CN114699527A - Lipopolysaccharide/indocyanine green/oxaliplatin-loaded nanoparticle and preparation method thereof - Google Patents
Lipopolysaccharide/indocyanine green/oxaliplatin-loaded nanoparticle and preparation method thereof Download PDFInfo
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- CN114699527A CN114699527A CN202210386458.XA CN202210386458A CN114699527A CN 114699527 A CN114699527 A CN 114699527A CN 202210386458 A CN202210386458 A CN 202210386458A CN 114699527 A CN114699527 A CN 114699527A
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- lipopolysaccharide
- oxaliplatin
- indocyanine green
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Abstract
The invention discloses a lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle, wherein a PLGA inner core is loaded with oxygen-carrying perfluoropentane, indocyanine green and oxaliplatin molecules, a lipid membrane consisting of dipalmitoylphosphatidylcholine, 1, 2-palmitoylphosphatidylglycerol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and cholesterol is tightly wrapped outside the PLGA inner core, and lipopolysaccharide is loaded on the surface of the lipid membrane. The preparation method is simple. The oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle has good particle size potential, dispersibility, encapsulation efficiency, drug-loading capacity and optical properties, has no obvious toxic effect on organisms, and has great popularization and application values in improving tumor immune microenvironment and antitumor immunotherapy.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle and a preparation method and application thereof.
Background
Cancer immunotherapy has changed cancer therapy as a revolutionary therapeutic approach by exploiting the specificity of the immune system to exert anti-tumor immune effects. However, there is a major obstacle to ovarian cancer immunotherapy, namely the immunosuppressive state of the tumor microenvironment, which leads to immune escape, which is an important factor in tumor recurrence and metastasis. The tumor immune microenvironment participates in many immunosuppressive functional pathways that block the anti-tumor immune response, including altering stromal cell function to promote tumor growth, altering angiogenesis patterns, existence of a multi-differentiated state, providing chronic antigenic stimulation and recruitment of immunosuppressive cells. Furthermore, the tumor immune microenvironment also creates a different metabolic environment than normal cells, such as hypoxia, lactic acidosis, hypoglycemia, and essential amino acid depletion. In addition, the tumor cells can change the microenvironment by reprogramming the host cells, thereby creating an environment favorable for the growth and development of the tumor cells. Evidence suggests that ovarian cancer cells have a metastatic propensity specific to the omentum majus, characterized by a highly vascularized immune cell structure, and that this process of metastasis to the omentum and peritoneum is maintained by the tumor microenvironment. Because the antigen presenting cell APC is the key to the initiation of an anti-tumor immune response, APC infiltrated in the tumor immune microenvironment, such as dendritic cells and macrophages, are ideal therapeutic targets. Based on the strategy of modulating immunity and stimulating antigen-presenting cells in the immune microenvironment to improve immunotherapy efficacy, TLRs have been shown to reverse the immune escape of tumors, while Lipopolysaccharide (LPS) is the drug of choice, LPS is the major component of the gram-negative bacterial outer membrane, and it can activate APC and induce the release of a large number of inflammatory factors, stimulate CD8+ T cell activation, trigger Th1 type cellular immune responses, and produce anti-tumor immune effects.
Recent studies have shown that induction of immunogenic death of tumor cells ICD can trigger the immunogenicity of tumor cells. Unlike the death of normal cells, tumor cells in the ICD process release immunostimulatory molecules with the eat-me signal to the tumor immune microenvironment, collectively referred to as DAMPs such as HMGB1, CRT, ATP. They can stimulate the activation of effector T cells following APC, enhancing the presentation of tumor antigens, and thereby reversing the suppressive state of the tumor immune microenvironment. There is increasing evidence that some platinum-based anti-tumor chemotherapeutic agents have the ability to induce ICD. Among them, OXP has been approved by FDA for chemotherapy of malignant tumors, and unlike other platinum-based chemotherapeutic drugs, OXP has a specific spectrum of activity that reduces resistance to ovarian cancer. However, OXP also has neurotoxic, hematologic and gastrointestinal toxicity, neutropenia, nausea and vomiting, among other side effects that limit the range of doses that can be used. The combined application of the OXP and the nano drug-carrying system reduces the toxic and side effects of the OXP and the nano drug-carrying system, and generates strong anti-tumor immune response mainly because the OXP-mediated ICD plays a role. OXP has become a potent ICD-inducing drug with clinical potential.
Photodynamic therapy PDT is a unique treatment for cancer and is approved for clinical use because it is non-invasive, selectively destroys tumor tissue, and is non-injurious to adjacent healthy tissue. PDT is composed of three basic elements, a photosensitizer, a light source and oxygen. Under the action of light, the photosensitizer generates ROS, which directly leads to the death of tumor cells. But the low penetration depth and tumor hypoxia limits its use. Sonodynamic therapy SDT is a novel non-invasive treatment, with sonosensitizers activated by low intensity ultrasound to generate ROS to kill tumor cells. SDT combined with other therapeutic strategies has stronger anticancer activity than monotherapy. The photodynamic combined sonodynamic treatment strategy has a good application prospect because the photodynamic combined sonodynamic treatment strategy has deeper penetration depth and no toxic or side effects and can supplement the deficiency of photodynamic therapy. ICG is a near infrared fluorescent dye which is approved to be applied to clinic, can be used as a fluorescent probe for radiography and imaging, can also be used for accurate positioning of certain tumors, has the characteristics of a photosensitizer and a sonosensitizer, and can be used in a combined treatment method of the photoacoustic dynamic PSDT. There is also evidence that both PDT and SDT have the potential to induce ICD. In addition, perfluoro compounds (PFCs) as highly oxygen-soluble compounds can be used to carry oxygen to improve the hypoxic state of the tumor microenvironment and enhance the anti-tumor immune effect. And the liquid-gas phase change of the PFCs under the irradiation of light or sound can also be used for ultrasonic or photoacoustic imaging.
Disclosure of Invention
In view of the above, the invention aims to provide oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles and a preparation method and application thereof.
In order to achieve the above purpose, the inventor of the present invention provides a technical solution of the present invention through long-term research and a great deal of practice, and the specific implementation process is as follows:
an oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle is characterized in that the nanoparticle is a lipid membrane wrapping PLGA spherical core structure, the lipid membrane is a lipid membrane formed by dipalmitoyl phosphatidylcholine (DPPC), 1, 2-palmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG 2000) and cholesterol, lipopolysaccharide is loaded on the surface of the lipid membrane, and perfluoropentane, indocyanine green and oxaliplatin molecules carrying oxygen are loaded in a PLGA core.
Further, the particle size of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle is 253.6 +/-11.90 nm.
Further, the potential of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle is-36.17 +/-11.40 mV.
The preparation method of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle adopts a double emulsification method in combination with a rotary evaporation film forming method, and comprises the following steps: s1, dissolving DPPC, DPPG, DSPE-mPEG2000 and cholesterol in a ratio of 5:2:2:1 in 10ml of mixed organic solution of trichloromethane and methanol (4:1, v/v);
s2, rotationally evaporating in a water bath at 52 ℃ and at the rotating speed of 100rpm until the organic solvent in the flask is completely volatilized completely, and leaving a layer of uniform and transparent lipid film on the flask;
s3, eluting and hydrating by using a 0.1mg/ml lipopolysaccharide aqueous solution, and resuspending for later use;
s4, under the conditions of ice bath and light protection, filling 200 mu l of perfluoropentane into oxygen until saturation, and then dropping the oxygen-carrying perfluoropentane into 6mg/ml oxaliplatin aqueous solution and then performing acoustic vibration for 30 seconds;
s5, adding the mixture in the S4 into dichloromethane in which 1.5mg of indocyanine green and 50mg of PLGA are dissolved, and performing acoustic vibration for 3 minutes again to obtain PLGA (poly (lactic-co-glycolic acid)) spherical core initial liquid carrying the indocyanine green and the oxaliplatin;
s6, mixing the lipid membrane of S3 and the PLGA spherical core initial solution in S5, then vibrating for 3 minutes, magnetically stirring for 4-6 hours until the organic solvent is completely volatilized, centrifuging for 5 minutes by using a high-speed low-temperature centrifuge (4 ℃, 12000rpm), and then re-suspending with double distilled water to obtain the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles.
The invention also provides a killing effect and a mechanism of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle on a mouse ovarian cancer cell ID8 cell.
Advantageous effects
The invention provides a method for preparing a phase-change contrast agent by using PLGA as a carrier, perfluoropentane PFP as a phase-change contrast agent and ICG as a photosensitizer, wherein the phase-change contrast agent and ICG are loaded with nanoparticles of lipopolysaccharide and oxaliplatin serving as chemotherapeutic drugs, and the phase-change contrast agent and ICG effectively kill tumor cells, improve the tumor immune microenvironment and enhance the anti-tumor immune effect under the synergistic effect of photoacoustic power, thereby laying the foundation for the diagnosis and treatment integration of ovarian cancer.
Drawings
FIG. 1 is a representation diagram of an oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle of the invention;
FIG. 2 is a confocal laser microscopy image and an optical property image of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle of the invention;
fig. 3 is an in vitro photoacoustic imaging graph of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle of the present invention; (paired t-tests before and after irradiation for comparison, significance level is expressed as # P <0.001)
FIG. 4 is a confocal laser microscope used for observing the uptake of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles of the invention by macrophages;
fig. 5 shows the immune activation characteristics of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles of the present invention; (comparison between groups Using Dunnett's test Single factor analysis of variance (ANOVA). comparison between groups uses t-test,. P < 0.01 vs control,. P <0.001 vs control,. P < 0.0001 vs control,. P < 0.05 vs control,. P # # P <0.001, ns indicates no significant difference)
FIG. 6 is a confocal laser microscope observation showing the in vitro uptake, killing and apoptosis effects of mouse ID8 cells on oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles; (comparison between groups using Dunnett's test Single factor analysis of variance (ANOVA). comparison between groups uses t-test,. P < 0.05 compared to control,. P < 0.01 compared to control,. P <0.001 compared to control,. P < 0.0001 compared to control,. P < 0.05 between groups,. # # P <0.001, ns indicates no significant difference)
FIG. 7 is a confocal laser scanning microscope used for observing the translocation of CRT and HMGB1 in mouse ID8 cells;
FIG. 8 shows that ATP release of mouse ID8 cells is detected by the ATP determination kit; (comparison between groups Using Dunnett's one-way analysis of variance (ANOVA) test comparison between groups Using t-test, # P < 0.05 vs control, # P < 0.0001 vs control, # P <0.001, ns indicates no significant difference)
The Control group corresponds to a blank Control group, the PSDT corresponds to a photoacoustic group, the OXP corresponds to an oxaliplatin group, the LOI _ NPs corresponds to an oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle group, the LI _ NPs + PSDT corresponds to an oxygen-carrying lipopolysaccharide/indocyanine green nanoparticle combined photoacoustic group, the OI _ NPs + PSDT corresponds to an oxygen-carrying oxaliplatin/indocyanine green nanoparticle combined photoacoustic group, and the LOI _ NPs + PSDT corresponds to an oxygen-carrying lipopolysaccharide/oxaliplatin/indocyanine green nanoparticle combined photoacoustic group.
Detailed Description
The present invention is further illustrated by the following specific examples so that those skilled in the art can better understand the present invention and can practice it, but the examples are not intended to limit the present invention.
Example 1
The specific preparation process of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle of the embodiment is as follows:
s1, weighing 5g of DPPC, 2g of DPPG, 2g of DSPE-mPEG2000 and 1g of cholesterol, and dissolving in 10ml of mixed organic solution (4:1, v/v) of trichloromethane and methanol to obtain the compound;
s2, rotationally evaporating the product obtained in the step S1 under the conditions of water bath at 52 ℃ and the rotating speed of 100rpm until the organic solvent in the flask is completely volatilized completely and a layer of uniform and transparent lipid film is left on the flask, and thus obtaining the lipid film;
s3, eluting and hydrating with 10ml of aqueous solution containing 0.1mg/ml lipopolysaccharide, and resuspending for later use;
s4, under the ice-bath and dark conditions, filling 200 mu l of perfluoropentane into oxygen until saturation, then dropping the oxygen-carrying perfluoropentane into 6mg/ml oxaliplatin aqueous solution, and then performing acoustic vibration for 30 seconds to obtain the product;
s5, adding the product obtained in the step S4 into 2ml of dichloromethane dissolved with 1.5mg of indocyanine green and 50mg of PLGA, and performing ultrasonic vibration for 3 minutes again (120W, 5 seconds of ultrasonic vibration and 10 seconds of pause) to obtain PLGA spherical core initial solution carrying oxygen and indocyanine green and oxaliplatin;
s6, mixing the lipid membrane of S3 and the PLGA spherical core initial solution obtained from S5, then performing acoustic vibration for 3 minutes (120W, 5 seconds of acoustic vibration and 10 seconds of pause), magnetically stirring for 4-6 hours until the organic solvent is completely volatilized, centrifuging for 5 minutes by using a high-speed low-temperature centrifuge (4 ℃, 12000rpm), and then performing double-distilled water resuspension to obtain the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles.
The results of detection and analysis of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles prepared in example 1 are as follows:
characterization of oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles
The specific operation is as follows: 1) diluting the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles by PBS for a certain time (which can be adjusted based on obtaining a better visual field under a microscope), and observing the shape of the nanoparticles under a fluorescence microscope; 2) diluting the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles by PBS by a certain multiple, and observing the fine morphology of the nanoparticles by a transmission electron microscope; 3) detecting the particle size, distribution and potential of the particles by using a Malvern particle size instrument; 4) diluting the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles by PBS by a certain multiple, and observing the form of the nanoparticles by a laser confocal microscope; 5) the absorption spectra of indocyanine green and the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles were detected using a UV-Vis spectrophotometer (scanning wavelength range 550nm to 850 nm). The absorbance (at 780 nm) of indocyanine green and oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles was measured every 3 days for 15 days to evaluate their optical stability.
The results are shown in fig. 1 and 2, and 1) the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles are in a spherical shape with good dispersion, uniformity and consistency, and show red fluorescence (fig. 1A and B), which indicates that ICG is successfully encapsulated in the nanoparticles. 2) As can be seen from FIG. 1C, the transmission electron microscope shows that the single nanoparticle has an obvious shell-core structure; 3) as can be seen from FIGS. 1D and 1E, the particle size of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles is 253.6 +/-11.90 nm, and the potential is-36.17 +/-11.40 mV; 4) as can be seen from fig. 2A, the Dil dye and FITC-lipopolysaccharide labeled oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles can see co-localization of red fluorescence and green fluorescence, indicating that LPS is successfully loaded on the nanoparticles; 5) as can be seen from fig. 2B, the absorption peak of indocyanine green encapsulated in the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle is approximately red-shifted by 17 nm. As can be seen from fig. 2C, in the 15-day observation period, the adsorption intensity of indocyanine green in the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles was reduced by about 38.1% of the initial intensity, and the free ICG was significantly reduced by 55% of the initial intensity, indicating that the optical characteristics of ICG in PLGA were not changed, and the optical stability was significantly improved.
② in-vitro photoacoustic imaging of oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles
Preparing the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles into an in-vitro imaging gel model, and specifically operating the steps as follows: dissolving agarose gel powder in degassed water at 2% W/V, heating, stirring with glass rod to obtain viscous material, slowly pouring into freezing box or gun head box, inserting into freezing tube or 200ul gun head to generate holes, standing, cooling, taking out, and coating with preservative filmWrapping in a refrigerator at 4 deg.C. The experiment was divided into 4 groups, control group (PBS group); indocyanine green group (ICG); a group of perfluoropentane nanoparticles not encapsulating lipopolysaccharide, indocyanine green and oxaliplatin (Blank _ NPs group); nanoparticle groups (LOI _ NPs) encapsulating lipopolysaccharide, indocyanine green and oxaliplatin, respectively, were placed in the gel pores, B-Mode and CEUS imaging was performed using an ultrasonic diagnostic imager, followed by 1.5W/cm with a 808nm laser2The pictures were again imaged after 2 minutes of irradiation. The Echo Intensities (EI) at B-Mode and CEUS were quantitatively analyzed by DFY software (developed by the ultrasound imaging research institute of Chongqing university of medicine). And (3) acquiring photoacoustic imaging of the 4 groups of samples before and after 808nm laser irradiation by using a VEVOLASR photoacoustic imaging system and analyzing the result.
The results are shown in FIG. 3: in photoacoustic imaging fig. 3A, the PA signals of the PBS group and the Blank NPs group did not change significantly before and after 808nm laser irradiation. PBS and Blank NPs without ICG entrapped do not show enhanced capability of photoacoustic imaging since they do not have light absorbing properties in the near infrared range. The PA value of the ICG group decreased from 0.25. + -. 0.05 to 0.07. + -. 0.02 after irradiation with near infrared light (FIG. 3D), and ICG was quenched, probably due to the absence of PLGA entrapment. In contrast, after 808nm laser irradiation, the photoacoustic signal of the LOI _ NPs group was significantly enhanced from 0.24 + -0.04 to 0.71 + -0.03 (FIG. 3D) compared with the other groups, and the EI value of 25.34 + -1.12 in the B-mode was increased to 50.95 + -0.91 in FIG. 3C. In the ultrasound imaging in fig. 3B, there was no imaging enhancement in both the PBS and ICG groups in the B and CEUS modes after laser irradiation. After 2 minutes of laser irradiation at 808nm, the EI in the Blank NPs group of ultrasound imaging B-mode increased from 17.14 + -1.43 to 35.39 + -2.99 (FIG. 3E), and the EI in CEUS increased from 5.46 + -0.91 to 13.95 + -4.10 (FIG. 3F). This increase may be due to a slight heat after irradiation with the near infrared laser, which causes a certain degree of phase change in the PFP in the Blank NPs. While the EI of the LOI _ NPs group was significantly enhanced compared to the other groups, from 35.18 ± 3.20 to 54.90 ± 2.93 in B mode (fig. 3E), and from 17.50 ± 1.86 to 47.92 ± 1.39 in CEUS (fig. 3F). The results show that the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles show enhanced capabilities of photoacoustic imaging and ultrasonic imaging under the excitation of near-infrared light.
③ immunological activation characteristic of oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle
The specific operation is as follows: 1) RAW264.7 macrophages were seeded onto 6-well plates (density 1x10 per well)6Individual cells) and cultured overnight before replacing the original medium with medium containing Dil dye and FITC-lipopolysaccharide labeled oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles (containing oxaliplatin at 10.0 μ g/mL) and incubating for 6 h. Subsequently, the cells were washed in sterile PBS and fixed with 4% Paraformaldehyde (PFA) for 15 min. Then, the cells were washed 3 times with DAPI staining solution staining cell nucleus PBS, 5 minutes each time, and then mounted with an anti-fluorescence attenuation mounting medium to prepare cell slide. Cellular uptake capacity was assessed by confocal laser microscopy. 2) Macrophage or BMDC at 7x10 respectively3Inoculating each well into a 96-well plate for overnight culture, replacing the original culture medium with a culture medium containing oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles (containing oxaliplatin at 10 mu g/ml) on the next day, continuously incubating at constant temperature for 24h, then respectively adding 10 mu l of CCK8 solution into each well, placing the well plate into an incubator for incubation for 1-4 h, measuring the absorbance value at 450nm by using a microplate reader, and calculating the cell survival rate. BMDC at 4x105Inoculating each well into a 24-well plate for overnight culture, replacing the original culture medium with a culture medium containing oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles (containing oxaliplatin at 10 mu g/ml) the next day, continuing to incubate overnight at constant temperature, washing away excess drugs by PBS, replacing with a new serum-containing culture medium, adding anti-CD 80 and anti-CD 86 antibodies for incubation for 2h, digesting and collecting cells by using trypsin, and detecting the expression conditions of CD80 and CD86 by using flow cytometry. Mix 4x105RAW264.7 macrophages or BMDCs were inoculated into 24-well plates, respectively, and the experiments were divided into 5 groups: (1) a control group; (2) low concentration lipopolysaccharide group (10 μ g/ml); (3) high concentration lipopolysaccharide group (30 μ g/ml); (4) an oxygen-carrying indocyanine green/oxaliplatin-containing nanoparticle group; (5) the nanoparticle group containing the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin. After overnight incubation with various factors, supernatants were collected and assayed for standard cytokine profile (TNF-. alpha., IL-12, and IL-6) and for cytokine (TNF-. alpha., IL-12, and IL-6) content in each group of supernatants, as determined by enzyme-linked immunosorbent assay kit (ELISA) manufacturer's instructions.
The results are shown in FIGS. 4 and 5: 1) fig. 4 shows that the macrophage has strong internalization degree on the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles, and the co-localization of red fluorescence and green fluorescence indicates that the entrapped lipopolysaccharide of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles is still combined on the nanoparticle structure after the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles are taken up by the macrophage, and the basic structure remains unchanged. 2) From the analysis in fig. 5, it can be seen that the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles have good biosafety (fig. 5A), can promote the significant up-regulation of the co-stimulation markers CD80 and CD86 on the surface of BMDC (fig. 5B), and have strong induction capability on the secretion of cytokines (TNF- α, IL-12 and IL-6) by macrophages (fig. 5C) and BMDC (fig. 5D).
The killing effect and mechanism of the photoacoustic synergetic oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles on ovarian cancer cells are grouped as follows: the experiments were divided into the following 7 groups: control group (Control); a photoacoustic group (PSDT); oxaliplatin group (OXP); an oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle set (LOI _ NPs); the preparation method comprises the following steps of (1) carrying oxygen-carrying lipopolysaccharide/indocyanine green nanoparticles and a photoacoustic group (LI _ NPs + PSDT); the preparation method comprises the following steps of carrying oxygen-carrying indocyanine green/oxaliplatin nanoparticles and a photoacoustic group (OI _ NPs + PSDT); oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle + photoacoustic group (LOI _ NPs + PSDT)
The specific operation is as follows: 1) mouse ovarian cancer cell ID8 cells were seeded onto 6-well plates (density 1X10 per well)6Individual cells) and incubated overnight, then the original medium was replaced with Dil dye and a medium of FITC-lipopolysaccharide labeled oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles (containing oxaliplatin at 10.0 μ g/mL), and incubated for 6 h. Subsequently, cells were washed in sterile PBS and fixed with 4% Paraformaldehyde (PFA) for 15 min. Then, the cells were washed 3 times with DAPI staining solution staining cell nucleus PBS, 5 minutes each time, and then mounted with an anti-fluorescence attenuation mounting medium to prepare cell slide. Cellular uptake capacity was assessed by confocal laser microscopy. 2) ID8 cells in logarithmic growth phase were cultured at 7X103Inoculating into 96-well plate, culturing overnight, replacing with nanoparticles containing oxaliplatin 10.0 μ g/ml or free drug culture solution according to groups, incubating for 6h, washing off excessive drug with sterile PBS, replacing with new culture solution, and laser (1.5W/cm)2808nm，2min) and low intensity ultrasound (1.0W/cm)21min), detecting the survival rate of each group of cells by using a CCK8 reagent after 24h, repeating the experiment three times, and avoiding light all the time. 3) ID8 cells at 1x106Inoculating each well into a 6-well plate, culturing overnight, performing grouping treatment as before, performing photoacoustic irradiation treatment for 24h, collecting ID8 cells subjected to different treatments by trypsinization, performing annexin V-FITC/PI double staining for 15min, and detecting apoptosis by a flow cytometer. 4) Log phase of ID8 cells at 7x103Inoculating into a confocal dish, culturing overnight, replacing with nanoparticle or free drug culture solution containing OXP10.0 μ g/ml, incubating for 6 hr, washing with sterile PBS to remove excessive drug, replacing with new culture solution, and laser (1.5W/cm)2808nm, 2min) and low intensity ultrasound (1.0W/cm)21min), and then the cells were washed 3 times with PBS, and the cells were incubated with an anti-CRT antibody or an anti-HMGB 1 antibody (dilution 1: 100) after 2h incubation, DAPI staining, PSB washing excess dye, and CRT and HMGB1 translocation were observed under a confocal laser microscope. 5) The logarithmic growth phase of ID8 cells was seeded in 24-well plates and the supernatants from each group were collected after 24h of treatment of ID8 cells with different factors. The amount of ATP secreted in the supernatant was measured using an ATP assay kit. ATP assay solutions were prepared according to the manufacturer's instructions. And (3) preparing ATP standard substance solutions with different concentrations to draw a standard curve, adding the collected supernatant sample into the ATP analysis solution, incubating for 20 minutes at room temperature, and detecting the chemiluminescence intensity. The concentration of ATP in the supernatant was calculated from the plotted standard curve.
The results are shown in FIGS. 6, 7 and 8: 1) as can be seen from fig. 6A, a large amount of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles are phagocytosed by ID8 cells, so that the fluorescence signal is enhanced, and the internalization degree is high; 2) as can be seen from fig. 6C, the cell survival rate of the oxygen-carrying lipo-indocyanine green/oxaliplatin nanoparticle + photoacoustic group is 31.25 ± 3.91%, the cell survival rate of the oxygen-carrying lipo-polysaccharide/indocyanine green/oxaliplatin nanoparticle + photoacoustic group is 29.78 ± 6.69%, the cell survival rates of the two groups are significantly reduced, and the differences are statistically significant (P <0.001) compared with all other groups, and the results show that the oxygen-carrying lipo-polysaccharide/indocyanine green/oxaliplatin nanoparticle + photoacoustic has the capability of inhibiting the activity of the ID8 cell in vitro (P > 0.05); 3) as can be seen from fig. 6B and D, the apoptosis rates of the oxygen-carrying indocyanine green/oxaliplatin nanoparticle + photoacoustic group (with the apoptosis rate of 71.75 ± 2.86%) and the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle + photoacoustic group (with the apoptosis rate of 75.06 ± 4.21%) were significantly higher than those of the other groups, and the difference was statistically significant (P <0.001), and the difference in apoptosis rate between the two groups was not statistically significant (P > 0.05), indicating that the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle + photoacoustic can effectively induce the apoptosis of ID8 cells. 4) As can be seen from fig. 7, the green fluorescence of the control group is distributed substantially in the cytoplasm, while the green fluorescence of the LOI _ NPs + PSDT group is mainly concentrated on the cell membrane, the fluorescence intensity in the cytoplasm is reduced, and a significant position change occurs to illustrate that CRT translocates from the cytoplasm to the cell membrane, and the HMGB1 is also translocated, and the red fluorescence of the control group is mainly concentrated in the blue nucleus, while the red fluorescence of the LOI _ NPs + PSDT group is mainly distributed in the cytoplasm, and a change from the nucleus to the cytoplasm occurs, so that compared with other groups, the degree of translocation of CRT and HMGB1 of the LOI _ NPs + PSDT group is also most significant. 5) As can be seen from FIG. 8, the ATP secretion induced by the LOI _ NPs + PSDT group and the OI _ NPs + PSDT group is obviously higher than that induced by the other groups, and the surface oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles + photoacoustic have stronger ATP secretion inducing ability of the ID8 cells.
Claims (4)
1. An oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle is characterized in that: the nanoparticles are lipid membranes wrapping PLGA spherical core structures, the lipid membranes are lipid membranes formed by dipalmitoyl phosphatidylcholine, 1, 2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol, lipopolysaccharides are loaded on the surfaces of the lipid membranes, and oxygen-carrying perfluoropentane, indocyanine green and oxaliplatin molecules are loaded in PLGA cores.
2. The oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle of claim 1, wherein the particle size of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle is 253.6 ± 11.90 nm.
3. The oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle of claim 1, wherein the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticle has a potential of-36.17 ± 11.40 mV.
4. The preparation method of the oxygen-carrying lipopolysaccharide/indocyanine green/oxaliplatin nanoparticles according to any one of claims 1 to 3, which is characterized in that a double emulsification method is adopted in combination with a rotary evaporation film forming method, and the method comprises the following steps: s1, dissolving DPPC, DPPG, DSPE-mPEG2000 and cholesterol in a ratio of 5:2:2:1 in 10ml of mixed organic solution of trichloromethane and methanol (4:1, v/v); s2, rotationally evaporating in a water bath at 52 ℃ and at the rotating speed of 100rpm until the organic solvent in the flask is completely volatilized completely, and leaving a layer of uniform and transparent lipid film on the flask; s3, eluting and hydrating by using a 0.1mg/ml lipopolysaccharide aqueous solution, and resuspending for later use; s4, under the conditions of ice bath and light protection, filling 200 mu l of perfluoropentane into oxygen until saturation, and then dropping the oxygen-carrying perfluoropentane into 6mg/ml oxaliplatin aqueous solution and then performing acoustic vibration for 30 seconds; s5, adding the mixture in the S4 into dichloromethane in which 1.5mg of indocyanine green and 50mg of PLGA are dissolved, and performing acoustic vibration for 3 minutes again to obtain PLGA (poly (lactic-co-glycolic acid)) spherical core initial liquid carrying the indocyanine green and the oxaliplatin; and S6, mixing the lipid membrane of S3 and the PLGA spherical nucleus initial solution in S5, performing acoustic vibration for 3 minutes, magnetically stirring for 4-6 hours until the organic solvent is completely volatilized, centrifuging for 5 minutes by using a high-speed low-temperature centrifuge (4 ℃, 12000rpm), and then performing double-distilled water resuspension to obtain the PLGA spherical nucleus aqueous solution.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114007649A (en) * | 2019-03-29 | 2022-02-01 | 布里格姆及妇女医院股份有限公司 | Targeted synergistic cancer immunotherapy |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114007649A (en) * | 2019-03-29 | 2022-02-01 | 布里格姆及妇女医院股份有限公司 | Targeted synergistic cancer immunotherapy |
Non-Patent Citations (3)
| Title |
|---|
| CHUNYAN CHEN等: ""A multifunctional-targeted nanoagent for dual-mode image-guided therapeutic effects on ovarian cancer cells"", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
| WAN XIE等: ""The Destruction Of Laser-Induced Phase-Transition Nanoparticles Triggered By Low-Intensity Ultrasound： An Innovative Modality To Enhance The Immunological Treatment Of Ovarian Cancer Cells"", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
| 赵光宗: ""光声联合携氧载脂多糖/吲哚菁绿液态氟碳纳米粒对小鼠宫颈移植瘤的抑制及免疫诱导作用研究"", 《重庆医科大学硕士学位论文》 * |
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