Man-PFH-ICG (at) PLGA nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses Man-PFH-ICG (at) PLGA nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of composite material preparation. According to the invention, PLGA is used as a carrier, the surface of the PLGA is marked with mannose by using polylactic acid-glycolic acid (PLGA) nanoparticles (NPs), and ICG and oxygen-carrying PFH are embedded in the PLGA nanoparticles, so that the obtained Man-PFH-ICG-PLGA nanoparticles have an excellent targeting effect on a mannose receptor overexpressed on the surface of a tumor cell, and obviously promote effective endocytosis of the cell and tumor enrichment in vivo in vitro; the hypoxic environment of tumors is directly relieved; in addition, oxygen can be supplemented endogenously, ROS generated by cells is used for activating TRPA1 channels over-expressed on the surfaces of tumor cells, and therefore cell respiration is inhibited, oxygen consumption is reduced, the tumor hypoxia environment is indirectly relieved, tumor cell growth is effectively inhibited, and a new thought is added for clinical exploration of anti-tumor treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of composite material preparation, and specifically 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) produced by PDT is the main factor in the direct death of tumor cells. However, since its occurrence is highly dependent on oxygen in the surrounding environment, the technology still has many limitations. In existing studies, some technologies enhance PDT by carrying oxygen in the material to directly supplement the oxygen required for PDT. However, since the material itself needs to pass through the systemic circulatory system to reach the tumor, the amount of oxygen carried by the material that can reach the tumor site is also very limited, so the effect of enhancing PDT is limited. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a Man-PFH-ICG@PLGA nanoparticle and a preparation method and application thereof, so as to solve the technical problem that the hypoxic environment of the tumor leads to poor therapeutic effect of the drug.

[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 0.5-1.5 h, centrifugation, and resuspending the carboxylated PLGA in alkaline MES buffer. The resuspended product was then mixed with Mannose-PEG-NH 2 The solution was mixed and reacted for 10-12 hours to obtain PLGA-PEG-Mannose; N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, carboxylated PLGA and Mannose-PEG-NH 2 The ratio of the solution is 25-35 mg: 80-100 mg: 40-60 mg: 0.5-1.5 mL;

[0006] N-hydroxysuccinimide is a chemical substance containing N=C=N functional groups, which can be hydrolyzed in water to generate urea derivatives, and is mainly used to activate carboxyl groups and promote the generation of amides and esters. The carboxyl groups and amino groups in the present invention can undergo a dehydration reaction like an esterification reaction to form a peptide bond "-C-NH-" (amide bond), so that the empty shell PLGA is labeled with mannose, wherein Mannose can actively recognize the mannose receptors overexpressed on the surface of tumor cells, achieve tumor targeting, and improve the enrichment of nanoparticles at the tumor site. 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, ultrasonicate 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 by a typical double emulsion method (water / oil / water). The principle of the double emulsion method (water / oil / water) mainly involves a two-step emulsification process. Different emulsifiers are used to form the internal phase and the external phase in two stages, and finally an emulsion with a dual continuous phase is constructed. The first stage: a W / O type emulsion is prepared using a lipophilic emulsifier. This step is to form the internal phase, that is, the primary emulsion of oil-in-water. The second stage: the W / O type emulsion obtained in the first stage is added to the aqueous phase containing a hydrophilic emulsifier, and a W / O / W type emulsion is formed by appropriate stirring and mixing. This step is to form the external phase, that is, a double emulsion of water-in-oil-in-water. In this way, the final W / O / W type emulsion has a dual continuous phase, that is, the internal structure is oil-in-water, and the external structure is water-in-oil, thereby achieving 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 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 is 5-6, and the pH value of the alkaline MES buffer is 7-9.

[0011] Furthermore, Mannose-PEG-NH 2 The concentration of the solution is 8-12g / 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 isopropanol 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 application 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 the present invention have excellent targeting effects on mannose receptors overexpressed on the surface of tumor cells, significantly promoting effective endocytosis of cells in vitro and tumor enrichment in vivo, further improving the PDT effect under 808nm laser irradiation; 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 a foundation for the clinical application of PDT.

[0020] 2. The present invention utilizes the excellent oxygen carrying capacity of PFH itself, and uses the high affinity of PFH for oxygen to carry oxygen through exogenous oxygen supplementation, and enters tumor cells to directly alleviate the hypoxic environment of the tumor; it also uses endogenous oxygen supplementation to activate the TRPA1 channel overexpressed on the surface of tumor cells with ROS produced by cells, resulting in continuous calcium ion influx, affecting mitochondrial respiratory enzymes, thereby inhibiting cell respiration, reducing oxygen consumption, and indirectly alleviating the hypoxic environment of the tumor. Direct and indirect effects jointly improve the hypoxic microenvironment of the tumor, enhance the effectiveness of PDT, and thus effectively inhibit the growth of tumor cells, adding new ideas for clinical exploration of anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The transmission electron microscopy 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 It is the UV-visible absorption spectrum curve of ICG with 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] Fig. 9 Comparison of the ability of different nanoparticles to produce ROS;

[0030] Fig.10 Laser confocal images of PI@P NPs and Man-PI@P NPs taken up by 4T1 cells;

[0031] Fig.11 The results of quantitative analysis of fluorescence intensity;

[0032] Fig.12 Uptake of PI@P NPs and Man-PI@P NPs by 4T1 cells;

[0033] Fig.13 This is the result of in vitro CCK-8 cell safety test;

[0034] Fig.14 The results of in vitro CCK-8 cell PDT toxicity test;

[0035] Fig.15 Live and dead cell staining images after different treatments;

[0036] Fig.16 This is a laser confocal image of ROS production in 4T1 cells;

[0037] Fig.17 is the percentage of ROS in cells after different treatments;

[0038] Fig.18 is the intracellular Ca 2+ Laser confocal image;

[0039] Fig.19 Ca after different treatments 2+ Percentage in cells;

[0040] Fig. 20 The intracellular Ca2+ of 4T1 cells was measured after pretreatment with TRPA1 channel inhibitor HC. 2+ Laser confocal image;

[0041] Fig.21 Comparison of mean fluorescence intensity after different treatments;

[0042] Fig. 22 This is a confocal laser image of the mitochondrial localization of free calcium ions;

[0043] Fig.23 is the quantitative analysis of mean fluorescence intensity;

[0044] Fig.24 To detect the activity of mitochondrial respiratory enzyme complex 1;

[0045] Fig.25 Laser confocal images of HIF-1α factor after 4T1 cells were treated for different groups. DETAILED DESCRIPTION

[0046] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0047] PLGA, carboxylated PLGA (50:50, molecular weight: 12000Da) and Mannose-PEG-NH 2 PEG (molecular weight: 2000Da) was provided by Shaanxi Ruixi Biotechnology Co., Ltd., ICG and PFH were provided by Shanghai Maijin Biotechnology Co., Ltd., and dichloromethane (CH 2 Cl 2) and dimethyl sulfoxide (DMSO) were provided by Chongqing Chuandong Chemical Co., Ltd., Cell Counting Kit-8 (CCK-8) was provided by GlpBio, Montclair, California, Minute TM 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 Biolon 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 NHS and 80 mg EDC in 1 mL acidic MES buffer (pH = 6). After complete dissolution, add 60 mg carboxylated PLGA to react for 0.5 h, centrifuge, resuspend the carboxylated PLGA in MES buffer (pH = 7), and then mix the resuspended product with 1.5 mL of 8 g / L Mannose-PEG-NH 2 The solutions were mixed and reacted for 10 h, and finally centrifuged at 12000 rpm for 5 min using a high-speed centrifuge 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 3 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 NHS and 100 mg EDC in 1 mL acidic MES buffer (pH = 5). After complete dissolution, add 40 mg carboxylated PLGA and react for 1.5 h. Centrifuge. 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-NH 2 The solutions were mixed and reacted for 12 h, and finally centrifuged at 12,000 rpm for 5 min using a high-speed centrifuge 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 3 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 NHS and 90 mg EDC in 1 mL acidic MES buffer (pH = 5.5). After complete dissolution, add 50 mg carboxylated PLGA and react for 1 h. Centrifuge. Resuspend the carboxylated PLGA in alkaline MES buffer (pH = 8). Then, mix the resuspended product with 1 mL of 10 g / L Mannose-PEG-NH 2 The solutions were mixed and reacted for 11 h, and finally centrifuged at 12000 rpm for 5 min using a high-speed centrifuge 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 3 times and separate to obtain Man-PFH-ICG@PLGA nanoparticles (Man-PI@P).

[0060] Comparative Example 1

[0061] The present invention is different from Example 3 in that Mannose-PEG-NH 2 The remaining components and preparation steps were the same as those in Example 3 to obtain PFH-ICG@PLGA (PI@P).

[0062] Comparative Example 2

[0063] The difference between the present invention and Example 3 is 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 difference between the present invention and Example 3 is 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 The morphology and structure of the Man-PFH-ICG@PLGA nanoparticles prepared in Example 3 were analyzed by transmission electron microscopy (Hitachi H-7600, Japan). Figure 1 As shown, Man-PFH-ICG@PLGA nanoparticles are spherical in shape 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 of the two nanoparticle solutions with a concentration of 5 mg / mL 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 the 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, and 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-vis spectrophotometer from Shimadzu (Japan). The results are shown in Figure 2. 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 had 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. Figure 7 As shown, 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 content (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 Evaluation of oxygen loading capacity

[0078] (1) After the test samples Man-PFH-ICG@PLGA (Man-PI@P), PFH-ICG@PLGA (PI@P), Man-ICG@PLGA (Man-I@P) nanoparticles and PBS were completely oxidized, equal amounts of samples were placed in an oxygen-free water environment, the liquid surface was sealed with paraffin oil to prevent air from entering, and the oxygen concentration in the water was 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 the four groups of samples were fully oxygenated, compared with the control group PBS, the PFH-loaded PI@P group increased from 2.43 mg / mL to 6.68 mg / mL, and the Man-PI@P group increased from 2.61 mg / mL to 6.88 mg / mL, and maintained a high saturation concentration until 12 hours; while the Man-I@P group loaded with only ICG only increased from 1.79 mg / mL to 2.73 mg / mL, and began to decline after 3 hours, and the control group PBS had no obvious upward trend. 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 SOSG solution with a concentration of 10 μg / mL. 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 0.75 W / cm 2 ) irradiated the mixture, and the fluorescence intensity was measured using a fluorescence plate reader at irradiation times of 30, 60, 90, 120, 150, and 180 s. Fig. 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 of 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, which was attributed to the oxygen-carrying effect of PFH, which enhanced the effect of PDT and generated more ROS.

[0081] Experimental Example 4 Specific Identification

[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% CO 2 environment.

[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 10 cells / mL in a confocal compatible culture dish and incubated at 37°C, 5% CO 2 The cells were cultured under the same conditions for 24 h, and 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 h). After co-culture, the culture solution was removed, the cells were gently washed three times with PBS, and then the cells were fixed in 4% paraformaldehyde for 10 min, and finally stained with DAPI, and the cells were directly observed by confocal laser scanning microscopy (CLSM). The observation results are shown in Figure 2. Fig.10 As shown in the figure, it can be seen that the red FL signal in cells co-incubated with PI@P NPs has no significant change with the extension of incubation time, while the red FL signal in cells co-incubated with Man-PI@P NPs increases significantly with the extension of 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%. 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.

[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. Fig.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) ( Fig.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 and contribute 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. Different concentrations of Man-PFH-ICG@PLGA nanoparticles (PLGA concentrations were 100, 200, 300, 400, and 500 μg / mL, respectively) were suspended in RPMI 1640 medium and co-cultured with the cells for 24 h. Cell viability was evaluated using the CCK-8 method.

[0090] CCK-8 results ( Fig.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 medium was added to the plate; (2) Laser Only group: in addition to 1640 medium, 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 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) for 4 h in an ice bath and then illuminated with 808 nm (0.75 W / cm 2 ) laser irradiation for 10 min, and the cell viability was tested using the CCK-8 method. In addition, the cells were seeded in a 6-well plate at a density of 100,000 cells per well and treated with the same conditions as described above. Finally, the live cells were stained with Calcein-AM, the dead cells were labeled with PI, and observed using a fluorescence microscope.

[0093] like Fig.14 As shown in Figure 2, only about 44.2% of the viability was maintained after treatment with Man-PFH-ICG@PLGA nanoparticles (concentration 200 μg / mL), and the cell survival rate was low. The Man-ICG@PLGA (Man-I@P) nanoparticle group also showed high cytotoxicity. In contrast, the group treated with PFH-ICG@PLGA (PI@P) nanoparticles showed lower cytotoxicity, as shown in Figure 2. Fig.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] After co-culturing 4T1 cells with the five experimental groups (Control, Laser Only, PI@P+Laser, Man-I@P+Laser, and Man-PI@P+Laser) in Experimental Example 5 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 free radicals (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 Ca 2+Fluorescence intensity was assessed.

[0096] like Fig.16 and Fig.17 As shown in the figure, the confocal results showed that the control group (Control) and the single laser group (Laser Only) had no ability to generate ROS autonomously. The other three groups all had LF signals due to the presence of ICG, and all had the ability to generate ROS. Among them, the PFH-ICG@PLGA group (PI@P+Laser) had 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) produced more ROS because PFH carried oxygen, which promoted the PDT effect to a greater extent, so the FL signal was the strongest.

[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. Fig.18 and Fig.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 signal was 100%, while the PFH-ICG@PLGA, Man-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticle groups showed more and more 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 results.

[0098] Then, 4T1 cells were pretreated with HC-030031 (HC), an inhibitor of TRPA1 channel, and confocal images showed ( Fig. 20 ), the PFH-ICG@PLGA, Man-ICG@PLGA and Man-PFH-ICG@PLGA nanoparticle groups showed a significant decrease in the intracellular FL signal compared to the group without the use of blockers. The semi-quantitative analysis results of mean fluorescence intensity ( Fig.21 ) is consistent with the confocal microscope. All the above results show that ROS generated in 4T1 cells can activate TRPA1 channels on the surface of 4T1 cells, causing extracellular Ca 2+ influx, leading to intracellular Ca 2+ Continued increase, causing Ca 2+If the TRPA1 channel is blocked with a blocker at the beginning, it will not cause 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 a control group and an 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 an 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 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, which can achieve cellular Ca 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 ( Fig. 22 ), compared with the control group, the experimental group used agonists to open TRPA1 channels, resulting in Ca 2+ The influx of Ca is shown as green FL signal and overlaps with mitochondria showing red FL signal, indicating the influx of Ca 2 + Aggregated to mitochondria, mean fluorescence intensity semi-quantitative analysis results ( Fig.23 ) is consistent with the confocal.

[0103] (2) Detection of mitochondrial complex I activity

[0104] To investigate the accumulation of Ca in mitochondria 2+ Effects of 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 about 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. The activity of mitochondrial complex I was subsequently assessed using a mitochondrial complex I detection kit according to the manufacturer's instructions.

[0105] The results show that Fig.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 1×10 5 4T1 cells were cultured in 12-well plates at a volume of 100 μg / well. 4T1 cells were cultured for 12 h under normoxic and hypoxic conditions (hypoxic conditions were set up: after 8 h of cell adhesion, they were placed in a hypoxic sealed box for further incubation, and the anaerobic gas-generating bags were purchased from Mitsubishi Corporation of Japan), and the control groups in normoxic and hypoxic environments were kept untreated. The experimental group treated 4T1 cells with JT010 (10 μmol) for 6 h in a hypoxic environment, fixed the cells with 4% paraformaldehyde for 10 min to make the cells permeable, and then incubated with primary and secondary antibodies, incubated with DAPA for 10 min in the dark, mounted, and observed HIF-1α immunofluorescence staining in the cells with CLSM.

[0108] The expression of HIF-1α in 4T1 cells was investigated by immunofluorescence. Fig.25 The results showed that in a normoxic environment, the expression of HIF-1α protein was very low, while in an oxygen-depleted environment, the expression of HIF-1α in 4T1 cells treated with JT010 was significantly lower than that in the control group, indicating that under hypoxic conditions, the intervention of JT010 in cells can significantly downregulate HIF-1α protein; the semi-quantitative analysis of mean fluorescence intensity also showed the same result. 2+ The influx further induces downregulation of HIF-1α expression by inhibiting the activity of respiratory enzymes.

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 an acidic MES buffer, then add carboxylated PLGA to react for 0.5-1.5 hours, centrifuge, resuspend the carboxylated PLGA with an alkaline MES buffer, and then mix the resuspended product with a Mannose-PEG-NH2 solution to 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, then add an activator, ultrasonicate at 40-50w for 3-7min, then add isopropanol solution and stir for 2.5-3.5h, and 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.

2. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, characterized in that: The pH value of the acidic MES buffer is 5-6, and the pH value of the alkaline MES buffer is 7-9.

3. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, characterized in that: 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, characterized in that: The organic solvent is dichloromethane, dichloroethane, dichloropropane or trichloroethane.

5. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, characterized in that: The photosensitizer is ICG, and the activator is a PVA solution with a mass fraction of 3-5%.

6. The method for preparing Man-PFH-ICG@PLGA nanoparticles according to claim 1, characterized in that: The mass fraction of the isopropanol solution is 1-3%.

7. A Man-PFH-ICG@PLGA nanoparticle, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Man-PFH-ICG@PLGA nanoparticles according to claim 7 in the preparation of drugs for treating tumors.

9. The use according to claim 8, characterized in that: The tumor is breast cancer.

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