Man-pfh-icg@plga nanoparticles, a preparation method and application thereof
By preparing Man-PFH-ICG@PLGA nanoparticles, tumor targeting and oxygen supplementation were achieved, which solved the problem of poor PDT effect in the tumor hypoxic environment, enhanced the therapeutic effect of PDT and provided photoacoustic imaging capabilities.
Patent Information
- Application Number
- CN202411470031.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-10-10
- Estimated Expiration
- 2044-10-21
AI Technical Summary
The hypoxic environment of tumors leads to poor efficacy of photodynamic therapy (PDT) drugs. Existing materials have limited oxygen carrying capacity and cannot effectively enhance the PDT effect.
Man-PFH-ICG@PLGA nanoparticles were prepared. Mannose-PEG-NH2 was modified on the surface of PLGA nanoparticles to achieve tumor targeting. Oxygen was supplemented endogenously and exogenously. The oxygen-carrying capacity of PFH was utilized and combined with the photosensitizer ICG for PDT. A double emulsification method was used to form a W/O/W emulsion to improve stability and sustained release effect.
It improves the targeting effect and PDT efficacy of tumor cells, alleviates the tumor hypoxia environment, enhances the PDT effect, has photoacoustic imaging capabilities, and provides new ideas for tumor treatment.
Smart Images
Figure CN119345354B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of composite material preparation, and particularly relates to Man-PFH-ICG@PLGA nanoparticles and a preparation method and application thereof. Background Art
[0002] Photodynamic therapy (PDT) is an emerging technology for treating breast cancer. The reactive oxygen species (ROS) it produces are the primary cause of tumor cell death. However, because their production is highly dependent on oxygen in the surrounding environment, the technology still has many limitations. Previous studies have investigated enhancing PDT by incorporating oxygen into materials, directly supplementing the oxygen required for PDT exogenously. However, because the materials themselves must circulate through the systemic circulatory system to reach the tumor, the amount of oxygen they carry that can reach the tumor is also very limited, limiting their effectiveness. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide Man-PFH-ICG@PLGA nanoparticles and a preparation method and application thereof, so as to solve the technical problem that the hypoxic environment of tumors leads to poor therapeutic effects of drugs.
[0004] To achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing Man-PFH-ICG@PLGA nanoparticles, comprising the following steps:
[0005] S1. N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in acidic MES buffer, followed by addition of carboxylated PLGA for reaction for 0.5-1.5 h, centrifugation, and resuspending the carboxylated PLGA in alkaline MES buffer. The resuspended product was then mixed with a Mannose-PEG-NH2 solution for reaction for 10-12 h to obtain PLGA-PEG-Mannose; the ratio of N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, carboxylated PLGA, and Mannose-PEG-NH2 solution was 25-35 mg: 80-100 mg: 40-60 mg: 0.5-1.5 mL;
[0006] N-hydroxysuccinimide is a chemical substance containing an N=C=N functional group. It hydrolyzes in water to form a urea derivative, which is mainly used to activate carboxyl groups and promote the formation of amides and esters. The carboxyl groups and amino groups in the present invention can undergo a dehydration reaction similar to an esterification reaction to form a peptide bond "-C-NH-" (amide bond), allowing the empty shell PLGA to be labeled with mannose. Mannose can actively recognize mannose receptors overexpressed on the surface of tumor cells, achieve tumor targeting, and improve the enrichment of nanoparticles in tumors. PEG can improve the stability of nanoparticles in the blood circulation.
[0007] S2. Dissolve PLGA-PEG-Mannose, photosensitizer and PFH in an organic solvent. PFH, as an oxygen-carrying carrier, needs to be mixed with oxygen in a closed space overnight so that PFH can carry enough oxygen to react with subsequent materials. Then add the activator, sonicate at 40-50w for 3-7min, then add isopropanol solution (to remove the organic solvent) and stir for 2.5-3.5h. Finally, separate to obtain Man-PFH-ICG@PLGA nanoparticles. The ratio of PLGA-PEG-Mannose, photosensitizer, PFH, activator and isopropanol solution is 45-55mg:2-4mg:140-160μL:6-10mL:8-12mL.
[0008] Man-PFH-ICG@PLGA nanoparticles were synthesized using a typical double emulsion method (water / oil / water). This method involves a two-step emulsification process, using different emulsifiers to form the inner and outer phases, respectively, ultimately creating an emulsion with a dual continuous phase. In the first stage, a W / O emulsion is prepared using a lipophilic emulsifier. This step forms the inner phase, a primary emulsion of water-in-oil. In the second stage, the W / O emulsion obtained in the first stage is added to an aqueous phase containing a hydrophilic emulsifier. Through appropriate stirring and mixing, a W / O / W emulsion is formed. This step forms the outer phase, a double emulsion of water-in-oil-in-water. This method results in a W / O / W emulsion with a dual continuous phase structure: an inner phase of water-in-oil and an outer phase of oil-in-water, enabling the encapsulation and sustained release of oil-soluble active ingredients. This emulsification technology not only retains the excellent emollient effect of W / O emulsion, but also makes the system more stable due to the presence of O / W and W / O double interface membranes, and can effectively divide the system into three mutually immiscible regions (oil phase / water phase / oil phase).
[0009] On the basis of the above technical solution, the present invention can also be improved as follows:
[0010] Furthermore, the pH value of the acidic MES buffer solution is 5-6, and the pH value of the alkaline MES buffer solution is 7-9.
[0011] Furthermore, the concentration of the Mannose-PEG-NH2 solution is 8-12 g / L.
[0012] Furthermore, the organic solvent is dichloromethane.
[0013] Furthermore, the photosensitizer is ICG (indocyanine green), and the activator is a PVA solution with a mass fraction of 3-5% (to make the material easier to combine with PFH and ICG).
[0014] Furthermore, the mass fraction of the isopropyl alcohol solution is 1-3%.
[0015] The invention also discloses Man-PFH-ICG@PLGA nanoparticles prepared by the preparation method.
[0016] The invention also discloses the use of Man-PFH-ICG@PLGA nanoparticles in preparing drugs for treating tumors.
[0017] Furthermore, the tumor is breast cancer.
[0018] The beneficial effects of the present invention are:
[0019] 1. The Man-PFH-ICG@PLGA nanoparticles prepared in this invention have excellent targeting effects on mannose receptors overexpressed on the surface of tumor cells, significantly promoting effective cell endocytosis in vitro and tumor accumulation in vivo, further enhancing the PDT efficacy under 808nm laser irradiation. Man-PFH-ICG@PLGA nanoparticles also have excellent photoacoustic (PA) imaging capabilities, which can effectively detect the real-time dynamic distribution of nanoparticles in tumor tissue, laying a foundation for the clinical application of PDT.
[0020] 2. This invention leverages PFH's inherently excellent oxygen-carrying capacity. Through exogenous oxygen supplementation, PFH's high affinity for oxygen allows oxygen to enter tumor cells and directly alleviate the tumor's hypoxic environment. Furthermore, through endogenous oxygen supplementation, cellular-generated ROS activates overexpressed TRPA1 channels on the tumor cell surface, leading to a sustained influx of calcium ions, affecting mitochondrial respiratory enzymes, thereby inhibiting cellular respiration, reducing oxygen consumption, and indirectly alleviating the tumor's hypoxic environment. These direct and indirect effects jointly improve the tumor's hypoxic microenvironment, enhance the efficacy of PDT, and effectively inhibit tumor cell growth, providing new insights into clinical anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Transmission and scanning electron microscopy analysis images of Man-PFH-ICG@PLGA nanoparticles prepared in Example 3;
[0022] Figure 2 This is the particle size distribution diagram of Man-PFH-ICG@PLGA nanoparticles prepared in Example 3;
[0023] Figure 3 is the particle size distribution diagram of PFH-ICG@PLGA;
[0024] Figure 4 Potential diagrams of PFH-ICG@PLGA and Man-PFH-ICG@PLGA;
[0025] Figure 5 is the UV-visible absorption spectrum curve of ICG at different concentrations;
[0026] Figure 6 UV-visible absorption spectra of ICG, PI@P, Man-P@P, and Man-I@P NPs;
[0027] Figure 7 In vivo photoacoustic imaging of PI@P and Man-PI@P NPs;
[0028] Figure 8 Comparison of oxygen loading capacity of different nanoparticles;
[0029] Figure 9 Comparison of the ability of different nanoparticles to produce ROS;
[0030] Figure 10 Laser confocal images of PI@P NPs and Man-PI@P NPs taken up by 4T1 cells;
[0031] Figure 11 is the quantitative analysis result of fluorescence intensity;
[0032] Figure 12 Uptake of PI@P NPs and Man-PI@P NPs by 4T1 cells;
[0033] Figure 13 This is the result of in vitro CCK-8 cell safety test;
[0034] Figure 14 The results of in vitro CCK-8 cell PDT toxicity test;
[0035] Figure 15 Live and dead cell staining images after different treatments;
[0036] Figure 16 This is a laser confocal image of ROS production in 4T1 cells;
[0037] Figure 17 is the percentage of ROS in cells after different treatments;
[0038] Figure 18 is the intracellular Ca 2+ Laser confocal image;
[0039] Figure 19 Ca after different treatments 2+ Percentage within cells;
[0040] Figure 20 is the intracellular Ca2+ of 4T1 cells after pretreatment with TRPA1 channel inhibitor HC. 2+ Laser confocal image;
[0041] Figure 21 Comparison of mean fluorescence intensity after different treatments;
[0042] Figure 22 Confocal laser scanning image showing the mitochondrial localization of free calcium ions;
[0043] Figure 23 is the quantitative analysis of mean fluorescence intensity;
[0044] Figure 24 To detect the activity of mitochondrial respiratory enzyme complex 1;
[0045] Figure 25 Laser confocal images of HIF-1α after 4T1 cells were treated with different groups. DETAILED DESCRIPTION
[0046] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.
[0047] PLGA, carboxylated PLGA (50:50, molecular weight: 12000 Da), and Mannose-PEG-NH2 (PEG molecular weight: 2000 Da) were provided by Shaanxi Ruixi Biotechnology Co., Ltd., ICG and PFH were provided by Shanghai Maijin Biotechnology Co., Ltd., dichloromethane (CH2Cl2) and dimethyl sulfoxide (DMSO) were provided by Chongqing Chuandong Chemical Co., Ltd., Cell Counting Kit-8 (CCK-8) was provided by GlpBio, Montclair, California, and Minute TMThe Mitochondrial Isolation Kit for Mammalian Cells and Tissues was provided by Invent Biotechnologies, Inc., Eden Prairie, Minnesota. HC-0 HIF-1-alpha (9U11) rabbit monoclonal antibody was purchased from Biolong Technology Co., Ltd., Suzhou, China. Fluo-8 AM, Cell Permeant was purchased from Shanghai Maokang Biotechnology Co., Ltd., Singlet Oxygen Sensor Green (SOSG) was purchased from Dalian Meilun Biotechnology Co., Ltd., and FITC goat anti-rabbit IgG (H+L) was purchased from Shanghai Biotech Technology Co., Ltd.
[0048] Example 1
[0049] A method for preparing Man-PFH-ICG@PLGA nanoparticles comprises the following steps:
[0050] S1. Dissolve 35 mg of NHS and 80 mg of EDC in 1 mL of acidic MES buffer (pH = 6). After complete dissolution, add 60 mg of carboxylated PLGA and react for 0.5 h. Centrifuge and resuspend the carboxylated PLGA in MES buffer (pH = 7). Then, mix the resuspended product with 1.5 mL of 8 g / L Mannose-PEG-NH2 solution and react for 10 h. Finally, centrifuge at 12,000 rpm for 5 min to obtain PLGA-PEG-Mannose.
[0051] S2. Dissolve 45 mg PLGA-PEG-Mannose, 4 mg ICG and 140 μL PFH in 2 mL of dichloromethane, ultrasonically emulsify at a power of 60 W for 5 min, then add 6 mL of 5 wt% PVA solution, ultrasonicate at 40 W for 7 min, then add 8 mL of 3 wt% isopropanol solution and stir for 2.5 h. Finally, centrifuge at 10,000 rpm for 5 min. Repeat the centrifugation three times and separate to obtain Man-PFH-ICG@PLGA nanoparticles.
[0052] Example 2
[0053] A method for preparing Man-PFH-ICG@PLGA nanoparticles comprises the following steps:
[0054] S1. Dissolve 25 mg of NHS and 100 mg of EDC in 1 mL of acidic MES buffer (pH = 5). After complete dissolution, add 40 mg of carboxylated PLGA and react for 1.5 h. Centrifuge and resuspend the carboxylated PLGA in alkaline MES buffer (pH = 9). Then, mix the resuspended product with 0.5 mL of 12 g / L Mannose-PEG-NH2 solution and react for 12 h. Finally, centrifuge at 12,000 rpm for 5 min to obtain PLGA-PEG-Mannose.
[0055] S2. Dissolve 55 mg PLGA-PEG-Mannose, 2 mg ICG and 160 μL PFH in 2 mL of dichloromethane, ultrasonically emulsify at a power of 60 W for 5 min, then add 10 mL of 3 wt% PVA solution, ultrasonicate at 50 W for 3 min, then add 12 mL of 1 wt% isopropanol solution and stir for 3.5 h. Finally, centrifuge at 10,000 rpm for 5 min. Repeat the centrifugation three times and separate to obtain Man-PFH-ICG@PLGA nanoparticles.
[0056] Example 3
[0057] A method for preparing Man-PFH-ICG@PLGA nanoparticles comprises the following steps:
[0058] S1. Dissolve 30 mg of NHS and 90 mg of EDC in 1 mL of acidic MES buffer (pH = 5.5). After complete dissolution, add 50 mg of carboxylated PLGA and react for 1 hour. Centrifuge and resuspend the carboxylated PLGA in alkaline MES buffer (pH = 8). Then, mix the resuspended product with 1 mL of 10 g / L Mannose-PEG-NH2 solution and react for 11 hours. Finally, centrifuge at 12,000 rpm for 5 minutes to obtain PLGA-PEG-Mannose.
[0059] S2. Dissolve 50 mg PLGA-PEG-Mannose, 3 mg ICG and 150 μL PFH in 2 mL of dichloromethane, ultrasonically emulsify at a power of 60 W for 5 min, then add 8 mL of 4 wt% PVA solution, ultrasonicate at 45 W for 5 min, then add 10 mL of 2 wt% isopropanol solution and stir for 2 h. Finally, centrifuge at 10,000 rpm for 5 min. Repeat the centrifugation three times and separate to obtain Man-PFH-ICG@PLGA nanoparticles (Man-PI@P).
[0060] Comparative Example 1
[0061] The difference between the present invention and Example 3 is that Mannose-PEG-NH2 is omitted, and the remaining components and preparation steps are the same as those in Example 3 to obtain PFH-ICG@PLGA (PI@P).
[0062] Comparative Example 2
[0063] The present invention differs from Example 3 in that ICG is omitted, and the remaining components and preparation steps are the same as those in Example 3, to obtain Man-PFH@PLGA (Man-P@P).
[0064] Comparative Example 3
[0065] The present invention is different from Example 3 in that PFH is omitted, and the remaining components and preparation steps are the same as those in Example 3 to obtain Man-ICG@PLGA (Man-I@P).
[0066] Experimental Example 1 Structural Characterization
[0067] Scanning electron microscopy (Zeiss SUPRA TM 55, Germany) and transmission electron microscopy (Hitachi H-7600, Japan) were used to analyze the morphology and structure of the Man-PFH-ICG@PLGA nanoparticles prepared in Example 3. The results are shown in Figure 5. Figure 1 As shown in the figure, Man-PFH-ICG@PLGA nanoparticles are spherical with uneven size and distribution.
[0068] The particle size distribution and zeta potential of PFH-ICG@PLGA (Comparative Example 1) and Man-PFH-ICG@PLGA (Example 3) nanoparticles were evaluated using a dynamic light scattering (DLS) instrument from Malvern Instruments (UK). The specific steps were as follows: 100 μL of each 5 mg / mL nanoparticle solution was placed in a 1 mL quartz cuvette, then diluted with distilled water until clear and transparent, and finally placed in the instrument to detect particle size and potential.
[0069] The results are as follows Figure 2 and Figure 3 As shown, PFH-ICG@PLGA( Figure 3 ) and Man-PFH-ICG@PLGA( Figure 2 ) nanoparticles have an average particle size of 210.3±1.8nm and 234.5±4.2nm, respectively. The Zeta potential ( Figure 4, PI@P represents PFH-ICG@PLGA, Man-PI@P represents Man-PFH-ICG@PLGA) were -18.2±0.15mV and -11.3±0.41mV respectively. The positive Zeta potential during the detection process indicated that mannose was successfully labeled on the surface of the nanoparticles.
[0070] The absorption spectra of free ICG at different concentrations (0.5, 1.5, 2.5, 3.5, and 4.5 μg / mL) and different NPs (ICG represents ICG (indocyanine green) at a concentration of 0.68 μg / mL, PI@P represents PFH-ICG@PLGA, Man-P@P represents Man-PFH@PLGA, and Man-PI@P represents Man-PFH-ICG@PLGA) were measured using a UV-visible (UV-vis) spectrophotometer from Shimadzu (Japan). Figure 5 As shown in Figure 2, the absorbance of ICG changes in a concentration-dependent manner as determined by UV-visible spectroscopy. Figure 6 As shown, compared with Man-P@P NPs loaded with PFH only, Man-PI@P and PI@P NPs have characteristic absorption peaks at 780 nm, further indicating that ICG was successfully encapsulated.
[0071] The photoacoustic (PA) imaging ability of PFH-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticles was tested, and the results were as follows: Figure 7 As shown in the figure, it shows that Man-PFH-ICG@PLGA nanoparticles also have good photoacoustic (PA) imaging capabilities, which can well detect the real-time dynamic distribution of nanoparticles in tumor tissues, laying the foundation for the clinical application of PDT.
[0072] Experimental Example 2 Calculation of encapsulation efficiency and drug loading
[0073] The RP-HPLC method was used to determine and calculate the encapsulation efficiency of the nanoparticles. The encapsulation efficiency (EE) and encapsulation capacity (EC) of ICG in Man-PFH-ICG@PLGA nanoparticles were evaluated by establishing a standard curve of free ICG (indocyanine green) concentration. The calculation formula is as follows:
[0074]
[0075] Among them, m I Indicates the mass of ICG, g; m' I Indicates the mass of supernatant ICG, g; m P represents the mass of Man-PFH-ICG@PLGA nanoparticles, g.
[0076] Further calculations showed that the ICG encapsulation efficiency was 55% and the drug loading was 3.4%.
[0077] Experimental Example 3 Oxygen Loading Capacity Evaluation
[0078] (1) After the test samples Man-PFH-ICG@PLGA (Man-PI@P), PFH-ICG@PLGA (PI@P), and Man-ICG@PLGA (Man-I@P) nanoparticles and PBS were completely oxidized, equal amounts of samples were placed in an oxygen-free water environment, and the liquid surface was sealed with paraffin oil to prevent air from entering. The oxygen concentration in the water was then measured using a portable dissolved oxygen meter to evaluate the oxygen loading capacity of the nanoparticle samples.
[0079] The results are as follows Figure 8 As shown, within 30 minutes after full oxygenation, the four groups of samples increased from 2.43 mg / mL to 6.68 mg / mL in the PI@P group loaded with PFH, and increased from 2.61 mg / mL to 6.88 mg / mL in the Man-PI@P group, compared with the control group PBS, and maintained a high saturation concentration until 12 hours; while the Man-I@P group loaded only with ICG only increased from 1.79 mg / mL to 2.73 mg / mL, and began to decline after 3 hours. There was no obvious upward trend in the control group PBS. It can be seen that PFH encapsulated in PLGA nanoparticles is a good oxygen carrier.
[0080] (2) Using SOSG as a detection probe, the ability of Man-PFH-ICG@PLGA (Man-PI@P) and Man-ICG@PLGA (Man-I@P) nanoparticles to generate reactive oxygen species (ROS) in aqueous solution was evaluated, and free ICG was used as a control group. SOSG reagent was diluted with methanol to a concentration of 10 μg / mL SOSG solution, and free ICG, Man-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticles were prepared into a solution, and the ICG concentration was maintained at 6.25 μg / mL. Subsequently, 10 μL of SOSG solution was added to the nanoparticle solution, and then the solution was illuminated by 808 nm laser (power density of 0.75 W / cm 2 ) irradiate the mixture, and detect the fluorescence intensity using a fluorescence plate reader at irradiation times of 30, 60, 90, 120, 150, and 180 s. Figure 9 As shown in the figure, at each time period, the fluorescence intensity of Man-PFH-ICG@PLGA nanoparticles was always higher than that of free ICG and Man-ICG@PLGA nanoparticles with the same concentration, indicating that the ability of Man-PFH-ICG@PLGA nanoparticles to generate ROS was stronger than that of Man-ICG@PLGA nanoparticles and free ICG groups. This was attributed to the oxygen-carrying effect of PFH, which enhanced the effect of PDT and generated more ROS.
[0081] Experimental Example 4 Specific Recognition
[0082] (1) Cell culture
[0083] The 4T1 mouse breast cancer cell line was provided by Wuhan Pusai Bioscience and Technology Co., Ltd., China. The cells were cultured in complete medium containing RPMI-1640 and Dulbecco's modified EMBO medium (DMEM) at 37°C and 5% CO2.
[0084] (2) Evaluation of nanoparticle endocytosis
[0085] Confocal laser scanning microscopy (CLSM) produced by Nikon was used to evaluate the internalization of PFH-ICG@PLGA (PI@P) and Man-PFH-ICG@PLGA (Man-PI@P) nanoparticles by 4T1 cells. 5 The cells were seeded at a density of 100 cells / mL in a confocal compatible culture dish and cultured at 37°C and 5% CO2 for 24 hours. Then, DiI-labeled PFH-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticle culture solutions were added to the culture dishes and co-cultured with 4T1 cells for different time periods (0.5, 1, and 2 hours). After co-culture, the culture solution was removed and the cells were gently washed three times with PBS. The cells were then fixed in 4% paraformaldehyde for 10 minutes and finally stained with DAPI. The cells were directly observed by confocal laser scanning microscopy (CLSM). The observation results are shown in Figure 2. Figure 10 As shown in the figure, the red FL signal in cells co-incubated with PI@P NPs remained unchanged with prolonged incubation time, whereas the red FL signal in cells co-incubated with Man-PI@P NPs increased significantly with prolonged incubation time. After 2 hours of co-incubation with 4T1 cells, the intracellular uptake rate of the Man-PI@P NPs group reached 56.8%, while the intracellular uptake rate of PI@P NPs was only 11.5%. These results demonstrate that mannose-modified nanoparticles can specifically recognize the mannose receptor (MR) on the surface of tumor cells, facilitating efficient endocytosis.
[0086] The fluorescence intensity of DiI-labeled PFH-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticles in 4T1 cells was quantitatively analyzed using a BD FacAdvantage SE microscope ( Figure 11). Confocal images showed that the red FL signal in cells co-incubated with PFH-ICG@PLGA nanoparticles did not change significantly with the extension of incubation time, while the red FL signal in cells co-incubated with Man-PFH-ICG@PLGA nanoparticles increased significantly with the extension of incubation time. At the same time, quantitative analysis was performed by flow cytometry (FC) ( Figure 12 ), also verified a similar trend in cellular uptake performance. The above results all indicate that nanoparticles modified with mannose can specifically recognize the mannose receptor (MR) on the surface of tumor cells, which contributes to effective cellular endocytosis.
[0087] Experimental Example 5 Evaluation of biocompatibility and PDT cytotoxicity of Man-PFH-ICG@PLGA nanoparticles
[0088] (1) Biocompatibility
[0089] In order to evaluate the biocompatibility of Man-PFH-ICG@PLGA nanoparticles with 4T1 cells, 10 4 Cells were cultured in each well of a 96-well plate for 24 h. Man-PFH-ICG@PLGA nanoparticles at different concentrations (PLGA concentrations were 100, 200, 300, 400, and 500 μg / mL) were suspended in RPMI 1640 medium and co-cultured with the cells for 24 h. Cell viability was assessed using the CCK-8 assay.
[0090] CCK-8 results ( Figure 13 ) showed that when the concentration of Man-PFH-ICG@PLGA nanoparticles was 500 μg / mL, the survival rate of 4T1 cells remained above 85%, indicating that the nanoparticles had high biocompatibility.
[0091] (2) PDT cytotoxicity
[0092] In order to evaluate the in vitro PDT toxicity of Man-PFH-ICG@PLGA nanoparticles on 4T1 cells, 10 4 The cells were seeded at a density of 100 cells / mL in a 96-well plate and randomly divided into 5 groups: (1) Control group: only 1640 culture medium was added to the plate; (2) Laser Only group: 1640 culture medium was added to the plate and the cells were laser-stimulated with 808 nm laser (0.75 W / cm 2 ) laser irradiation for 10 min; (3) PI@P+Laser group: 200 μL of 0.1 mg / mL PFH-ICG@PLGA was added and irradiated with 808 nm laser (0.75 W / cm 2) laser irradiation for 10 min; (4) Man-I@P+Laser group: 200 μL of 0.1 mg / mL Man-ICG@PLGA was added and irradiated with 808 nm laser (0.75 W / cm 2 ) laser irradiation for 10 min; (5) Man-PI@P+Laser group: 200 μL of 0.1 mg / mL Man-PFH-ICG@PLGA was added and irradiated with 808 nm laser (0.75 W / cm 2 ) laser irradiation for 10 min. Each cell group was exposed to different concentrations of nanoparticles (PLGA concentrations of 100, 200, and 500 μg / mL) and cultured for 4 h under ice bath conditions, and then the cells were illuminated with 808 nm (0.75 W / cm 2 ) laser irradiation for 10 minutes, and cell viability was assessed using the CCK-8 assay. Furthermore, cells were seeded at a density of 100,000 cells per well in 6-well plates and treated with the same conditions as described above. Live cells were stained with Calcein-AM, and dead cells were labeled with PI, and observed using a fluorescence microscope.
[0093] like Figure 14 As shown in Figure 2, after treatment with Man-PFH-ICG@PLGA nanoparticles (concentration 200 μg / mL), only about 44.2% of the cell viability was maintained, indicating a low cell survival rate. The Man-ICG@PLGA (Man-I@P) nanoparticle group also exhibited high cytotoxicity. In contrast, the group treated with PFH-ICG@PLGA (PI@P) nanoparticles showed lower cytotoxicity, as shown in Figure 2. Figure 15 As shown, live-dead double-staining images also showed the same trend, which was consistent with the CCK8 results.
[0094] Experimental Example 6 Intracellular ROS and Ca 2+ Detection
[0095] The five experimental groups (Control, Laser Only, PI@P+Laser, Man-I@P+Laser, and Man-PI@P+Laser) set in Experimental Example 5 were co-cultured with 4T1 cells for 4 h. The cells were placed in an ice bath and illuminated with 808 nm (0.75 W / cm 2 ) laser irradiation for 10 min, followed by the addition of 20 μM DCFH-DA to the culture dish for confocal laser microscopy. After incubation for 30 min, the fluorescence intensity of reactive oxygen species (ROS) in each group was quantified using CLSM. After exposure to 808 nm laser, 1 μM Fluo-8AM was added to the culture dish for confocal laser scanning microscopy. Finally, after a 30-min incubation period, the Ca2+ concentration of each group of cells was quantified using CLSM. 2+Fluorescence intensity was assessed.
[0096] like Figure 16 and Figure 17 As shown in the figure, the confocal microscopy results show that the control group (Control) and the single laser group (Laser Only) have no ability to generate ROS autonomously. The other three groups all have LF signals due to the presence of ICG, and all have the ability to generate ROS. Among them, the PFH-ICG@PLGA group (PI@P+Laser) has the weakest FL signal due to the lack of labeled mannose to promote cell endocytosis; compared with the Man-ICG@PLGA group (Man-I@P+Laser), the Man-PFH-ICG@PLGA group (Man-PI@P+Laser) produces more ROS because PFH carries oxygen, which promotes the PDT effect to a greater extent, and thus has the strongest FL signal.
[0097] At the same time, flow cytometry was used for quantitative analysis and statistical analysis of flow cytometry results, and the results were consistent with those of confocal microscopy. Figure 18 and Figure 19 As shown, confocal images showed that the control group and the single laser group did not represent Ca 2+ The green fluorescence of the PFH-ICG@PLGA, Man-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticle groups showed an increasing amount of green FL signals, which was consistent with the DCFH-DA results. The above results indicate that the generation of ROS helps to promote the opening of TRPA1 channels and induce extracellular Ca 2+ In addition, the brightest green fluorescence was observed in the Man-PFH-ICG@PLGA nanoparticle group, indicating that more ROS generation helped to further increase the opening of TRPA1 channels and trigger more Ca 2+ Similarly, quantitative analysis by flow cytometry showed consistent results with the confocal microscopy.
[0098] Then, 4T1 cells were pretreated with the TRPA1 channel inhibitor HC-030031 (HC), and confocal images showed ( Figure 20 ), the FL signal in the cells of PFH-ICG@PLGA, Man-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticle groups was greatly reduced compared with the group without blocker. The semi-quantitative analysis results of mean fluorescence intensity ( Figure 21 ) is consistent with the confocal microscope. All the above results indicate that ROS generated in 4T1 cells can activate TRPA1 channels on the surface of 4T1 cells, 2+ influx of intracellular Ca 2+ Continued increase, causing Ca 2+If TRPA1 channels are blocked with blockers at the outset, extracellular Ca 2+ internal flow.
[0099] Experimental Example 7 ROS stimulation points
[0100] (1) Blocking and activating TRPA1 channels, and Ca 2+ Accumulates in mitochondria
[0101] 4T1(1x10 5 The cells were divided into control group and experimental group. The cells in the control group were only added with RPMI-1640 medium, while the cells in the experimental group were added with RPMI-1640 medium + TRPA1 channel agonist JT010 (1 μmol) and placed in the incubator for 4 hours. After the TRPA1 channel was activated by the TRPA1 channel agonist JT010, a continuous influx of calcium ions occurred, and calcium ions accumulated specifically in the mitochondria. Calcium ions were labeled with Fluo-8AM (1 μmol) and mitochondria were labeled with mitoRed probe (120 nmol). The distribution of calcium ions in biological tissues was observed using CLSM. Cellular Ca2+ can be achieved by opening the cell surface TRPA1 channel. 2+ internal flow.
[0102] To further explore the influx of Ca 2+ To investigate the relationship between TRPA1 and respiratory enzymes, 4T1 cells were pretreated with JT010, an agonist of TRPA1 channels, to open the channels. Then, Ca was labeled with Fluo-8 AM. 2+ , MitoRed labeled mitochondria and observed by CLSM. The results showed that ( Figure 22 ), compared with the control group, the experimental group used agonists to open TRPA1 channels, resulting in Ca 2+ Influx, shown as green FL signal, is overlapped with mitochondria showing red FL signal, indicating the influx of Ca 2 + Aggregated to mitochondria, the semi-quantitative analysis results of mean fluorescence intensity ( Figure 23 ) is consistent with confocal microscopy.
[0103] (2) Detection of mitochondrial complex I activity
[0104] To investigate the accumulation of Ca in mitochondria 2+ Effects on the activity of mitochondrial respiratory chain complex I, 3×10 54T1 cells were seeded into 10-cm cell culture dishes to establish a control group and a JT010-treated group. After the cells reached approximately 80% confluence, they were pre-treated with the TRPA1 agonist JT010 (10 μmol) for 6 h and then centrifuged at 4°C for 5 min to collect the cells for mitochondrial extraction. Subsequently, the activity of mitochondrial complex I was assessed using a mitochondrial complex I detection kit according to the manufacturer's instructions.
[0105] The results show that ( Figure 24 ), compared with the control group (expressed as 1.0), after 4T1 cells were pretreated with the TRPA1 channel agonist JT010 for 6 h, the activity of complex I was reduced to 0.11. This indicates that the influx of Ca 2+ The activity of complex I is greatly reduced, and the reduction of complex I activity will inhibit cellular respiration and reduce oxygen consumption.
[0106] (3) Intracellular evaluation of HIF-1α
[0107] 4T1 cells were cultured at a rate of 1 × 10 5 4T1 cells were cultured in 12-well plates at a concentration of 10 cells / well. 4T1 cells were cultured for 12 hours under normoxic and hypoxic conditions (hypoxic conditions were set up: after 8 hours of cell attachment, cells were placed in a hypoxic sealed box for further incubation. Anaerobic gas generation bags were purchased from Mitsubishi Corporation, Japan). Control groups in normoxic and hypoxic environments remained untreated. In the experimental group, 4T1 cells were pre-treated with JT010 (10 μmol) for 6 hours in a hypoxic environment. Cells were fixed with 4% paraformaldehyde for 10 minutes to permeabilize the cells, then incubated with primary and secondary antibodies. The cells were incubated with DAPA in the dark for 10 minutes. The slides were mounted and HIF-1α immunofluorescence staining in the cells was observed using CLSM.
[0108] The expression of HIF-1α in 4T1 cells was investigated by immunofluorescence. Figure 25 The results showed that in a normoxic environment, the expression of HIF-1α protein was very low, while in a hypoxic environment, the expression of HIF-1α in 4T1 cells treated with JT010 was significantly lower than that in the control group. This indicates that under hypoxic conditions, intervention with JT010 can significantly downregulate HIF-1α protein. The semi-quantitative analysis of mean fluorescence intensity also showed the same result. 2+ Influx further induces downregulation of HIF-1α expression by inhibiting respiratory enzyme activity.
Claims
1. A method for preparing Man-PFH-ICG@PLGA nanoparticles, characterized in that: The following steps are involved: S1. Dissolve N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in acidic MES buffer, then add carboxylated PLGA and react for 0.5-1.5 hours. Centrifuge, resuspend the carboxylated PLGA in alkaline MES buffer, and then mix the resuspended product with mannose-PEG-NH2 solution and react for 10-12 hours to obtain PLGA-PEG-mannose; the ratio of N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, carboxylated PLGA, and mannose-PEG-NH2 solution is 25-35 mg:80-100 mg:40-60 mg:0.5-1.5 mL; S2. Dissolve PLGA-PEG-mannose, photosensitizer and PFH in an organic solvent. PFH, as an oxygen-carrying carrier, needs to be mixed with oxygen in a closed space overnight so that PFH carries enough oxygen to react with subsequent materials. Then add an activator, sonicate at 40-50w for 3-7min, then add isopropanol solution and stir for 2.5-3.5h. Finally, separate to obtain Man-PFH-ICG@PLGA nanoparticles. The ratio of the PLGA-PEG-mannose, photosensitizer, PFH, activator and isopropanol solution is 45-55mg:2-4mg:140-160μL:6-10mL:8-12mL. The photosensitizer is ICG, and the activator is a PVA solution with a mass fraction of 3-5%.
2. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, wherein: The pH value of the acidic MES buffer solution is 5-6, and the pH value of the alkaline MES buffer solution is 7-9.
3. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, wherein: The concentration of the mannose-PEG-NH2 solution is 8-12 g / L.
4. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, wherein The organic solvent is dichloromethane, dichloroethane, dichloropropane or trichloroethane.
5. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, wherein: The mass fraction of the isopropanol solution is 1-3%.
6. A Man-PFH-ICG@PLGA nanoparticle, characterized in that: The method is prepared according to any one of claims 1 to 5.
7. Use of the Man-PFH-ICG@PLGA nanoparticles according to claim 6 in the preparation of drugs for photodynamic therapy of tumors.
8. The use according to claim 7, characterized in that The tumor is breast cancer.