Preparation and application of biomimetic cell membrane modified photosensitizer

Abstract

The invention discloses preparation and application of a biomimetic cell membrane modified photosensitizer. A tumor cell membrane bionic camouflage technology is adopted, the surface of the photosensitizer nanoparticle is coated with the tumor cell membrane through repeated membrane extrusion or ultrasound, the photosensitizer-coated tumor cell membrane bionic nanoparticle is prepared, the preparation process is simple, the cost is low, and a complex synthesis process is not needed. The bionic nanoparticle disclosed by the invention has the functions of homologous targeting and immune escape, and can realize near-infrared two-region imaging. In addition, the bionic nanoparticles can kill tumor cells through photo-thermal and photodynamic effects, and have the advantages of good curative effect and low toxic and side effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation of photosensitizer nanoparticles coated on tumor cell membranes. These biomimetic nanoparticles have homologous targeting and immune escape functions, and can achieve near-infrared II (NIR-II) imaging. They can also synergistically kill tumors through photothermal and photodynamic effects. Background Technology

[0002] Optical therapy for tumor treatment relies on the active or passive targeted delivery of photosensitizers to the tumor site. Under near-infrared laser excitation, light energy is effectively converted into heat energy to raise the local temperature and kill cancer cells (photothermal therapy) or reactive oxygen species (ROS) are generated at the tumor site to directly kill tumor cells (photodynamic therapy). Compared with surgical treatment, radiotherapy, and chemotherapy, photothermal therapy and photodynamic therapy have higher tumor ablation efficiency and relatively fewer side effects. Fluorescence imaging refers to the ability to track the in vivo circulation process of nanomaterials through their own fluorescence properties, enabling non-destructive, real-time in vivo imaging at the cellular and molecular levels. Excitation light in the second near-infrared (NIR-II) window effectively avoids interference from autofluorescence and tissue light scattering, providing higher spatial resolution and signal-to-noise ratio, making near-infrared imaging a promising technology in the biomedical field.

[0003] Tumor cell membranes possess homologous binding properties, attributed to surface plasma membrane proteins, including neurotrophic proteins, galactosidase-binding proteins, and epithelial cell adhesion molecules. Photosensitizers modified with tumor cell membranes retain their original structural properties while incorporating the biological characteristics of the cell membrane. On one hand, laser irradiation can rupture the cell membrane, releasing nanoparticles and drugs at the tumor site; on the other hand, they can evade immune clearance, highly specifically targeting tumor tissue and increasing accumulation at the tumor site. Depending on the disease, specific therapeutic drugs and one or more biological cell membranes can be selected, and this flexible combination strategy demonstrates enormous application potential. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing and applying a tumor cell membrane modification photosensitizer.

[0005] Specifically, the present invention is achieved through the following technical solution:

[0006] The first aspect of the present invention provides a photosensitizer having the structure shown in formula (I):

[0007]

[0008] in:

[0009] n represents the degree of polymerization of the polymer molecule, and its value is in the range of 2 to 10, preferably 3 to 5;

[0010] x is 1-50, preferably 5-20, more preferably 5-10, and even more preferably 5-8.

[0011] The present invention also provides a specific photosensitizer with the structure shown in TPB-TT, wherein x = 6 and n = 3 ~ 5:

[0012] .

[0013] A second aspect of the present invention provides a tumor cell membrane biomimetic nanoparticle loaded with a photosensitizer, wherein the photosensitizer described in the present invention is coated with a tumor cell membrane to form the tumor cell membrane biomimetic nanoparticle.

[0014] The tumor cell membrane described in this invention can be adaptively selected according to the specific needs of tumor treatment, including but not limited to one or more of the following: breast cancer cell membrane, lung cancer cell membrane, bladder cancer cell membrane, melanoma cell membrane, cervical cancer cell membrane, colorectal cancer cell membrane, or gastric cancer cell membrane. The melanoma cell membrane can be the choroidal melanoma cell membrane. Adaptive selection of the cell membrane can enable the nanoparticles to possess homologous targeting and immune evasion functions, helping to prolong in vivo circulation time and achieve higher tumor tissue targeting.

[0015] The third aspect of the present invention provides a method for preparing a photosensitizer-loaded tumor cell membrane biomimetic nanoparticle, wherein the photosensitizer described in the present invention is co-precipitated with a surfactant to form photosensitizer nanoparticles, which are then mixed with a tumor cell membrane in a certain proportion and encapsulated using membrane encapsulation technology to form the photosensitizer-loaded tumor cell membrane biomimetic nanoparticle.

[0016] The surfactant used in the preparation method of this invention can be a commonly used surfactant for the preparation and functionalization of nanoparticles in the art, such as, but not limited to, DSPE-mPEG2000. The specific method for forming the photosensitizer aqueous solution can be a conventional method in the art. For example, in a specific example, the steps are as follows: a certain proportion of photosensitizer and surfactant are thoroughly mixed, and then dropped into ultrapure water under stirring. After the organic solvent has fully evaporated, the mixture is filtered using a 0.22 µm aqueous filter membrane. The filtrate is concentrated by a 100 kDa ultrafiltration centrifuge tube to obtain photosensitizer nanoparticles.

[0017] In some embodiments, the weight ratio of the photosensitizer to the surfactant described in this invention is 1:(5~15); preferably 1:10.

[0018] In some embodiments, the mass ratio of the photosensitizer nanoparticles to the cell membrane of the present invention is 1:(1~100), preferably 1:(1~10); more preferably 1:5.

[0019] The membrane encapsulation technology described in this invention can be a conventional technology in the field, such as membrane extrusion or ultrasound. The method can be a conventional method in the field. For example, in a specific example, it includes the steps of: mixing photosensitizer nanoparticles and tumor cell membranes in a certain proportion and then sonicating in a water bath; then extruding the above mixture in a 400 nm polycarbonate membrane using a liposome extruder; and finally extruding in a 200 nm polycarbonate membrane to obtain photosensitizer nanoparticles coated with tumor cell membranes.

[0020] The method for preparing photosensitizer-loaded biomimetic nanoparticles for tumor cell membranes according to the present invention, wherein the preparation of the tumor cell membrane can be a conventional method in the art, for example including the steps of: digesting and collecting cells in the logarithmic growth phase, lysing and sonicating them, and then passing the lysate through a 4-channel filter. o Centrifuge at low speed (700 g × 10 min) at C to remove precipitate; supernatant is then subjected to 4... o Centrifuge at 4°C (10000 g × 20 min) to remove precipitate; supernatant is then subjected to 4°C high-speed centrifugation. o Centrifuge at 150,000 g for 40 min, collect the precipitate (cell membrane), wash with deionized water, resuspend the cell membrane precipitate in deionized water, aliquot and store at -80°C. o Save C for later use.

[0021] The fourth aspect of this invention also provides the application of the photosensitizer described in this invention or the tumor cell membrane biomimetic nanoparticles loaded with the photosensitizer described in this invention in near-infrared II imaging and anti-tumor activity.

[0022] The present invention also provides the application of the photosensitizer described in the present invention or the tumor cell membrane biomimetic nanoparticles loaded with the photosensitizer described in the present invention in the field of vascular imaging.

[0023] The fifth aspect of the present invention also provides the use of the photosensitizer described above or the tumor cell membrane biomimetic nanoparticles containing the photosensitizer described above in the preparation of drugs for treating tumors.

[0024] The sixth aspect of this invention also provides the application of the aforementioned photosensitizer or the tumor cell membrane biomimetic nanoparticles loaded with the photosensitizer described in this invention, in combination with photothermal or photodynamic therapy, in the preparation of drugs for treating tumors. These biomimetic nanoparticles can enhance targeting to tumor tissues using the EPR effect and homology targeting, and subsequently, through the photothermal and photodynamic effects of the photosensitizer, effectively inhibit tumor growth and proliferation.

[0025] The tumors described in this invention include breast cancer, lung cancer, bladder cancer, melanoma, cervical cancer, colorectal cancer, and gastric cancer. Specifically, the tumor cell membrane biomimetic camouflage technology of this invention can be used to coat the cell membrane of the target tumor onto the surface of photosensitizer nanoparticles through membrane encapsulation technology, thereby obtaining tumor cell membrane biomimetic nanoparticles loaded with photosensitizer.

[0026] The present invention has the following beneficial results:

[0027] (1) The preparation process of the photosensitizer-loaded biomimetic nanoparticles described in this invention is simple, low-cost, and does not require a complex synthesis process. The cell membrane encapsulation process is simple, and the stability and reproducibility are high.

[0028] (2) The biomimetic photosensitizer nanoparticles described in this invention, after being coated with tumor cell membranes, have the functions of homologous targeting and immune escape, which helps to prolong the in vivo circulation time and achieve high tumor tissue targeting.

[0029] (3) The biomimetic photosensitizer nanoparticles described in this invention can achieve near-infrared II imaging, enabling imaging of blood vessels throughout the body. They have advantages such as being non-invasive, highly sensitive, and easy to operate, and have important clinical application value: a. Surgical navigation prospects: With excellent imaging signal-to-noise ratio, they can clearly distinguish living microcapillaries, which is expected to promote the development of new surgical navigation contrast agents; b. Pharmacokinetic analysis: They can monitor the dynamic distribution and metabolic process of drugs in animals in real time; c. Diagnosis of ophthalmic diseases: With the advantage of high resolution, they are expected to achieve retinal microvascular imaging, which can help the early diagnosis and follow-up of diseases such as diabetic retinopathy and age-related macular degeneration; d. Drug safety evaluation: They can monitor the potential toxicity of drugs to the microcirculation of important organs such as the liver and kidneys, providing a basis for preclinical safety assessment.

[0030] (4) The biomimetic nanoparticles loaded with photosensitizers described in this invention can kill tumors synergistically through photothermal and photodynamic effects, and have the advantages of good efficacy and low toxicity. Attached Figure Description

[0031] Figure 1 This is the synthesis route diagram for TPB-TT;

[0032] Figure 2 These are particle size distribution diagrams for TPB-TT NPs and CM-TPB-TT NPs;

[0033] Figure 3 These are potential diagrams for TPB-TT NPs and CM-TPB-TT NPs;

[0034] Figure 4 It is the particle size stability of CM-TPB-TT NPs;

[0035] Figure 5 This is a graph showing the membrane protein assays of TPB-TT NPs and CM-TPB-TT NPs;

[0036] Figure 6 These are the absorption spectra of TPB-TT NPs and CM-TPB-TT NPs;

[0037] Figure 7 These are the fluorescence spectra of TPB-TT NPs and CM-TPB-TT NPs;

[0038] Figure 8 This is a graph showing the photothermal capabilities of TPB-TT NPs and CM-TPB-TT NPs;

[0039] Figure 9 The photo-induced reactive oxygen species generation performance of TPB-TT NPs and CM-TPB-TT NPs;

[0040] Figure 10 It refers to the cellular uptake of TPB-TT NPs and CM-TPB-TT NPs;

[0041] Figure 11 The dark toxicity and phototoxicity of TPB-TT NPs and CM-TPB-TT NPs;

[0042] Figure 12 It is an in vivo fluorescence imaging image;

[0043] Figure 13 This is a graph showing changes in tumor volume;

[0044] Figure 14 It's a survival rate chart. Detailed Implementation

[0045] 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.

[0046] Example 1: Synthesis of compound TPB-TT

[0047] Compound 1 (112 mg) and compound 2 (36 mg) were dissolved in 15 mL of ultra-dry toluene in a three-necked flask. After removing oxygen from the solvent by three gas purgings, the catalyst tetratetraphenylphosphine palladium (Pd(PPh3)4, 4.8 mg) was rapidly added to the three-necked flask, and the product TPB-TT was synthesized and purified by Stille coupling reaction. Figure 1), where x = 6 and n = 3 ~ 5. 1 H NMR (400 MHz, CDCl3) δ 8.67 (s, 4H), 8.19 – 7.60 (m, 4H), 7.48 (d, J =30.0 Hz, 4H), 4.69 (s, 4H), 3.44 – 2.60 (m, 4H), 1.94 (d, J = 70.4 Hz, 15H), 1.25 (s, 94H), 0.98 – 0.79 (m, 21H), 0.72 (d, J = 7.5 Hz, 12H). GPC: M n =4056, M w = 4186, PDI=1.03.

[0048] Example 2: Preparation and characterization of CM-TPB-TT NPs

[0049] Compound TPB-TT (0.5 mg) and DSPE-mPEG 2000 (5 mg) were co-precipitated to obtain a clear aqueous solution of TPB-TT nanoparticles NPs. The nanoparticles were then concentrated by ultrafiltration and placed at 4°C. o Preservation. Collect choroidal melanoma cells (MUM-2B) in the logarithmic growth phase, lyse the cells by sonication, and then collect the cell membrane CMs by low-temperature differential centrifugation and low-temperature ultracentrifugation. Resuspend the cell membrane in PBS (5 mg / mL) and fuse it with TPB-TT NPs in PBS solution (1 mg / mL) by sonication. Then, remove large particles by low-temperature low-speed centrifugation. Collect the precipitate CM-TPB-TT NPs by low-temperature ultracentrifugation, wash with PBS, and incubate at -80°C. o C-aliquots were stored. Particle size analysis showed that the particle size increased by 27 nm after coating the MUM-2B cell membrane with CMs. Figure 2 Both surfaces are negatively charged. Figure 3 The particle size of CM-TPB-TT NPs did not change significantly within 10 days, indicating that CM-TPB-TT NPs have good stability. Figure 4 Protein electrophoresis analysis revealed that the cell membrane-encapsulated nanoparticles CM-TPB-TT NPs contained proteins essentially identical to those of CMs, while TPB-TT NPs alone were not detected. This indicates that CMs successfully encapsulated TPB-TT NPs nanoparticles. Figure 5 ).

[0050] Example 3: Absorption Spectroscopy Measurement

[0051] The THF solution of TPB-TT exhibits near-infrared absorption peaks, with the highest absorption peak located at 696 nm. After preparation into nanoparticles, the absorption peak of the TPB-TT NPs aqueous solution shows a significant red shift relative to the TPB-TT molecules and splits into a doublet, demonstrating that molecular aggregation occurs in the aqueous solution, and intermolecular charge transfer interactions lead to the change in absorption peaks. Furthermore, the absorption spectra of CM-TPB-TT NPs are essentially consistent with those of TPB-TT NPs, exhibiting the highest absorption peak at 808 nm. Figure 6 ).

[0052] Example 4: Emission Spectroscopy Measurement

[0053] Fluorescence emission spectroscopy showed that after absorbing light energy, TPB-TT NPs release a portion of the energy through fluorescence, generating a fluorescence signal located in the near-infrared II region at 930 nm, providing support for in vivo fluorescence imaging-guided phototherapy. The fluorescence spectra of CM-TPB-TT NPs were basically consistent with those of TPB-TT NPs, indicating that the cell membrane coating of MUM-2B does not affect its spectral properties. Figure 7 ).

[0054] Example 5: Photothermal Performance Testing

[0055] Prepare 500 μL of a 50 μg / mL aqueous solution of nanoparticles in a 0.65 mL EP tube, and then use an 808 nm laser at a power density of 1.0 W / cm². 2 Irradiate vertically and record the solution temperature change every 30 seconds. For example... Figure 8 As shown: The highest temperature of the aqueous solution of CM-TPB-TT NPs nanoparticles can reach 80°C. o C, and the photothermal properties of TPB-TT NPs are not affected after cell membrane coating.

[0056] Example 6: Measurement of Photoinduced Reactive Oxygen Species Generation Performance

[0057] Take 2 mL of activated DCFH probe solution, and add appropriate volumes of ICG (indocyanine green), TPB-TT NPs, and CM-TPB-TT NPs nanoparticle solutions respectively, to a final concentration of 20 µg / mL. Measure the fluorescence spectra before illumination using a 0.33 W / cm² solution. 2 After irradiation with an 808 nm laser, the solution was uniformly agitated and measured immediately, with measurements taken every 1 minute until the 5th minute. The fluorescence intensity value at 525 nm was recorded, and the relative change in fluorescence intensity It / I0 was used as the probe fluorescence activation rate. The obtained data were analyzed using Origin 2021. Figure 9As shown, ICG, TPB-TT NPs, and CM-TPB-TT NPs nanoparticle solutions can all effectively generate reactive oxygen species after irradiation with an 808nm laser. The reactive oxygen species generation rate of the CM-TPB-TT NPs nanoparticle aqueous solution is higher than that of the control ICG.

[0058] Example 7: Cellular uptake

[0059] MUM-2B cells in the logarithmic growth phase were seeded in 24-well plates (1×10⁻⁶ cells per well). 4 cells / well), 37 o After culturing at C for 12 hours and allowing cells to adhere, the culture medium was discarded. 1 mL of TPB-TT NPs or CM-TPB-TT NPs containing 20 μg / mL fluorescein-labeled NPs was added to each well, along with a PBS control group. After incubation for 1 hour, the culture medium was discarded, and the cells were washed three times with PBS buffer. The fluorescence intensity of FITC excited at 488 nm was then measured using flow cytometry. Figure 10 As shown, the fluorescence intensity inside cells of the CM-TPB-TT NPs group treated with cell membrane-coated nanoparticles was significantly stronger than that of the TPB-TT NPs group without cell membrane coating, indicating that cell membrane coating of MUM-2B enables TPB-TT NPs to have homologous targeting function and increases the selective uptake of TPB-TT NPs by MUM-2B.

[0060] Example 8: Dark toxicity and phototoxicity

[0061] MUM-2B cells in the logarithmic growth phase were seeded in 96-well plates (1×10⁻⁶ cells / well). 4 Cells / well were incubated for 12 h to allow them to adhere. The experiment was divided into PBS, TPB-TT NPs, CM-TPB-TT NPs, TPB-TT NPs+Laser, and CM-TPB-TT NPs+Laser groups. Each group had 6 concentrations, and each concentration required 3 replicates to calculate the average. After adding the drug, each group was incubated for 4 h, the culture medium was changed, and the cells were washed 3 times with PBS buffer. The TPB-TT NPs+Laser and CM-TPB-TT NPs+Laser groups were subjected to a 1.0 W / cm² incubation. 2 Irradiate with an 808 nm laser for 5 min. Continue incubation for 24 h, discard the culture medium, add CCK8-containing medium and incubate for 4 h, then measure the absorbance at 450 nm using a microplate reader. Figure 11As shown, without excitation light irradiation, TPB-TT NPs and CM-TPB-TT NPs had no effect on cell growth within the tested concentration range. Under excitation light irradiation, both TPB-TT NPs and CM-TPB-TT NPs exhibited significant cytotoxic effects, especially at concentrations of 2.5 and 5 μg / mL, where the cytotoxic effect of CM-TPB-TT NPs was higher than that of TPB-TT NPs. This indicates that cell membrane encapsulation enhanced the MUM-2B cell targeting effect of TPB-TT NPs.

[0062] Example 9: In vivo fluorescence imaging

[0063] Three mice were randomly selected and subjected to NIR-II fluorescence imaging using a 1400 nm filter under 808 nm laser excitation. Subsequently, 100 μL of either 1.0 mg / mL ICG or CM-TPB-TT NPs solution was injected via tail vein, and the vascular fluorescence signal was continuously detected and photographed. Figure 12 It can be seen that CM-TPB-TT NPs can image blood vessels throughout the body, and their signal-to-noise ratio for capillary imaging is significantly higher than that of the control ICG. This demonstrates that CM-TPB-TT NPs exhibit excellent imaging signal-to-noise ratio, clearly distinguishing tiny capillaries in living organisms, and holds promise for advancing the development of novel surgical navigation contrast agents. Furthermore, it can monitor the dynamic distribution and metabolic processes of drugs in animals in real time; with its high-resolution advantage, it holds promise for retinal microvascular imaging, aiding in the early diagnosis and follow-up of diseases such as diabetic retinopathy and age-related macular degeneration; and it can monitor the potential toxicity of drugs to the microcirculation of vital organs such as the liver and kidneys, providing a basis for preclinical safety assessments.

[0064] Example 10: In vivo anti-tumor experiment

[0065] A MUM-2B subcutaneous tumor model was constructed and divided into four groups: PBS group, PBS+Laser group, CM-TPB-TT NPs group, CM-TPB-TT NPs+Laser group, and DTIC (dacarbazine) group. Six animals in each group received a tail vein injection of PBS or 100 μL of 1.0 mg / mL CM-TPB-TT NPs solution. The DTIC group received an intraperitoneal injection of 80 mg / kg of dacarbazine. An 808 nm laser (0.75 W / cm²) was used. 2 The tumor sites of mice in the PBS+Laser group and the CM-TPB-TT NPs+Laser group were irradiated with laser for 10 min. Tumor size changes in each mouse were recorded during the treatment period (40 days). Data were processed using Origin2021 and survival curves were plotted. Figure 13Changes in tumor volume indicate that CM-TPB-TT NPs combined with 808 nm excitation light can achieve complete ablation of the tumor volume. During the 40-day observation period, the survival rate of the CM-TPB-TT NPs+Laser group reached 100%, which was higher than that of the DTIC positive control group. Figure 14 ).

Claims

1. A photosensitizer having the structure shown in formula (I): in: n represents the degree of polymerization of the polymer molecule, which takes a value of 2 to 10, preferably 3 to 5; x is 1 to 50, preferably 5 to 20, more preferably 5 to 10, and even more preferably 5 to 8.

2. A photosensitizer having the structure shown in TPB-TT, wherein x = 6 and n = 3 ~ 5: 。 3. A tumor cell membrane biomimetic nanoparticle loaded with a photosensitizer, characterized in that, The photosensitizer of claim 1 or 2 is coated with a tumor cell membrane to form the tumor cell membrane biomimetic nanoparticles.

4. The tumor cell membrane biomimetic nanoparticles loaded with photosensitizer according to claim 3, characterized in that, The tumor cell membrane is selected from one or more of the following: breast cancer cell membrane, lung cancer cell membrane, bladder cancer cell membrane, melanoma cell membrane, cervical cancer cell membrane, lung cancer cell membrane, colorectal cancer cell membrane, or gastric cancer cell membrane; preferably, the melanoma cell membrane is the choroidal melanoma cell membrane.

5. A method for preparing tumor cell membrane biomimetic nanoparticles loaded with photosensitizer as described in claim 3 or 4, characterized in that, The photosensitizer described in claim 1 or 2 is co-precipitated with a surfactant to form photosensitizer nanoparticles. The photosensitizer nanoparticles are then mixed with tumor cell membranes in a certain proportion and encapsulated using membrane encapsulation technology to form the tumor cell membrane biomimetic nanoparticles loaded with photosensitizers.

6. The preparation method according to claim 5, characterized in that, The surfactant is selected from DSPE-mPEG2000; preferably, the membrane encapsulation technology is membrane extrusion or ultrasound.

7. The preparation method according to claim 5, characterized in that, The weight ratio of photosensitizer to surfactant is 1:(5~15); preferably 1:10; the mass ratio of photosensitizer nanoparticles to cell membrane is 1:(1~100), preferably 1:(1~10); more preferably 1:

5.

8. The application of the photosensitizer according to claim 1 or 2 or the tumor cell membrane biomimetic nanoparticles loaded with the photosensitizer according to claim 3 or 4 in near-infrared II imaging, preferably in the field of vascular imaging.

9. The use of the photosensitizer according to claim 1 or 2 or the tumor cell membrane biomimetic nanoparticles carrying the photosensitizer according to claim 3 or 4 in the preparation of a drug for treating tumors; preferably, the tumors include breast cancer, lung cancer, bladder cancer, melanoma, cervical cancer, colorectal cancer, and gastric cancer.

10. The use of the photosensitizer of claim 1 or 2 or the tumor cell membrane biomimetic nanoparticles carrying the photosensitizer of claim 3 or 4 in combination with photothermal or photodynamic therapy in the preparation of a drug for treating tumors; preferably, the tumor includes breast cancer, lung cancer, bladder cancer, melanoma, cervical cancer, colorectal cancer, and gastric cancer.

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