bonding in NBs-O2 and the control group NBs-SF6 (sulfur hexafluoride) showed that the hydrogen bonding absorption peak appeared at 3000–3500 cm -1 Department ( Figure 2 D), therefore, hydrogen bonding interactions contribute to the stability of the bubbles.

[0050] Next, the present invention studied the changes in oxygen content within one week after dilution with degassed water. 75%, 50%, and 25% represent different dilutions, specifically: the volume ratio of NBs-O2 to degassed water is 75%, 50%, and 25%. As the ratio of degassed water to NBs-O2 water increases, the oxygen reduction rate gradually decreases ( Figure 2 E). Meanwhile, the average particle size of NBs-O2 becomes larger, especially for 50% NBs-O2 ( Figure 2 F). However, despite the relatively low oxygen concentration, the size change in the 75% NBs-O2 group was minimal, indicating that gas supersaturation helps stabilize the bubbles. Therefore, greater gas supersaturation makes gas escape more easily but does not significantly change bubble size. In contrast, the more diluted 50% and 25% NBs-O2 groups both had significant effects on particle size, indicating that bubble stability is affected by the bubble density within the system. Particle size reached a minimum at 72 hours ( Figure 2 F), which may be due to the balance between interfacial electrostatic forces and internal pressure, it can be inferred that the larger particle size caused by dilution increases the incidence of embolism during intravenous infusion.

[0051] The bubble stability can also be attributed to the formation of a negative double layer. At pH 9, the NB surface is negatively charged because a large number of hydroxyl groups are adsorbed on the NB surface, which may help to slow down the release of oxygen. At 24 hours, the oxygen content at pH 9 is slightly higher than that at other pH values ​​( Figure 2 G), indicating that the negative surface charge helps maintain bubble size and reduces excessive oxygen release. The addition of acid also slightly slows down the release of oxygen ( Figure 2 G), which is presumably due to the formation of an ionic bond membrane on the NB surface. However, the stability of the bubble is determined by the combined effect of the ionic membrane structure and the surface potential.

[0052] To verify the effect of the formed membrane structure on the bubble stability, positively charged polyethyleneimine (PEI) in aqueous solution was added to NBs-O2 water, and the oxygen content was monitored for one week ( Figure 2 H). After adding PEI, the oxygen content in NBs-O2 water was higher, which shows that the positively charged hydrophilic polymer material has a stabilizing effect on NBs-O2. This stabilizing effect may be due to the formation of a polymer film through ion adsorption. The effects of hydrophilic negatively charged excipients (including 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-RAC-glycerol) (DPPG), polyethylene glycol 4000 (PEG4000), glycerol and distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 (DSPE-MPEG2000)) on maintaining the oxygen content in NBs-O2 water were also studied. Figure 2 As shown in Figure 1, the oxygen content decreased significantly after 72 hours of DPPG addition, indicating that DPPG negatively impacted the long-term stability of oxygen concentration in NBs-O2 water. Similar results were observed for polyethylene glycol 4000 and glycerol. In summary, we speculate that the addition of negatively charged hydrophilic supplements at near-neutral pH is detrimental to long-term oxygen retention in NBs, possibly due to competition with BNB for hydroxyl groups in aqueous solution. However, considering that DPPG contributes to particle stability in the dispersed system and provides some support for lyophilization, and that its short-term effect on oxygen content in bubbles (within two days) is not significant, the inclusion of DPPG in the formulation is still considered. Furthermore, the oxygen content remained similar to that of the NBs-O2 group after the addition of DSPE-MPEG2000. DSPE-MPEG2000 is hydrophobic and tends to be positively charged in aqueous solution. Therefore, the hydrophobic material can mitigate the adverse effects of PEG on the oxygen stability of NBs-O2 water.

[0053] 2. Characterization of Pro@Lipo-PS-NBs-O2:

[0054] Reference Figure 3 .in,

[0055] Figure 3A is the average hydrodynamic size of different systems. After coating, the particle size of Lipo-NBs-O2 is still about 135.6±4.4nm, indicating that the in vivo circulation may be prolonged.

[0056] Figure 3 B is the zeta potential of different drug delivery systems. Due to the introduction of DSPE-PEG2000, the Lipo-NBs-O2 potential becomes more negative (22.5±1.2 mV), which reduces particle collisions and enhances the stability between particles.

[0057] Figure 3 C shows the oxygen release curves from Lipo and Lipo-NBs-O2 over a 3-day period. Lipo-NBs-O2 continuously released oxygen with first-order kinetics over a 3-day period, and the oxygen level was significantly higher than that in the control group (blank liposome Lipo). This indicates that Lipo-NBs-O2 can carry oxygen and that oxygen is absorbed in the form of nanoparticles.

[0058] Figure 3 D is based on near-infrared radiation (808 nm, 100 mW / cm 2 ) during NIR irradiation. DPBF absorption was recorded in the DD-water, NBs-O2+PS, and DD-water+PS systems after 0.5, 1, 2, 3, 5, and 7 minutes of NIR irradiation. The NBs-O2+PS group exhibited the greatest change in absorbance and the highest ROS yield during irradiation. Furthermore, the system temperature remained unchanged during NIR irradiation, indicating minimal photothermal interference.

[0059] Figure 3 E is the change in the ζ potential of Pro@Lipo-PS-NBs-O2 with different concentrations of fusion protein, indicating that the absolute value of the carrier potential decreases with increasing protein concentration.

[0060] Figure 3 F is a super-resolution fluorescence image of Pro@Lipo-PS-NBs-O2. The red fluorescence indicates rhodamine (RhoB)-labeled liposomes, and the green fluorescence corresponds to the fluorescein isothiocyanate (FITC)-labeled fusion protein. The scale bar in the large image on the left is 100 nm, and the small image on the right is obtained by magnifying it at the same scale. The lower right image (yellow image) is an overlay of the upper two images (red and green), showing the co-localization of protein and liposomes. Figure 3 F shows overlapping red and green fluorescence, demonstrating that adsorption occurs between the fusion protein and the PEGylated carrier. Anti-HER2 scFv-nCytc is adsorbed onto the nanocarrier, forming a protein corona-like structure that is expected to play a role in in vivo tumor targeting.

[0061] Figure 3 G is the determination of the molecular interaction between Lipo-NBs-O2 and Anti-HER2 scFv-nCytc using microscale thermophoresis. Here, microscale thermophoresis is used to characterize the dissociation constant (K) between the protein and the nanoparticles. d ).like Figure 3 G, K d The value is in the nanomolar (364 nM) range, indicating that the protein has a strong binding affinity to the carrier in vitro and is expected to exhibit good stability in vivo.

[0062] Figure 3 H is a TEM image of the micromorphology of Pro@Lipo-PS-NBs-O2 (120 kV). Scale bar, 1 μm. The micromorphology of Pro@Lipo-PS-NBs-O2 was examined by transmission electron microscopy (TEM) before and after NIR irradiation. After negative staining with phosphotungstic acid, Pro@Lipo-PS-NBs-O2 was spherical or quasi-spherical.

[0063] Figure 3 Figure 1 shows the microscopic morphological changes of Pro@Lipo-PS-NBs-O2 after near-infrared irradiation. In the large image on the left, the red circles indicate the residual lipids after irradiation (scale bar = 500 nm), and the upper and lower panels on the right show the aggregation of PS after irradiation. Because PS is an electron-rich substance, it appears dark black under TEM. However, after irradiation, PS clearly separated from the lipid carrier, and some PS aggregated. Therefore, NIR may affect the distribution of PS in the carrier, which may facilitate the release or redistribution of the loaded drug when NIR irradiation is performed at the target site.

[0064] 3. Pro@Lipo-PS-NBs-O2 and HER2 + Breast cancer cell function research

[0065] 1. Pro@Lipo-PS-NBs-O2 selectively binds to HER2 + breast cancer cells and taken up via receptor-mediated endocytosis.

[0066] Human breast cancer cell lines SK-BR-3, T47, MDA-MB-23, and MCF-7 were obtained from ATCC and authenticated according to ATCC guidelines. All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Gibco) and a 1% penicillin and streptomycin mixture (Invitrogen). Mycoplasma contamination was routinely tested for the cells and no contamination was found. All cells were grown at 37°C in a humidified atmosphere containing 5% CO2.

[0067] Since Lipo-PS-NBs-O2 has been modified with anti-HER2 scFv-nCytc, the targeting activity of Pro@Lipo-PS-NBs-O2 was investigated by immunofluorescence analysis. MDA-MB-231 and MCF-7 cell lines represent HER2 - cells, while SK-BR-3 and T47D cell lines represent HER2 + All cells were treated with equal concentrations of Pro@Lipo-PS-NBs-O2 for 30 min at 4°C, incubated with FITC to label anti-HER2scFv-nCytc, and incubated with DAPI (4',6-diamidino-2-phenylindole) to image cell nuclei. Figure 4 As shown in A, obvious green color was observed in cells of SK-BR-3 and T47D groups, but not in MDA-MB-231 and MCF-7 groups (p<0.0001, HER2 + HER2 - Compared with HER2 + Cell-specific binding and HER2 + Internalized in cells.

[0068] RhoB was incorporated into Pro@Lipo-PS-NBs-O2 to verify the selective binding and delivery of the fusion protein. The efficiency of RhoB entry into SK-BR-3 cells was monitored. Red fluorescence was clearly observed in the Pro@Lipo-PS-NBs-O2 group, but not in the unmodified groups (Lipo-PS-NBs-O2 and CDDP@Lipo-PS-NBs-O2; Figure 4 B; p < 0.0001) No red fluorescence was observed. Therefore, the carrier modified with the fusion protein promoted the uptake of the loaded drug in the target area.

[0069] Next, the present invention investigated whether near-infrared radiation affects targeting ability or internalization. After incubation with RhoB in Pro@Lipo-PS-NBs-O2 or Lipo-PS-NBs-O2 for 30 min, SK-BR-3 cells were irradiated with near-infrared radiation (808 nm, 100 mW / cm 2 , 2 min). The results showed that irradiation had no negative impact on cell targeting ability or internalization; on the contrary, the intracellular fluorescence intensity was enhanced after irradiation ( Figure 4 C), indicating that the endocytosis efficiency is improved. The present invention also monitors the cell temperature during the entire irradiation process, indicating that the temperature fluctuation is not large, considering Figure 3 The results shown in I suggest that PDT may promote drug uptake.

[0070] Then, the present invention studied the internalization pathway of Pro@Lipo-PS-NBs-O2. The main cellular uptake mechanisms of nanoparticles include phagocytosis, microcytotoxicity, clathrin-mediated endocytosis (CME), caveolae-dependent endocytosis, and clathrin / caveolae-independent endocytosis. The CME pathway is the most well-known endocytosis pathway, also known as receptor-mediated endocytosis. This study used methyl-β-cyclodextrin and nystatin as inhibitors of caveolae-mediated endocytosis, amiloride to inhibit microcytosis, and chlorpromazine as a chemical inhibitor of CME. The results are shown in Figure 2. Figure 4 D. Chlorpromazine caused a significant decrease in red fluorescence intensity (p < 0.0001), indicating that CME is the main mechanism of Pro@Lipo-PS-NBs-O2 internalization. These findings indicate that Pro@Lipo-PS-NBs-O2 can be internalized by HER2 through the CME pathway based on the specific recognition and binding of the fusion protein. + The loaded drug can then be internalized into breast cells in a targeted manner before NIR irradiation.

[0071] 2. After Pro@Lipo-PS-NBs-O2 treatment and NIR irradiation, HER2 + Breast cancer cells undergo apoptosis.

[0072] The present invention studies the pharmacodynamics and mechanism of cell death after Pro@Lipo-PS-NBs-O2 treatment.

[0073] In SK-BR-3 cells, cell survival / death was assessed by Calcein-AM / PI co-staining. Calcein-AM labels live cells with green fluorescence, while PI (propidium iodide) labels dead cells with red fluorescence. Figure 5 A shows the cell survival / death results after different interventions (Lipo-PS-NBs-O2, fusion protein, CDDP@Lipo-PS-NBs-O2, and Pro@Lipo-PS-NBs-O2). No significant cell death was observed in the control group receiving the same dose of NIR irradiation, indicating that irradiation is safe. Furthermore, all intervention groups exhibited some degree of cell death.

[0074] Next, acridine orange (AO) / ethidium bromide (EB) staining was used to determine whether cell death occurred through apoptosis or necrosis. After AO / EB staining, normal cells showed uniform green fluorescence ( Figure 5 control group and Lipo-PS-NBs-O2 group in B), while apoptotic cells showed dense green fluorescence or granules ( Figure 5The fusion protein and Pro@Lipo-PS-NBs-O2 groups in B). To more clearly distinguish apoptosis from necrosis, saponin was used as a positive control for necrosis. The saponin and CDDP@Lipo-PS-NBs-O2 groups were stained with EB, further confirming that Pro@Lipo-PS-NBs-O2 did not kill breast cells by necrosis. Flow cytometry further confirmed that the apoptotic pathway occurred in the Pro@Lipo-PS-NBs-O2 group ( Figure 5 C) In Figure 5 In C, the cell population of Q2 represents early and late apoptotic events, and is the highest in the Pro@Lipo-PS-NBs-O2 group among all experimental groups.

[0075] The present invention also examined the levels of inflammatory factors produced after intervention. There was no statistically significant difference in the levels of IL-1β, IL-6, and TNF-α between the Lipo-PS and Lipo-PS-NBs-O2 groups, indicating that NBs-O2 itself did not increase inflammation during irradiation ( Figure 5 D). Meanwhile, the IL-1β, IL-6 and TNF-α levels in the fusion protein group and Pro@Lipo-PS-NBs-O2 group were significantly lower than those in the control group, CDDP@Lipo-PS-NBs-O2 group and Lipo-PS-NBs-O2 group ( Figure 5 D), indicating that Pro@Lipo-PS-NBs-O2 alleviated the inflammatory response during irradiation.

[0076] Finally, JC-1 staining was used to investigate the ability of Pro@Lipo-PS-NBs-O2 treatment and near-infrared radiation treatment to induce apoptosis in SK-BR-3 cells at the mitochondrial level. Figure 5 As shown in Figure E, compared with the control group and the CDDP@Lipo-PS-NBs-O2 group, the red-green fluorescence intensity ratio of the fusion protein group and the Pro@Lipo-PS-NBs-O2 group was significantly reduced (p<0.0001), indicating that the mitochondrial membrane potential, which plays an important role in cell apoptosis, decreased after drug and irradiation treatment. These results indicate that the combination of Pro@Lipo-PS-NBs-O2 and PDT induced cancer cell apoptosis and reduced inflammatory effects at the cellular level.

[0077] 3. The combination of Pro@Lipo-PS-NBs-O2 and PDT has a synergistic lethal effect on breast cancer cells.

[0078] DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) was used to monitor the intracellular ROS level after near-infrared irradiation. In the Pro@Lipo-PS-NBs-O2 group, the ROS level increased significantly after near-infrared irradiation compared with that before irradiation ( Figure 6A; p < 0.001), indicating the contribution of encapsulated oxygen. In addition, there was a significant difference between Pro@Lipo-PS-NBs-O2 and Lipo-PS-NBs-O2, indicating that NBs modified with the fusion protein produced more ROS after irradiation than NBs without the fusion protein. Figure 4 The results shown in C and 4D attribute this finding to improved cellular endocytosis by HER2 antibody modification.

[0079] The fusion protein not only promoted targeted binding and endocytosis, but also induced cell apoptosis (IC50 = 70.74 nM; Figure 6 B). The present invention also studies the relationship between the efficacy of PDT and the irradiation time, such as Figure 6 As shown in C, it shows that PDT promotes cancer cell death by generating ROS. In addition, the present invention uses SynergyFinder to study whether there is a synergistic effect between PDT and carrier fusion protein. Using the HSA reference model, the average synergy score is +12.76 ( Figure 6 D), indicating the existence of a synergistic effect, demonstrating the rationality and superiority of the combined use of fusion protein-modified vectors and PDT.

[0080] 4. Modification with anti-HER2 scFv-nCytc can improve the distribution of Pro@Lipo-PS-NBs-O2 in tumors and inhibit tumor proliferation in vivo.

[0081] In this study, we prepared indocyanine green (ICG)-loaded carriers to track the distribution of Pro@Lipo-PS-NBs-O2 in tumor-bearing mice. Twenty-four hours after administration, the mice were sacrificed, and heart, liver, spleen, lung, kidney, and tumor tissues were isolated for in vitro imaging. Figure 7 The ICG concentration in each tissue is shown. Among all the groups, the tumor tissue of the Pro@Lipo-PS-NBs-O2 group showed the highest fluorescence intensity, indicating that the modification with anti-HER2scFv-nCytc enhanced the tumor targeting ability of Pro@Lipo-PS-NBs-O2. Figure 7 Based on the results shown, we speculate that the anti-HER2 scFv fragment in the fusion protein of Pro@Lipo-PS-NBs-O2 promotes its own binding to HER2 + The protein is then internalized into the cells and concentrated in the tumor area.

[0082] SK-BR-3 cells were inoculated subcutaneously into mice (1×10 6 / mouse, 100 μL), a tumor-bearing mouse model was established to study the in vivo anti-tumor effect of Pro@Lipo-PS-NBs-O2. 3The mice were randomly divided into 6 groups: saline group, Lipo-PS group, Lipo-PS-NBs-O2 group, anti-HER2 scFv-nCytc group, CDDP@Lipo-PS-NBs-O2 group, and Pro@Lipo-PS-NBs-O2 group. 8 h after intravenous injection, the tumors in each group were irradiated with NIR (808 nm, 100 mW / cm 2 )2min. The treatment process is as follows Figure 8 As shown in A, the tumor growth curve after intervention is as follows Figure 8 As shown in Figure B. The tumor volume in the saline group increased rapidly within 21 days. In contrast, treatment with the fusion proteins Lipo-PS, Lipo-PS-NBs-O2, CDDP@Lipo-PS-NBs-O2, and Pro@Lipo-PS-NBs-O2 inhibited tumor overgrowth. Among them, Pro@Lipo-PS-NBs-O2 had the most significant antitumor effect. The tumor volume growth in the Pro@Lipo-PS-NBs-O2 group was significantly lower than that in the fusion protein and Lipo-PS-NBs-O2 groups (p < 0.001), indicating the significant efficacy of targeted apoptosis induction and PDT. In fact, the tumor inhibitory effect of PDT in the Lipo-PS-NBs-O2 group was stronger than that in the Lipo-PS group, which demonstrates the role of oxygen in Lipo-PS-NBs-O2.

[0083] Figure 8 C and 8D show the size and weight of the tumors isolated on day 21, further verifying the ability of Pro@Lipo-PS-NBs-O2 to inhibit tumor proliferation. The body weight of mice was also monitored throughout the treatment process. Except for the CDDP@Lipo-PS-NBs-O2 group, no significant differences were observed between the groups, reflecting the relative safety of Pro@Lipo-PS-NBs-O2 treatment combined with PDT ( Figure 8 E). TUNEL staining of tumor tissues showed that apoptosis occurred in all drug groups after intervention, and obvious apoptosis occurred in the fusion protein group and Pro@Lipo-PS-NBs-O2 group ( Figure 8 F) To illustrate the effect of the drug delivery system on PDT, we monitored its ability to improve tumor hypoxia using a pimozole hydrochloride hypoxia probe to detect tumor hypoxia, as shown in Figure 5. Figure 8 As shown in G, Pro@Lipo-PS-NBs-O2 significantly improved the hypoxic state of TME rats (p<0.0001).

[0084] 5. The combined treatment of Pro@Lipo-PS-NBs-O2 and NIR irradiation causes almost no inflammation.

[0085] Tissue homogenates and cell lysates were assayed for IL-6, IL-1β, and TNF-α using high-sensitivity ELISA (mlbio, Shanghai, China). Intra-assay and inter-assay variability were 10% and 15%, respectively. Figure 9 As shown in A, the levels of IL-1β, IL-6, and TNF-α in the tumor tissues of mice in the fusion protein group and Pro@Lipo-PS-NBs-O2 group were significantly lower than those in the other groups.

[0086] The present invention also studied the changes in serum biochemical indicators, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine (Cre), and blood urea nitrogen (BUN), to evaluate heart, liver, and kidney function. It was found that there were no significant differences in these indicators among the groups ( Figure 9 B) HE staining further confirmed the relative safety of the treatment, with no significant abnormalities observed in any tissue. During NIR irradiation, body temperature was monitored for each mouse, and no significant thermal changes were observed. Therefore, the combined treatment of Pro@Lipo-PS-NBs-O2 and NIR irradiation induces little inflammation.

[0087] In summary, the Pro@Lipo-PS-NBs-O2 of the present invention has high oxygen carrying capacity, strong stability in aqueous solution, lasting for at least 7 days, and targeting HER2 + cells, enhancing the efficacy of PDT. During NIR irradiation, the loaded PS and lipid materials reassemble, promoting the local release of the fusion protein and possibly other loaded drugs, further enhancing the therapeutic effect. Pro@Lipo-PS-NBs-O2 combined with PDT synergistically enhances the anti-tumor effect by inducing cell apoptosis. This synergistically improves the therapeutic effect and alleviates tumor hypoxia in vivo without causing an inflammatory response, with a good safety profile.

[0088] The above are only preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should be regarded as the scope of protection of the present invention.

Claims

1. An oxygen nanobubble for inducing cell apoptosis and improving photodynamic therapy, characterized in that: The oxygen nanobubbles are modified with anti-HER2 scFv-nCytc, carry photosensitizers, and are lipid-encapsulated oxygen nanobubbles; the preparation method comprises: Preparation of S1, NBs-O2 water: Oxygen and water are introduced into a gas-liquid mixing pump to form a supersaturated gas solution, which is then introduced into a water storage tank through a pipeline. The supersaturated gas solution is decompressed under atmospheric pressure to generate bubble nucleation and generate NBs-O2, thereby obtaining NBs-O2 water. Preparation of S2, Pro@Lipo-PS-NBs-O2: The phospholipid mixture and photosensitizer are dissolved in an organic solvent, and the organic solvent is then removed by rotary evaporation to produce a thin film at the bottom of the bottle. NBs-O2 water is added for hydration, the bottle is sealed and incubated in the dark for 8 hours, the whole particles are filtered to make the particles uniform in size, the filtrate is taken, anti-HER2 scFv-nCytc is added, and the mixture is incubated in the dark for another 8 hours to obtain Pro@Lipo-PS-NBs-O2; the photosensitizer is a fat-soluble photosensitizer for PDT.

2. The oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy according to claim 1, characterized in that: In S1, the pressure of the gas-liquid mixing pump is 0.3-0.4 MPa.

3. The oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy according to claim 1, characterized in that: In the above S2, the filtering operation is specifically: filtering through a 0.45 μm filter membrane, or filtering through a 0.22 μm filter membrane, or filtering through a 0.45 μm filter membrane and a 0.22 μm filter membrane in sequence.

4. The oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy according to claim 1, characterized in that: In said S2, the phospholipid mixture includes DPPG, egg yolk lecithin and DSPE-MPEG2000, wherein, The weight ratio of DPPG, egg yolk lecithin and DSPE-MPEG2000 is 2:20:

1.

5. The oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy according to claim 1, characterized in that: In the S2, the usage ratio of the phospholipid mixture, the photosensitizer, the NBs-O2 water and the anti-HER2 scFv-nCytc is 128 mg: 0.25 mg: 25 ml: 265 μg.

6. The oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy according to claim 1, characterized in that: In the above-mentioned S2, the photosensitizer is copper phthalocyanine, zinc phthalocyanine, Ce6 or IR780.

7. Use of the oxygen nanobubbles for inducing cell apoptosis and improving photodynamic therapy according to claim 1 in the preparation of drugs for treating breast cancer.

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