Biomimetic photoactive nanofomulation for tumor therapy and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kind of bionic photoactive nano-preparation for tumor treatment and its preparation method and application, including nano calcium peroxide and polydopamine, iodide IR780 synthesis CaO2@PDA-IR780, again through cell membrane coating technology synthesis CaO2@PDA-IR780@M method.The CaO2@PDA-IR780@M obtained in the application is a kind of bionic photoactive nano-preparation, can utilize nano calcium peroxide in situ oxygen production to enhance the PDT effect of IR780 to improve the curative effect on tumor, utilize PDA acid response to realize the controllable release of preparation in tumor and utilize cell membrane to realize the functional modification of nanometer particles in vivo long cycle and tumor targeted delivery;And photodynamic and photothermal activity is high, has good tumor targeting property and safety, and has wide application prospect in the application of treating tumor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a biomimetic photoactive nano-formulation for tumor treatment, its preparation method and application. Background Technology

[0002] Cancer (also known as malignant tumors) is a major social, public health, and economic problem in the 21st century. The hypoxic tumor microenvironment (TME) is defined as a condition where the partial pressure of oxygen (pO2) is below 10 mmHg. Hypoxia-induced disorder and leakage of non-functional blood vessels within the tumor significantly reduce oxygen supply. Furthermore, in larger tumors, due to the limited diffusion distance of oxygen, tumor tissues farther from blood vessels experience poorer oxygen supply. TME is considered a major mechanism leading to resistance in tumors to various therapies, including chemotherapy, radiotherapy, and immunotherapy.

[0003] Photothermal therapy (PTT) is an emerging cancer treatment method that uses a photothermal agent (PTA) to convert light energy into heat energy, raising the local temperature of the tumor to destroy tumor cells. It is currently widely used in clinical treatment and laboratory scientific research. However, it still has unavoidable drawbacks: insufficient photothermal conversion efficiency of PTA, low tumor accumulation, tumor heat resistance, and the inability of single-modality therapy to completely eradicate tumors pose significant challenges to the future clinical translation of PTT.

[0004] Nano-calcium peroxide (CaO2) possesses multiple functions. It can provide hydrogen peroxide (H2O2) and oxygen (O2) to the tumor microenvironment without external stimulation, while simultaneously rapidly releasing a large amount of free calcium ions (Ca). 2+ Ca 2+ Involved in various cancer diagnosis and treatment events, including calcium homeostasis disorder caused by calcium overload, calcium channel dysregulation, mitochondrial dysfunction, calcium-related immune regulation, and cell / vascular / tumor calcification.

[0005] IR780 iodide exhibits good photostability, fluorescence intensity, and in vivo circulation time, enabling near-infrared fluorescence (NIRF) imaging with significant phototherapy-to-tumor (PTT) effects. Furthermore, IR780 can actively target and bind to the mitochondria of tumor cells, making it suitable as a targeting probe. It also demonstrates photodynamic therapy (PDT) efficacy under 808nm laser irradiation. However, IR780 suffers from drawbacks such as hydrophobicity, poor photostability, and aggregation-caused quenching (ACQ) effects. These significantly impact its PDT efficacy and limit its clinical application.

[0006] Polydopamine (PDA) can serve as a platform for combining with other materials to design core-shell structures, thereby creating nanoplatforms with excellent functions (such as high stability, stability, and controlled release). PDA exhibits the PTT effect, which can enhance the PTT effect; it also shows similar catalase (CAT) activity, capable of catalyzing H2O2 to H2O and O2, which may facilitate the decomposition of calcium peroxide in formulations.

[0007] Cell membrane coating technology, a biomimetic method for replicating cell membrane properties, is an active area of ​​research with applications in nanoscale biomedicine. Cell membrane-coated nanoparticles (NPs) combine the properties of natural cell membranes with those of artificial core materials. Coated NPs not only enhance their biocompatibility but also achieve efficient and prolonged circulation in vivo, enabling targeted delivery. Because the membrane coatings are structurally and functionally similar to host cells, they can express specific markers useful for NP delivery. Cancer cell membranes possess domains for adhesion to homologous cells and homology-binding proteins, which can provide tumor-targeting properties for nanoparticles coated with tumor cell membranes. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention proposes a biomimetic photoactive nanoparticle formulation for tumor therapy, its preparation method, and its application. The formulation combines nano-CaO2, PDA, and IR780 with cancer cell membranes to create a biomimetic nanoparticle formulation. This formulation enhances the photodynamic therapy (PDT) effect of IR780 by utilizing in-situ oxygen production from nano-calcium peroxide, thereby improving tumor efficacy. It also achieves controlled release of the formulation within the tumor through PDA acid response and utilizes cell membrane functionalization of the nanoparticles to achieve long-term in vivo circulation and tumor-targeted delivery. Furthermore, the resulting nanoparticle formulation exhibits high photodynamic and photothermal activity, good tumor-targeting characteristics and safety, and demonstrates good efficacy against tumors, showing broad prospects for application in tumor treatment.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A biomimetic photoactive nano-formulation CaO2@PDA-IR780@M (hereinafter collectively referred to as CPIC) for tumor treatment is characterized by having nano-CaO2 as the core, encapsulating PDA and loading IR780, and finally encapsulating it with a cell membrane.

[0011] The present invention also provides a method for preparing the aforementioned biomimetic photoactive nanoparticle formulation for tumor treatment, comprising the following steps:

[0012] S1: Dissolve CaCl2·6H2O in anhydrous ethanol, mix the ammonia solution with the CaCl2 solution by stirring; add hydrogen peroxide dropwise and continue stirring; after stirring is complete, collect the precipitate by centrifugation, wash it with anhydrous ethanol 2-4 times, and freeze-dry to obtain nano CaO2.

[0013] S2: After dispersing CaO2 in Tris-HCl buffer, add polydopamine reaction solution, and add iodide IR780 while stirring continuously. After centrifuging the mixture, collect the precipitate to obtain CaO2@PDA-IR780 (hereinafter collectively referred to as CPI).

[0014] S3: Culture 4T1 cells until they reach confluence. Add trypsin and follow digestion, digestion termination, and centrifugation steps. Collect the cells and resuspend them in a mixture of 1 / 4×PBS and 100×PMSF at a ratio of 100:1. Repeat the freeze-thaw cycle 4-6 times with the cell suspension. Centrifuge at low speed to remove any broken cells and organelles. Sonicate the supernatant containing the crude cell membrane. Shake the collected cell membrane suspension well, centrifuge, and collect the precipitate to obtain the purified cell membrane.

[0015] S4: Dissolve the purified cell membrane in PBS buffer, then redisperse the CPI obtained in step S2 in water, mix and stir continuously; after stirring, use an aqueous filter membrane with reduced pore size for continuous extrusion to obtain CPIC.

[0016] In one optional embodiment, in step S1, the mass ratio of CaCl2·6H2O, ammonia, and hydrogen peroxide is 1:(0.14~0.20):(0.32~0.36).

[0017] In one optional embodiment, in step S2, the mass ratio of IR780, CaO2 and polydopamine is 1:(7~11):(2~6).

[0018] In one optional embodiment, in step S2, the pH value of the Tris-HCl buffer solution is 8.3 to 8.7.

[0019] In one optional embodiment, in step S4, the continuous stirring time is 2-3 hours.

[0020] This invention also provides the application of nano-formulations in the preparation of drugs for treating tumors.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The biomimetic photoactive nano-formulation provided by the present invention can enhance the PDT efficacy of IR780, which is weakened by the hypoxic tumor microenvironment, by utilizing the in-situ oxygen production of nano-calcium peroxide, thereby improving the therapeutic effect on tumors.

[0023] (2) The biomimetic photoactive nano-formulation provided by the present invention utilizes PDA acid response to achieve controlled release in tumors, thereby improving bioavailability in tumor treatment.

[0024] (3) The biomimetic photoactive nanoparticles provided by the present invention utilize the cell membrane to functionalize the nanoparticles, giving them good biomimetic properties and enabling long-term circulation and tumor-targeted delivery in vivo. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the fabrication process of the CPIC of this invention;

[0026] Figure 2 These are TEM images of CaO2, CPIC, and phosphotungstic acid negatively stained CPIC according to the present invention;

[0027] Figure 3 This is a diagram showing the DLS particle size and ZETA potential of CaO2, CPI, and CPIC in this invention.

[0028] Figure 4 The DLS particle size distribution curves and FT-IR infrared transmission spectra of CaO2, CPI, and CPIC are shown.

[0029] Figure 5 A distribution diagram of CPIC's EDS elements;

[0030] Figure 6 This is a quantitative analysis chart of IR780, calcium, and protein contained in CPIC;

[0031] Figure 7 SDS-PAGE electrophoresis image of CPIC;

[0032] Figure 8 The image shows the UV and fluorescence spectra of the CPIC.

[0033] Figure 9 The ultraviolet spectrum of IR780 and CPIC after being mixed with DPBF and irradiated by an 808nm laser.

[0034] Figure 10 Real-time thermal imaging detection images of IR780 and CPIC;

[0035] Figure 11 Figure 1 shows the effect of different powers and concentrations on the photothermal activity of CPIC.

[0036] Figure 12 The absorption spectrum of the supernatant of the mixture of CPIC and red blood cell suspension;

[0037] Figure 13 Real-time thermal imaging detection images of CPI and CPIC;

[0038] Figure 14 This is a CPIC intracellular photothermal detection image;

[0039] Figure 15 The intracellular ROS oxygen fluorescence color of IR780, CPI, and CPIC;

[0040] Figure 16 In vivo NIR fluorescence imaging of mice after intravenous injection of CPIC;

[0041] Figure 17 Infrared images of mice after intravenous injection of IR780, CPI, and CPIC. Detailed Implementation

[0042] The technical solutions of the embodiments of this application will be further described clearly and completely below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0043] To make the inventive objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be further described in detail below with reference to the accompanying drawings: In order to better understand the above-mentioned objectives, features, and advantages of this invention, the advantages of this invention will be further illustrated below by comparing the embodiments with the accompanying drawings and specific implementation methods.

[0044] The preparation of the biomimetic photoactive nanoparticle formulation (CPIC) provided by this invention involves first preparing nano-CaO2 using CaCl2, NH3, and H2O2 as raw materials; then, combining CaO2 with PDA and IR780 to obtain CaO2@PDA-IR780; and finally, combining CPI with a cell membrane to obtain CPIC. The preparation route is as follows: Figure 1 As shown.

[0045] Example 1

[0046] This embodiment provides a method for preparing a biomimetic photoactive nanoparticle formulation that can be used for tumor treatment, including the following steps:

[0047] (1) Dissolve 0.1 g of calcium chloride hexahydrate (CaCl2·6H2O) in 150 mL of anhydrous ethanol. Mix 1.2 mL of 1 mol / L ammonia solution with the calcium chloride (CaCl2) solution under magnetic stirring for 10 min. Over 20 min, add 0.95 mL of hydrogen peroxide (1M) dropwise to the mixture. Continue stirring for 2 h while maintaining the stirring speed. After stirring, collect the precipitate by centrifugation (12000 rpm / min), wash twice with anhydrous ethanol, freeze-dry to obtain nano-CaO2, and disperse it in anhydrous ethanol.

[0048] (2) 580 μL of CaO2 (26.26 mM) was dispersed in 4.5 mL of Tris-HCl buffer (10 mM, pH 8.3), followed by the addition of 150 μL of polydopamine solution (4 mg / mL) and stirring at 1000 rpm. During continuous stirring, 20 μL of IR780 (5 mg / mL) was added. After 25 min, the product was collected by centrifugation at 12000 rpm for 10 min to obtain CPI. The CPI was redispersed in water for later use.

[0049] (3) Collect 1×10 8 Mouse triple-negative breast cancer cells (4T1 cells) were digested with trypsin for 3 minutes, and then culture medium was added to stop the digestion. After centrifugation at 1000 rpm for 3 minutes, the cell pellet was collected and resuspended in 1 mL of a 100:1 mixture of 1 / 4×PBS and 100×PMSF. The cell suspension was then frozen at -80°C for 20 minutes and thawed at 37°C, repeated 4 times. After repeated freeze-thaw cycles, the cells were centrifuged at 4°C at 500g for 5 minutes to remove unbroken cells and large aggregates of cell membrane pellet. The supernatant containing crude cell membrane was then used... After ultrasound with the probe (300W for 2 seconds, stop for 4 seconds), shake vigorously for 1 minute, centrifuge at 14000 rpm and 4℃ for 1 hour, and collect the precipitate to obtain the purified cell membrane.

[0050] (4) The purified cell membrane was dissolved in a small amount of PBS buffer (pH 7.4) and mixed with an equal volume of CPI aqueous dispersion. The mixture was stirred continuously for 2.5 h. After stirring, the CPIC was obtained by continuous extrusion using an aqueous filter membrane with reduced pore size (800 nm, 400 nm).

[0051] Example 2

[0052] This embodiment provides a method for preparing CPIC, a biomimetic photoactive nanoparticle formulation that can be used for tumor treatment, including the following steps:

[0053] (1) Dissolve 0.1 g CaCl2·6H2O in 150 mL of anhydrous ethanol. Mix 1 mL of 1 mol / L ammonia solution with the CaCl2 solution under magnetic stirring for 10 min. Over 20 min, add 1 mL of hydrogen peroxide (1 M) dropwise to the mixture. Continue stirring for 2 h while maintaining the stirring speed. After stirring, collect the precipitate by centrifugation (12000 rpm / min), wash it three times with anhydrous ethanol, and freeze-dry it to obtain nano-CaO2, which is then dispersed in anhydrous ethanol.

[0054] (2) Disperse 500 μL CaO2 (26.26 mM) in 4.5 mL Tris-HCl buffer (10 mM, pH 8.5), then add 100 μL polydopamine solution (4 mg / mL) and stir at 1000 rpm. During continuous stirring, add 20 μL IR780 (5 mg / mL). After 25 min, collect the precipitate by centrifugation at 12000 rpm for 10 min to obtain CPI. Redisperse CPI in water for later use.

[0055] (3) Collect 1×10 8 4T1 cells were digested with trypsin for 3 minutes, and then culture medium was added to stop the digestion. After centrifugation at 1000 rpm for 3 minutes, the cell pellet was collected and resuspended in 1 mL of a 100:1 mixture of 1 / 4×PBS and 100×PMSF. The cell suspension was then frozen at -80°C for 20 minutes and thawed at 37°C, repeated 5 times. After repeated freeze-thaw cycles, the cells were centrifuged at 500g for 5 minutes at 4°C to remove unbroken cells and large aggregates of cell membranes. The supernatant containing the crude cell membrane was then used... After ultrasound with the probe (300W for 2 seconds, stop for 4 seconds), shake vigorously for 1 minute, centrifuge at 14000 rpm and 4℃ for 1 hour, and collect the precipitate to obtain the purified cell membrane.

[0056] (4) The purified cell membrane was dissolved in a small amount of PBS buffer (pH 7.4) and mixed with an equal volume of CPI aqueous dispersion. The mixture was stirred continuously for 2 hours. After stirring, the CPIC was obtained by continuous extrusion using an aqueous filter membrane with reduced pore size (800 nm, 400 nm).

[0057] Example 3

[0058] This embodiment provides a method for preparing a biomimetic photoactive nanoformulation CaO2@PDA-IR780@M that can be used for tumor treatment, including the following steps:

[0059] (1) Dissolve 0.1 g CaCl2·6H2O in 150 mL of anhydrous ethanol. Mix 0.8 mL of 1 mol / L ammonia solution with the CaCl2 solution under magnetic stirring for 10 min. Over 20 min, add 1.05 mL of hydrogen peroxide (1 M) dropwise to the mixture. Maintain stirring speed and continue stirring for 2 h. Collect the precipitate after centrifugation (12000 rpm / min), wash it 4 times with anhydrous ethanol, and freeze-dry it to obtain nano-CaO2, which is then dispersed in anhydrous ethanol.

[0060] (2) 400 μL CaO2 (26.26 mM) was dispersed in 4.5 mL Tris-HCl buffer (10 mM, pH 8.7), followed by the addition of 50 μL polydopamine solution (4 mg / mL) and stirring at 1000 rpm. During continuous stirring, 20 μL IR780 (5 mg / mL) was added. After 25 min, the product was collected by centrifugation at 12000 rpm for 10 min to obtain CPI. The CPI was redispersed in water for later use.

[0061] (3) Collect 1×10 8 4T1 cells were digested with trypsin for 3 minutes, and then culture medium was added to stop the digestion. After centrifugation at 1000 rpm for 3 minutes, the cell pellet was collected and resuspended in 1 mL of a 100:1 mixture of 1 / 4×PBS and 100×PMSF. The cell suspension was then frozen at -80°C for 20 minutes and thawed at 37°C, repeated 6 times. After repeated freeze-thaw cycles, the cells were centrifuged at 500g for 5 minutes at 4°C to remove unbroken cells and large aggregates of cell membranes. The supernatant containing the crude cell membrane was then used... After ultrasound with the probe (300W for 2 seconds, stop for 4 seconds), shake vigorously for 1 minute, centrifuge at 14000 rpm and 4℃ for 1 hour, and collect the precipitate to obtain the purified cell membrane.

[0062] (4) The purified cell membrane was dissolved in a small amount of PBS buffer (pH 7.4), mixed with an equal volume of CPI aqueous dispersion, and stirred continuously for 3 hours. After stirring, the membrane was continuously extruded using an aqueous filter membrane with reduced pore size (800 nm, 400 nm) to obtain CPIC.

[0063] Examples 1-3 were subjected to CPIC characterization and related trials to assess their efficacy against tumors.

[0064] The specific contents of the experiment are as follows:

[0065] I. Transmission electron microscopy characterization of CPIC

[0066] After diluting the CaO2 and CPIC obtained in Example 2 to appropriate concentrations, 10 μL of the CaO2 suspension was dropped onto a copper grid; then 10 μL of the CPIC suspension was dropped onto two separate copper grids. 10 μL of phosphotungstic acid negative staining solution was added to one of the copper grids, and the grid was allowed to stand for 1 minute to allow the negative staining solution to fully cover and penetrate the sample. Excess negative staining solution was then absorbed again with filter paper. After drying, the copper grid containing the sample was placed inside a TEM machine, and its microstructure was observed.

[0067] Figure 2 Figure a shows a TEM image of CaO2, figure b shows a TEM image of CPIC, and figure c shows a partial image of CPIC stained with phosphotungstic acid. Figure a shows that the synthesized CaO2 has a stable structure and uniform size. After modification with PDA and IR780 and subsequent cell membrane coating, the resulting particles are shown in figure b. It can be seen that the modified particles have increased size and a significant contrast between the core and shell. Analysis of figure c shows that phosphotungstic acid negative staining enhances the contrast of the membrane structure, revealing that the particles exhibit a ring-shaped membrane structure.

[0068] II. Characterization of CPIC by DLS and Zeta potentials

[0069] The CaO2, CPI, and CPIC obtained in Example 2 were diluted to appropriate concentrations and then measured using a particle size analyzer.

[0070] Figure 3 Figure a shows the dynamic light scattering (DLS) particle size distribution of CaO2, CPI, and CPIC, and Figure b shows the Zeta potential distribution of CaO2, CPI, and CPIC. Figure 3 The average particle size of CaO2 is 37.67±6.93 nm, and its Zeta potential is 44.93±0.9182 mV; the average particle size of CPI is 177.8±11.191 nm, and its Zeta potential is -35.10±3.495 mV; the average particle size of CPIC is 236.67±22.0967 nm, and its Zeta potential is -31.56±7.635 mV. The Zeta potentials of CPI and CPIC are greater than 30 mV, indicating that the system is very stable and the particles are difficult to aggregate due to strong electrostatic repulsion.

[0071] III. Verification of CPIC synthesis process using DLS and FT-IR characterization

[0072] After diluting the CaO2, CPI, and CPIC obtained in Example 2 to appropriate concentrations, particle size distribution curves were obtained using a particle size analyzer, and the differences in particle size distribution were compared.

[0073] The CaO2, CPI, CPIC, and KBr obtained in Example 2 were weighed at a mass ratio of 1:50, and ground in an agate mortar for 10 minutes until the mixture was a uniform mist with no visible particles. The mixture was then placed into a mold and pressed under 6 tons of pressure for 23 minutes to form a transparent sheet. The prepared sheets were then sequentially placed in an FT-IR spectrometer to collect infrared transmission spectra.

[0074] Figure 4 Figure a shows the particle size distribution curves of CaO2, CPI, and CPIC, while Figure b shows the infrared transmission spectra of CaO2, CPI, and CPIC. Figure a shows that each synthesis step from CaO2 to CPI and then to CPIC leads to an increase in particle size, reflecting the controllable composite material composition in terms of physical size. Figure b shows that CaO2 and CPI each have characteristic infrared absorption peaks, representing their inherent chemical structures; the absorption peak of CPIC integrates the characteristic peaks of CaO2 and CPI, and also shows absorption by functional groups of cell membrane components, indicating that CPI has successfully composited with the cell membrane, and there is an interaction in their chemical structures.

[0075] IV. Determination of elemental composition and spatial distribution characterization of CPIC

[0076] The CPIC solution obtained in Example 2 was diluted, and a drop was placed on a carbon film copper grid. After the liquid evaporated, the morphology was observed under a transmission electron microscope. A Super X-ray spectrometer was turned on in the TEM system, and the electron beam was focused on the target nanoparticle region. Elemental distribution scanning was performed on the selected CPIC particle region, and characteristic X-ray signals of Ca, O, N, and Cl were acquired to generate single-element distribution images. These images were then superimposed to obtain a Merge plot, and the uniformity of elemental distribution and co-location within the particles were analyzed.

[0077] Figure 5 The energy dispersive spectroscopy (EDS) elemental distribution map of CPIC shows that the red region (Ca) exhibits a distinct spherical distribution, indicating that calcium is the core component of this material and is evenly distributed. The yellow region (O) highly overlaps with the distribution of Ca, and combined with the common compound forms of calcium, this indicates that oxygen and calcium form the main compound phase. The green spots (N) are dispersed within the spherical region, indicating that N exists in the material in a composite form. The purple region (Cl) is also evenly distributed within the spherical region, and combined with other elements, it can be seen that Cl exists in the form of calcium chloride, playing a modifying role. When the distributions of Ca, O, N, and Cl are superimposed, a complete spherical morphology is formed, indicating that these four elements are co-distributed within the micro-regions of CPIC. That is, CPIC is a composite nanomaterial composed of calcium, oxygen, nitrogen, and chlorine, with each element uniformly mixed at the microscale.

[0078] V. Quantification of the content of each component of CPIC

[0079] A series of IR780 concentrations were prepared, and a standard curve for IR780 content was established for subsequent IR780 quantification. For example... Figure 6 As shown, Figure 6 Figures a and b in the figure are the standard curve and UV-Vis absorption spectrum of IR780, respectively, showing a good linear relationship between absorbance and concentration.

[0080] Using a colorimetric assay kit for calcium content, the calcium content is quantified according to a calcium content standard curve. For example... Figure 6 As shown, Figure 6 Figure c in the figure shows the standard curve for calcium content, with optical density (OD) at a wavelength of 575 nm. 575 The α value is linearly correlated with calcium content.

[0081] Using the BCA protein concentration assay kit, the protein content was quantified according to the bovine serum albumin (BSA) standard curve, and then a subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment was performed based on the protein quantification. Figure 6 The d-plot in the figure is the BSA standard curve, which shows the absorbance at a wavelength of 562 nm (A). 562 The value is linearly correlated with the BSA concentration. Figure 7 The SDS-PAGE electrophoresis image shows that the coated nanoparticles have a similar band pattern to the original cell membrane protein bands, with molecular weights of around 53 kDa.

[0082] VI. Determination of CIPC UV and Fluorescence Characterization

[0083] The ultraviolet and fluorescence spectra of the CPIC obtained in Example 2 were detected using an ultraviolet spectrophotometer and a fluorescence spectrophotometer (Ex 750 nm).

[0084] like Figure 8 As shown, Figure 8Figure a shows the UV-Vis absorption spectra of CaO2, IR780, CPI, and CPIC, while Figure b shows the fluorescence emission spectra of IR780 and CPIC. Figure a shows that IR780 and CPIC exhibit strong absorption peaks around 750 nm, CPI's absorption peak is at a different position, and CaO2 shows almost no significant absorption. CPIC retains the near-infrared characteristic absorption of IR780, indicating that IR780 successfully participated in the construction of CPIC. Figure b shows that IR780 exhibits a characteristic fluorescence emission peak around 800 nm after excitation. After excitation, the fluorescence peak position of CPIC is basically consistent with that of IR780, and the fluorescence intensity is significantly higher than that of IR780, indicating that the fluorescence properties of IR780 in CPIC are effectively retained and enhanced due to the composite structure. In summary, it can be concluded that CPIC successfully composites with the near-infrared photosensitizer IR780, while retaining its near-infrared light absorption and fluorescence emission characteristics.

[0085] VII. Determination of the photodynamic and photothermal activity of CIPC

[0086] Using 1,3-diphenylisobenzofuran (DPBF) as a probe, deionized water (DW), IR780, and the CPIC obtained in Example 2 were mixed with DPBF, with DW serving as a negative control. An 808 nm laser (1 W / cm²) was used. 2 Irradiation was performed, and samples were taken at pre-set points to measure its absorption spectrum in the range of 350~500 nm. DPBF and singlet oxygen ( 1 When O2 reacts, it has a characteristic absorption peak at a specific wavelength (around 410 nm). The amount of oxygen produced in the system can be calculated by measuring the attenuation intensity of this characteristic peak. 1 The more O2 absorbs decrease, the more it produces. 1 The more O2, the better.

[0087] Figure 9 Figures a, b, and c show the UV spectra of DW, IR780, and CPIC mixed with DPBF after irradiation with an 808nm laser, respectively. It can be seen that the absorbance of the characteristic peak of the IR780 mixed with DPBF decreases significantly faster over time than that of the DW mixed with DPBF, and the decrease is more pronounced in the later stages, indicating good photodynamic activity. The characteristic peak of the CPIC mixed with DPBF decreases most rapidly over time compared to IR780 and DW, producing... 1 O2 is more efficient and has better photodynamic activity.

[0088] An infrared thermal imaging system was used to evaluate the photothermal properties of CPIC. A series of CPIC solutions at different concentrations (IR780 concentrations of 5, 10, 20, 30, 40, and 50 μg / mL) were prepared and analyzed using an 808 nm laser (1 W / cm²). 2Irradiation was performed, and the temperature of the solution was recorded every 30 seconds using an infrared thermal imager. The temperature change of DW under the same conditions was used as a negative control. Figure 10 This is a real-time thermal imaging detection image of CPIC, provided by... Figure 10 As shown, both the IR780 and CPIC heat up rapidly starting from 1 minute of laser irradiation, and the CPIC heats up at a higher and faster rate than the IR780.

[0089] In addition, an 808nm laser was used as the light source, with wavelengths of 0.6, 1, and 1.4 W / cm², respectively. 2 The CPIC solution was irradiated with high power, and the effect of laser intensity on the temperature change of the solution was recorded and analyzed. For example... Figure 11 As shown, Figure 11 Figure a shows the effect of different concentrations on the photothermal activity of CPIC, and Figure b shows the effect of different powers on the photothermal activity of CPIC. It can be seen that the concentration of CPIC is linearly positively correlated with the heating rate, and the heating rate increases linearly with increasing power. This indicates that the higher the concentration and the greater the laser power density, the more significant the photothermal heating effect of CPIC. Since photothermal conversion relies on non-radiative transitions, it is a good organic photothermal material.

[0090] VIII. Evaluation of CPIC in vitro safety

[0091] Healthy BALB / c mice of similar size and conformation were selected, and whole blood samples were collected from the orbital region of the mice. After collection, the blood samples were centrifuged at 3500 rpm for 15 min to separate the supernatant plasma. The collected red blood cells were washed twice with 1×PBS and diluted to a 4% suspension (v / v).

[0092] Each red blood cell suspension was mixed with 0.5 mL of 0.9% NaCl (negative control), 0.5 mL of DW (positive control), and 0.5 mL of a series of concentrations of CPIC obtained in Example 2. All samples were incubated in a laboratory water bath at 37°C for 3 h. Then, the samples were centrifuged at 4000 rpm for 10 min, and the supernatant was analyzed at 540 nm using a microplate reader (Thermo, USA).

[0093] The hemolysis rate (HR) is calculated using the following formula:

[0094]

[0095] Where A is the absorbance at 540 nm.

[0096] like Figure 12As shown, the absorption spectra of the supernatant after mixing CPIC with red blood cells at various concentrations are basically the same as those of the supernatant after mixing 0.9% NaCl with red blood cells, indicating that CPIC does not damage the red blood cell structure and induce hemolysis when it comes into contact with blood, and has good in vitro safety.

[0097] IX. Evaluation of the intracellular photothermal properties of CPIC

[0098] 4T1 cells (5×10) 5 Cells were seeded in 12-well plates and allowed to adhere overnight. The medium was then replaced with fresh medium containing CPI and CPIC (IR780 concentration 3 μg / mL) obtained in Example 2 and incubated for 12 h. Cells were then collected by trypsin digestion and resuspended in fresh RPMI 1640 medium. All cell suspensions were heated under an 808 nm laser (1 W / cm²). 2 Irradiate the solution for 3 minutes, and record the temperature of the solution every 30 seconds using an infrared thermal imager. Use the temperature change of the untreated group under the same conditions as a negative control.

[0099] Figure 13 The graph shows the intracellular photothermal activity of CPI and CPIC. As can be seen, the temperature of CPI increases with illumination time, reaching a plateau after 120 seconds of illumination; the temperature of CPIC increases rapidly, reaching approximately 55℃ after about 90 seconds of illumination, and then shows no significant decrease after reaching the plateau. This indicates that CPIC has higher photothermal conversion efficiency and better thermal stability, and its intracellular photothermal activity is significantly better than that of CPI.

[0100] Figure 14 The images show real-time thermal imaging detection of CPI and CPIC. As can be seen from the images, the color of CPI gradually transitions to orange with the increase of illumination time, and the range and brightness of the high-temperature area gradually stabilize. In contrast, the color of CPIC rapidly transitions to orange-yellow, and the brightness and range of the high-temperature area are more significant, indicating that the intracellular photothermal conversion efficiency of CPIC is significantly higher than that of CPI.

[0101] 10. Evaluation of intracellular photodynamic properties (ROS) of CPIC

[0102] 4T1 cells (3×10) 5 Cells (cells / well) were seeded in confocal dishes for 12 h. The cell culture medium was removed, and an appropriate volume of diluted DCFH-DA fluorescent probe was added. The cells were incubated at 37°C for 20 min, and washed three times with PBS to thoroughly remove any uncontaminated DCFH-DA. IR780, CPI obtained in Example 2, CPIC (IR780 concentration 3 μg / mL), and PBS were added, and the cells were incubated for 12 h. The cells were then treated with an 808 nm laser (1 W / cm²). 2Irradiate each well for 30 s. Fix cells with 4% paraformaldehyde for 7 min, and then irradiate with 1 mL of the DNA fluorescent dye DAPI (10 μg / mL). -1 The cells were then labeled. They were then washed twice and excited using a 488 nm excitation wavelength and a 525 nm emission wavelength.

[0103] Figure 15 The image shows the fluorescence color of intracellular reactive oxygen species (ROS) in IR780, CPI, and CPIC cells. The location of green fluorescence (DCFH-DA) indicates the ROS production area. Combined with the observation of blue fluorescence (DAPI), it can be seen that the fluorescence intensity of CPIC cells is significantly stronger than that of IR780 and CPI, indicating that CPIC can generate a large amount of intracellular ROS and has good photodynamic activity.

[0104] XI. Determination of CPIC targeting in mice

[0105] In vivo NIR fluorescence imaging was performed on BALB / c mice with tumors derived from 4T1 cells. When the tumor reached approximately 100 mm... 3 The CPIC (IR780 1.5 mg·kg) obtained in Example 2 was used. -1 The drug was intravenously injected into mice. Fluorescence images of the mice were acquired at different time points using the Kino system (excitation: 710 nm, emission: 770 nm, exposure time: 30 s). Figure 16 As shown, 4–10 hours after intravenous injection, CPIC is distributed throughout the body via blood circulation; 22–48 hours after injection, the targeted therapy of CPIC takes effect in the body, and non-targeted tissues begin to be cleared; 72–96 hours after injection, CPIC is gradually metabolized and cleared from the body. It can be concluded that CPIC has good targeting specificity, reasonable time-kinetic characteristics, and has the potential for clinical translation.

[0106] XII. Evaluation of the photothermal effect of CPIC in vivo

[0107] Select tumor-bearing mice of similar size and shape, and when the tumor volume reaches 100 mm... 3 At the same time, CPI, CPIC and IR780 (IR780, 1.5 mg·kg) obtained in Example 2 were administered intravenously. -1 Control group mice were treated with saline. 32 hours after tail vein administration, 808nm laser (1.2W / cm²) was applied. 2 Irradiate for 10 minutes, capturing infrared images every 5 seconds using an infrared imaging device (FLIR-PRO). Record temperature changes using a visual infrared thermometer (FLIR Tools).

[0108] Figure 17Infrared images of mice intravenously injected with IR780, CPI, and CPIC are shown. As can be seen from the images, both CPI and CPIC showed bright yellow high-temperature areas after 3 minutes of light exposure. The high-temperature areas expanded and the temperature increased over time, indicating that both CPI and CPIC have certain photothermal activity.

[0109] However, after 6 minutes of light exposure, the high-temperature region of CPIC was more concentrated and the temperature was higher, indicating that the photothermal activity of CPIC in vivo was significantly better than that of CPI. Furthermore, CPIC coated with tumor cell membranes could target tumors and had good tumor-targeting properties.

[0110] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0111] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A method for preparing a biomimetic photoactive nanoparticle formulation CaO2@PDA-IR780@M for tumor therapy, characterized in that, Includes the following steps: S1: Dissolve CaCl2·6H2O in 150mL of anhydrous ethanol, mix the ammonia solution with the CaCl2 solution by stirring; add hydrogen peroxide dropwise and continue stirring; after stirring is complete, collect the precipitate by centrifugation, wash it with anhydrous ethanol 2-4 times, and freeze-dry it to obtain nano-CaO2; wherein the mass ratio of CaCl2·6H2O, ammonia and hydrogen peroxide is 1:(0.14~0.20):(0.32~0.36); S2: CaO2 was dispersed in Tris-HCl buffer solution with a pH of 8.3~8.7 and then added to polydopamine reaction solution. Iodide IR780 was added while stirring continuously. After the reaction was completed, the precipitate was collected by centrifugation to obtain CaO2@PDA-IR780. The mass ratio of IR780, CaO2 and polydopamine was 1:(7~11):(2~6). S3: Culture 4T1 cells until they reach confluence. Add trypsin, digest and centrifuge, collect the cells, and resuspend them in a mixture of 1 / 4×PBS and 100×PMSF at a ratio of 100:

1. Repeat the freeze-thaw cycle 4-6 times, centrifuge at low speed to remove unbroken cells and organelles, and sonicate the supernatant containing crude cell membrane. Shake the collected cell membrane suspension well, centrifuge, collect the precipitate, and obtain the purified cell membrane. S4: Dissolve the purified cell membrane in PBS buffer, then redisperse the CaO2@PDA-IR780 obtained in step S2 in water, mix and stir continuously; after stirring, use an aqueous filter membrane with reduced pore size for continuous extrusion to obtain CaO2@PDA-IR780@M.

2. The preparation method of the biomimetic photoactive nano-formulation CaO2@PDA-IR780@M for tumor treatment according to claim 1, characterized in that, In step S4, the continuous stirring time is 2-3 hours.

3. The application of a biomimetic photoactive nanoparticle preparation obtained by any one of the preparation methods described in claims 1-2 in the preparation of drugs for treating tumors.

Images