CN112826932A - Preparation method and application of lipopolysaccharide-loaded composite nanoparticles - Google Patents
Preparation method and application of lipopolysaccharide-loaded composite nanoparticles Download PDFInfo
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Abstract
The invention discloses a preparation method and application of lipopolysaccharide-loaded composite nanoparticles, and belongs to the technical field of biomedicine. The lipopolysaccharide composite nanoparticle has a stable outer membrane, the outer membrane is made of polylactic-co-glycolic acid (PLGA), and a mixed aqueous solution consisting of liquid fluorocarbon (PFP), indocyanine green (ICG), Lipopolysaccharide (LPS) and water is encapsulated in the nanoparticle; in the composite nanoparticle, the loading capacity of ICG is 10.61% +/-0.12%, and the loading capacity of lipopolysaccharide is 1.70%. According to the lipopolysaccharide composite nanoparticles, on the basis of PLGA nanoparticles, immune adjuvants LPS, ICG, oxygen and oxygen-carrying phase-change material PFP are added, so that the stability and half-life period of ICG in the circulation process are improved, the ICG can be combined with photoacoustic power to induce the immune response of an organism to generate, an immune system is activated together with LPS, and the lipopolysaccharide composite nanoparticles have popularization and application values in the aspect of tumor immunotherapy.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method and application of lipopolysaccharide-loaded composite nanoparticles.
Background
Over the past decade, integration of diagnosis and treatment has become an aggressive area of cancer treatment research, which has led to a tighter integration of diagnosis and treatment, making treatment regimens more individualized. Phototherapy (such as photothermal therapy PTT and photodynamic therapy PDT) has great advantages in the emerging tumor treatment due to its advantages of small invasiveness, small side effects, easy control, etc. Sonodynamic therapy (SDT) is a new method of tumor treatment developed on the basis of photodynamic therapy (PDT). Different from PDT, PDT has poor tissue penetration, ultrasonic energy can penetrate into tissues, and tumor tissues are taken as targets to mediate cytotoxicity of the ultrasonic sensitizer. The photoacoustic combination therapy (SPDT) is a method which can exert respective advantages and enhance immune protection function synergistically.
Indocyanine green (ICG) is an FDA approved reagent for determining liver function and hepatic blood flow. In addition, ICG, as a dye with a spectral absorption peak around 800nm, can be used for PDT and PTT treatment. ICG can produce ROS under the mediation of light and sound, and ROS can induce Immunogenic Cell Death (ICD) of tumor cells, while releasing impaired associated molecular patterns (DAMP), including Calreticulin (CRT) and high mobility group box 1 (HMGB 1). The release of DAMP activates the immune system, particularly by inducing the maturation of Dendritic Cells (DCs) which eventually migrate to lymph nodes, and the interaction of antigens with naive T cells to make them differentiated CTLs (CD 8)+). In addition, when ICD occurs, the induced dead tumor cells may release tumor associated antigens. These dying tumor cells can be used as a whole cell cancer vaccine that induces immunity to all potential tumor antigens released. Therefore, ICG can generate 'tumor vaccine' at the tumor part under the action of light and sound, overcomes the heterogeneity of tumor antigens, and is a very effective and simple method for obtaining high-immunogenicity tumor vaccine and using the tumor vaccine for cancer immunotherapy.
Lipopolysaccharide (LPS) is a component of gram-negative outer membrane of bacteria, and Toll-like receptors 4(TLR4) has high expression in monocytes (such as DC and macrophages), lymphocytes and splenocytes of immune system, and can selectively recognize LPS. DCs are the most powerful Antigen Presenting Cells (APCs) and also the key mediators of the adaptive immune response. LPS promotes antigen processing and presentation by activating TLR4 to affect expression of APC surface costimulatory molecules and control antigen uptake, a key element in the induction and regulation of adaptive immunity. In addition, LPS and DAMP can promote the secretion of cytokines such as IFN-gamma, TNF-alpha, IL-2, IL-12 and IL-6 to increase, and the cytokines play an important role in antitumor treatment.
Disclosure of Invention
In view of the above, the present invention aims to provide a preparation method and an application of lipopolysaccharide-loaded composite nanoparticles.
Through research, the invention provides the following technical scheme:
1. a lipopolysaccharide-loaded composite nanoparticle having a stable outer membrane made of polylactic-co-glycolic acid (PLGA), the nanoparticle having encapsulated therein a mixed aqueous solution consisting of liquid fluorocarbon (PFP), indocyanine green (ICG), Lipopolysaccharide (LPS), and water; in the nanoparticles, the loading amount of indocyanine green is 10.61% +/-0.12%, and the loading amount of lipopolysaccharide is 1.70%.
Preferably, the nanoparticles are of a spherical structure, and the particle size of the nanoparticles is 195.24 +/-11.56 nm.
Preferably, the zeta potential of the nanoparticles is-38.45. + -. 0.59 mV.
Preferably, the liquid fluorocarbon is at least one of perfluoropentane and perfluorohexane.
2. The preparation method of the lipopolysaccharide-loaded composite nanoparticle comprises the following steps:
dissolving polylactic acid-glycolic acid copolymer (PLGA) in dichloromethane to obtain a mixed solution;
introducing oxygen into the liquid fluorocarbon (PFP) for 5-10 min to obtain oxygen-carrying liquid fluorocarbon (PFP);
adding oxygen-carrying liquid fluorocarbon (PFP), Lipopolysaccharide (LPS) and indocyanine green (ICG) into water, carrying out ultrasonic treatment for 0.5-1 min, and introducing oxygen into the mixture for 5-10 min to obtain an emulsion; the polylactic acid-glycolic acid copolymer (PLGA), dichloromethane, liquid fluorocarbon (PFP), Lipopolysaccharide (LPS) and indocyanine green (ICG) are 100-300: 15-20: 1-2: 5-10: 15-20 in terms of g: L: L: L: g;
and mixing the mixed solution with the emulsion, carrying out ultrasonic emulsification for 3-5 min, pouring into a polyvinyl alcohol (PVA) aqueous solution, carrying out ultrasonic treatment for 1-3 min, adding an isopropanol aqueous solution, stirring for 4-6 h, then centrifuging for 3-8 min, taking the precipitate, washing with water, centrifuging again, and suspending the obtained precipitate in oxygenated water.
Preferably, the polylactic-co-glycolic acid (PLGA), dichloromethane, liquid fluorocarbon (PFP), Lipopolysaccharide (LPS) and indocyanine green (ICG) are 100:15:2:5:15 in g: L: L: g.
Preferably, the steps of preparing the mixed solution, the oxygen-carrying liquid fluorocarbon, the emulsion and the precipitate are all carried out under the conditions of low temperature and light shielding.
Preferably, the amount of the polyvinyl alcohol (PVA) aqueous solution is 5mL, and the amount of the isopropanol aqueous solution is 10 mL.
Preferably, the concentration of the polyvinyl alcohol in the aqueous solution of polyvinyl alcohol (PVA) is 1%, and the volume percentage of the isopropanol in the aqueous solution of isopropanol is 2%.
3. The lipopolysaccharide-loaded composite nanoparticles are applied to the preparation of tumor drugs by being used as a sonosensitizer in ultrasound and combined with optoacoustic.
The invention has the beneficial effects that:
1) according to the lipopolysaccharide-loaded composite nanoparticles (OLI _ NPs), on the basis of PLGA nanoparticles, an immunologic adjuvant LPS, a photosensitizer ICG, oxygen and an oxygen-carrying phase-change material PFP are added, so that novel diagnosis and treatment integrated multifunctional nanoparticles with phase change type, namely oxygen-carrying lipopolysaccharide/ICG liquid fluorocarbon nanoparticles are constructed, wherein when ICG is irradiated by visible light with proper wavelength, the internal temperature of the nanoparticles can be increased along with the time, and the PFP undergoes phase change along with the further increase of the temperature, so that liquid is converted into gas, and Ultrasonic (US) and Photoacoustic (PA) imaging is facilitated; lipopolysaccharide (LPS) is a component of gram-negative bacteria outer membrane, can be selectively recognized by Toll-like receptor 4(TLR4), the receptor is highly expressed in monocyte cells (such as dendritic cells and macrophages) of immune system, and LPS has good effect in tumor immunotherapy by activating immune cells. When the tumor part is irradiated by combining with photoacoustic radiation, the tumor cells can be induced to generate immunogenic death, so that HMGB1 is released, CRT is expressed on the cell surface, LPS is released simultaneously by the cracked nanoparticles, APC can be gathered and matured at the tumor part, the antigen presentation efficiency is improved, and the aim of effectively activating immune response is fulfilled.
2) The preparation method of the lipopolysaccharide-loaded composite nanoparticles (OLI _ NPs) is simple and easy to operate, the used materials are low in price and high in feasibility, and the prepared lipopolysaccharide-loaded composite nanoparticles can be used as a contrast agent and a sensitizer at the same time, and have popularization and application values in the fields of ultrasonic imaging, photoacoustic imaging and tumor immunotherapy.
Drawings
FIG. 1 is a transmission electron microscope analysis chart of lipopolysaccharide-loaded composite nanoparticles of the present invention;
FIG. 2 is a graph showing the results of particle size analysis of the lipopolysaccharide-loaded composite nanoparticles of the present invention;
FIG. 3 is a diagram showing the results of the optical microscopic analysis of the lipopolysaccharide-loaded composite nanoparticles of the present invention;
FIG. 4 is a diagram showing the results of confocal microscopy analysis of lipopolysaccharide-loaded composite nanoparticles according to the present invention;
FIG. 5 is a graph showing the results of UV absorption spectroscopy analysis of the lipopolysaccharide-loaded composite nanoparticles of the present invention;
FIG. 6 is a graph showing the results of fluorescence spectroscopy analysis of lipopolysaccharide-loaded composite nanoparticles according to the present invention;
FIG. 7 is a graph of the results of in vitro ultrasound imaging analysis of lipopolysaccharide-loaded composite nanoparticles of the present invention;
FIG. 8 is a graph showing the results of in vitro photoacoustic imaging analysis of lipopolysaccharide-loaded composite nanoparticles of the present invention;
FIG. 9 is a graph of the results of in vivo photoacoustic imaging analysis of lipopolysaccharide-loaded composite nanoparticles of the present invention;
fig. 10 is a graph of the results of in vivo photoacoustic imaging comparative analysis of lipopolysaccharide-loaded composite nanoparticles and free indocyanine green according to the present invention;
FIG. 11 is a graph comparing photoacoustic imaging effects before and after injecting lipopolysaccharide-loaded composite nanoparticles;
FIG. 12 is a graph comparing the photoacoustic imaging effect of lipopolysaccharide-loaded composite nanoparticles injected with free ICG;
FIGS. 13 and 14 are graphs comparing DC cell maturation in tumors;
FIG. 15 is a graph comparing the killing of tumor cells by lymphocytes;
FIGS. 16 and 17 are graphs comparing tumor growth in mice.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In this embodiment, the preparation method of the lipopolysaccharide-loaded composite nanoparticle includes the following steps:
1) dissolving 100mg of polylactic-co-glycolic acid (PLGA) in 20mL of dichloromethane to obtain a mixed solution;
2) slowly introducing oxygen into liquid fluorocarbon (PFP) for 5min to obtain oxygen-carrying liquid fluorocarbon (PFP), adding 2mL of the oxygen-carrying liquid fluorocarbon (PFP), 5mL of Lipopolysaccharide (LPS) and 15mg of indocyanine green (ICG) into water, performing ultrasonic treatment for 0.5min, and introducing oxygen into the mixture for 5min to obtain an emulsion;
3) mixing the mixed solution obtained in the step 1) with the emulsified liquid obtained in the step 2), emulsifying for 3min by using an ultrasonic vibrometer, pouring into 5mL of PVA aqueous solution (1% w/v), performing vibro-acoustic treatment for 3min, adding 10mL of isopropanol aqueous solution (2% v/v), magnetically stirring for 4h to fully volatilize dichloromethane, finally performing high-speed centrifugation for 5min, removing supernatant, taking precipitate, fully washing by using double distilled water, centrifuging again, repeating for three times, re-suspending the obtained precipitate in 5mL of oxygenated double distilled water, and storing at 4 ℃ for later use. The steps are carried out under the conditions of low temperature and light protection.
Through determination, the average potential of the indocyanine green composite nanoparticles prepared in example 1 is-38.45 mV, the encapsulation efficiency of ICG is 84.86%, the encapsulation efficiency of LPS is about 40%, the loading capacity of indocyanine green is 10.61% ± 0.12%, and the loading capacity of lipopolysaccharide is 1.70%.
Detection assay
1) Transmission electron microscopy analysis
The specific operation is as follows: the lipopolysaccharide-loaded composite nanoparticles prepared in example 1 were diluted by a certain fold with double distilled water, and observed by a transmission electron microscope, and the results are shown in fig. 1.
As can be seen from the analysis in fig. 1, the indocyanine green composite nanoparticles have a smooth and spherical surface with a size of about 200nm, an outer layer coated with PLGA, and an inner portion composed of an aqueous solution of PFP mixed with ICG and LPS, wherein PFP is the higher density, and the aqueous solution is the lower density.
2) Particle size analysis
The specific operation is as follows: the particle size of the lipopolysaccharide-loaded composite nanoparticles prepared in example 1 was measured by a Malvern particle sizer, and the results are shown in FIG. 2.
As can be seen from the analysis in FIG. 2, the particle size distribution of the lipopolysaccharide-loaded composite nanoparticles prepared in example 1 is between 150 nm and 350nm, the symmetry is good, and the average particle size is 195.24 nm.
3) Optical mirror analysis
The specific operation is as follows: the lipopolysaccharide-loaded composite nanoparticles prepared in example 1 were diluted by a certain fold with double distilled water, and then analyzed by a light microscope, and the results are shown in fig. 3.
From the analysis in fig. 3, it can be seen that the indocyanine green composite nanoparticles prepared in example 1 have uniform size and good dispersibility under a light mirror, and after the nanoparticles are heated at a high temperature of 49 ℃ for 1min, the nanoparticles are significantly increased to generate a phase change phenomenon, thereby proving that the lipopolysaccharide-loaded composite nanoparticles are phase change nanoparticles.
4) Confocal microscopy analysis
The specific operation is as follows: the lipopolysaccharide-loaded composite nanoparticles prepared in example 1 were observed and analyzed under a confocal microscope, and the results are shown in fig. 4.
As can be seen from the analysis in fig. 4, under a confocal microscope, green light was LPS, red light was ICG, and the combined light was yellow.
5) Ultraviolet absorption spectroscopy and fluorescence spectroscopy
The specific operation of the ultraviolet absorption spectrum is as follows: ultraviolet absorption spectrum analysis is respectively carried out on the lipopolysaccharide-loaded composite nanoparticles and the indocyanine green prepared in the embodiment 1, so that the initial ultraviolet absorbances of the lipopolysaccharide-loaded composite nanoparticles and the indocyanine green are consistent, and the ultraviolet absorbances are observed once every three days.
The specific operation of the fluorescence spectrum analysis is as follows: respectively carrying out fluorescence spectrum analysis on the lipopolysaccharide-loaded composite nanoparticles and indocyanine green prepared in the embodiment 1 to enable the initial fluorescence absorbances of the lipopolysaccharide-loaded composite nanoparticles and the indocyanine green to be consistent, and observing the fluorescence absorbances once every three days. The results are shown in fig. 5, 6, 7 and 8.
From the comprehensive analysis in fig. 5 and fig. 7, it can be seen that the ultraviolet absorption peak and the fluorescence emission peak of the indocyanine green composite nanoparticles (OLI _ NPs) are substantially consistent with those of the free ICG solution. Indicating that the optical properties of ICG were not altered during nanoparticle preparation. In FIG. 6, by measuring the UV absorbance at 780nm every three days for OLI _ NPs and free ICG, the free ICG decreased dramatically compared to OLI _ NPs, the ICG absorption intensity in OLI _ NPs decreased by about 20% of the initial intensity, and the free ICG decreased significantly by 70% of the initial intensity, over a 15-day observation period. In FIG. 8, the fluorescence absorbance at 780nm was measured every three days for OLI _ NPs and free ICG, and both free ICG decreased dramatically compared to OLI _ NPs, with the ICG absorbance in OLI _ NPs decreasing by about 18% of the initial intensity and free ICG decreasing significantly by 74% of the initial intensity over a 15-day observation period. The results demonstrate that the optical stability of ICG in PLGA is significantly improved compared to free ICG.
6) In vitro bimodal imaging analysis
The specific operation is as follows: performing in-vitro photoacoustic imaging detection and analysis on the lipopolysaccharide-loaded composite nanoparticles prepared in the embodiment 1, adding 4g of agarose gel powder into 800ml of deionized water, gradually heating until the liquid is clear and has no bubbles, quickly pouring the liquid into a mold box, inserting a 1ml gun head or a 200 mu l gun head, and putting the mold into a refrigerator at 4 ℃ for later use after the mold is solidified into blocks. In ultrasound imaging, a mold with a 1ml per well size was used, in four groups: a PBS group; ② free ICG group (ICG 160. mu.g/ml, 1 ml); ③ Blank NPs group (PLGA 1.25mg/ml, 1 ml); OLE _ NPs group (ICG 160. mu.g/ml, 1 ml). In photoacoustic imaging, a mold with a pore size of 200 μ l was used, in four groups: a PBS group; ② a free ICG group (ICG 638. mu.g/ml, 200. mu.l); ③ Blank NPs group (PLGA 5mg/ml, 200. mu.l); OLE _ NPs group (ICG 638. mu.g/ml, 200. mu.l). The results are shown in FIGS. 9 and 10.
FIG. 9 is an ultrasonic imaging image before and after irradiation of 808nm laser (1.5w 5min) and after action of low-power focused ultrasound (LIFU, the equipment parameters are as follows: the ultrasonic output frequency is 650KHz +/-10%, the focal length of a first channel treatment head is 28mm +/-15%, and the focal length of a second channel treatment head is 12.5mm +/-15%), and FIG. 10 is an opto-acoustic imaging before and after irradiation of 808nm laser and after action of LIFU (3w 1 min).
As can be seen from the ultrasonic image of fig. 9, in both the B mode and CEUS mode after laser irradiation, there was no image enhancement in the PBS group and the free ICG group (free ICG). After irradiating for 5 minutes by using 808nm laser, B mode signals of Blank NPs group ultrasonic imaging are obviously enhanced. This growth may be due to phase transition of Blank NPs due to slight thermal radiation after irradiation with the near-infrared laser. While the ultrasonic signal B-mode and CEUS of the OLI _ NPs group are significantly enhanced compared to the other groups. The phase change occurs by the temperature rise of PFP caused by the excitation of ICG, which leads to the obvious enhancement of ultrasonic imaging. Thus, it is proved that the OLI _ NPs can be phase-changed by near infrared light irradiation and can become contrast agents for ultrasonic imaging.
From the analysis of the photoacoustic imaging chart in fig. 10, 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 encapsulated ICG do not show the capability of photoacoustic imaging enhancement since they do not have light absorption characteristics in the near infrared range. And after the laser irradiation of 808nm, the photoacoustic signal of the OLI _ NPs group is obviously enhanced compared with other groups. Thus, it is proved that the OLI _ NPs can change phase by irradiation of near infrared light and can become contrast agents for ultrasonic imaging, and the OLI _ NPs can also serve as a good photoacoustic imaging sensitizer.
In ultrasonic imaging or photoacoustic imaging, after laser irradiation, signals of the OLI _ NPs are obviously enhanced, and then after nanoparticles are irradiated by ultrasonic, the signals can be obviously weakened, so that the nanoparticles are cracked, and the step is also the key for releasing the drugs at tumor sites.
7) In vivo photoacoustic imaging analysis
The specific operation is as follows: lipopolysaccharide-loaded composite nanoparticles (ICG 638. mu.g/mL, 750. mu.L) prepared in example 1 and free ICG (ICG 638. mu.g/mL, 750. mu.L) were injected intravenously into the tail of tumor-bearing mice, and then the tumor parts at different time points were analyzed by photoacoustic imaging detection in vivo, and the results are shown in FIG. 11.
Fig. 11 is a graph comparing photoacoustic imaging effects before and after injecting lipopolysaccharide-loaded composite nanoparticles. As can be seen from the analysis in fig. 11, before injection, there was no photoacoustic signal at the tumor site, and after 2h of nanoparticle injection, photoacoustic signals appeared at the tumor site gradually, and the signals were very weak; the photoacoustic signal reaches the strongest after 4h, which indicates that the number of the nanoparticles reaches the maximum value at the moment, and the time point is the optimal imaging and treatment point; over time, the nanoparticle signal at the tumor site gradually diminished.
Fig. 12 is a graph comparing the photoacoustic imaging effect of lipopolysaccharide-loaded composite nanoparticles injected with free ICG. As can be seen from the analysis in fig. 12, no photoacoustic signal was observed in vivo for free ICG, either before or after injection. After the OLI _ NPs4h is injected, the imaging can be obviously carried out on the tumor part, and the photoacoustic signal is obviously enhanced after the 1.5w laser is irradiated for 5 min. After 3w ultrasonic action for 1min, the photoacoustic signal is obviously reduced, which indicates that the nanoparticles are cracked at the moment, and the physical effect generated by cracking is also one of the important influence factors for killing tumors.
8) Nanoparticle combined with photoacoustic for treating tumor and inducing immune response
The specific operation is as follows: subcutaneous nodules in the left dorsal part of the mouse with cell number1×105Four days later, equal cell numbers were seeded in the contralateral side until the left tumor grew to 100mm3And the method is divided into four groups: (1) saline group, (2) PSDT group, (3) OLI _ NPs group, (4) OLI _ NPs + PSDT group, left tumors were removed on day 7 after treatment, and intratumoral DC cell maturation was analyzed, with the results shown in fig. 13 and 14: spleen lymphocytes and tumor cells are taken for co-culture, and the killing condition of the tumor cells by the lymphocytes is observed, and the result is shown in figure 15; the rest mice continue to observe the growth of the tumor, and the growth trend of bilateral tumors is analyzed, and the result is shown in fig. 16 and 17.
From the analysis in fig. 13 and 14, the ratio of mature DC in the OLI _ NPs + PSDT group is the largest, and there is no difference in other 3 groups, thus demonstrating that lipopolysaccharide nanoparticle in combination with photoacoustic therapy can promote antigen-presenting cell maturation. From the analysis in fig. 15, it can be seen that spleen lymphocytes in the OLI _ NPs + PSDT group had the highest ability to kill tumor cells, demonstrating that the lipopolysaccharide nanoparticle in combination with photoacoustic therapy can induce CTL generation. It can be analyzed from fig. 16 and 17 that the lipopolysaccharide nanoparticle combined photoacoustic therapy can not only inhibit the growth of in-situ tumor, but also obviously inhibit the growth of distant tumor, so that the lipopolysaccharide-loaded composite nanoparticle combined photoacoustic therapy can effectively stimulate the immune system in the body of a mouse to achieve the purpose of controlling the growth of tumor.
The lipopolysaccharide-loaded composite nanoparticle prepared by the invention is a multifunctional nanoparticle for diagnosis and treatment integration, LPS is successfully wrapped by the nanoparticle, so that the lipopolysaccharide-loaded composite nanoparticle has the capability of activating immune cells such as dendritic cells, ICG in the nanoparticle not only keeps physical characteristics, but also is not easy to extract and kill, and has better stability compared with free ICG, PFP is used as a phase-change oxygen-carrying material, ultrasonic imaging is facilitated while the nanoparticle is subjected to phase change, the PFP and the ICG participate in photoacoustic imaging together, and accurate basis is provided for judging the time when the nanoparticle reaches a tumor part.
In the lipopolysaccharide-loaded composite nanoparticle, PFP takes oxygen carrying as assistance, mainly reflects the function in the aspect of imaging, and is used as a substance which can be subjected to phase change along with temperature rise and has a synergistic effect with ICG, so that the particle size of the nanoparticle is increased and even broken, and the effects of enhancing the imaging effect and killing tumor cells are achieved. Indocyanine green (ICG) is a near-infrared dye with high biocompatibility, but ICG is unstable in circulation and short in half-life, is easy to degrade after long-time near-infrared light irradiation, and not only solves the problem that ICG is easy to extract and kill after being coated in a protective manner by a nano structure, but also enhances the photoacoustic imaging effect of ICG by cooperating with PFP, so that the stability and half-life of ICG are improved by nanoparticles.
The killing means of the lipopolysaccharide-loaded composite nanoparticles to tumor tissues mainly comprises three aspects: the killing effect of the photothermal effect of the ICG on the tumor; the photodynamic action of ICG can induce the immunogenic death of tumor cells, which is the key point of the invention; c. the cavitation effect generated by the nanoparticle rupture can also kill tumor cells. Therefore, the lipopolysaccharide-loaded composite nanoparticles have multiple treatment ways on tumor tissues, and mainly treat the tumor tissues from the aspect of immunity.
In the lipopolysaccharide-loaded composite nanoparticle, the added immune adjuvant LPS can activate TLR4, and the antigen processing and presentation are promoted by influencing the expression of Antigen Presenting Cell (APC) surface costimulatory molecules and controlling antigen uptake, so that the lipopolysaccharide-loaded composite nanoparticle is a key medium for starting and regulating adaptive immune response. The photodynamic action of ICG can induce tumor cells to generate immunogenic death, thereby exposing tumor antigens, LPS can recruit APC to promote the processing and presentation of the antigens, and the synergistic effect of the APC and the LPS can effectively activate an immune system, thereby achieving the purposes of effectively radically treating tumors and preventing tumor recurrence.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A lipopolysaccharide-loaded composite nanoparticle having a stable outer membrane made of polylactic-co-glycolic acid (PLGA), the nanoparticle encapsulating a mixed aqueous solution consisting of liquid fluorocarbon (PFP), indocyanine green (ICG), Lipopolysaccharide (LPS), and water; in the nanoparticles, the loading amount of indocyanine green is 10.61% +/-0.12%, and the loading amount of lipopolysaccharide is 1.70%.
2. The lipopolysaccharide-loaded composite nanoparticles according to claim 1, wherein the nanoparticles are of spherical configuration, and the particle size of the nanoparticles is 195.24 ± 11.56 nm.
3. The lipopolysaccharide-loaded composite nanoparticles according to claim 1, wherein the zeta potential of the nanoparticles is-38.45 ± 0.59 mV.
4. The lipopolysaccharide-loaded composite nanoparticles according to claim 1, wherein the liquid fluorocarbon is at least one of perfluoropentane and perfluorohexane.
5. The method for preparing the lipopolysaccharide complex nanoparticle according to any one of claims 1 to 4, comprising the steps of:
dissolving polylactic acid-glycolic acid copolymer (PLGA) in dichloromethane to obtain a mixed solution;
introducing oxygen into the liquid fluorocarbon (PFP) for 5-10 min to obtain oxygen-carrying liquid fluorocarbon (PFP);
adding oxygen-carrying liquid fluorocarbon (PFP), Lipopolysaccharide (LPS) and indocyanine green (ICG) into water, carrying out ultrasonic treatment for 0.5-1 min, and introducing oxygen into the mixture for 5-10 min to obtain an emulsion; the polylactic acid-glycolic acid copolymer (PLGA), dichloromethane, liquid fluorocarbon (PFP), Lipopolysaccharide (LPS) and indocyanine green (ICG) are 100-300: 15-20: 1-2: 5-10: 15-20 in terms of g: L: L: L: g;
and mixing the mixed solution with the emulsion, carrying out ultrasonic emulsification for 3-5 min, pouring into a polyvinyl alcohol (PVA) aqueous solution, carrying out ultrasonic treatment for 1-3 min, adding an isopropanol aqueous solution, stirring for 4-6 h, then centrifuging for 3-8 min, taking the precipitate, washing with water, centrifuging again, and suspending the obtained precipitate in oxygenated water.
6. The preparation method of the lipopolysaccharide-loaded composite nanoparticle according to claim 5, wherein the polylactic-co-glycolic acid (PLGA), dichloromethane, liquid fluorocarbon (PFP), Lipopolysaccharide (LPS) and indocyanine green (ICG) are 100:15:2:5:15 in terms of g: L: L: L: g.
7. The preparation method of the lipopolysaccharide composite nanoparticles according to claim 5, wherein the steps of preparing the mixed solution, the oxygen-carrying liquid fluorocarbon, the emulsion and the precipitate are all performed at 0-4 ℃ in the absence of light.
8. The preparation method of the lipopolysaccharide composite nanoparticle according to claim 5, wherein the amount of the polyvinyl alcohol (PVA) aqueous solution is 5mL, and the amount of the isopropanol aqueous solution is 10 mL.
9. The method for preparing the lipopolysaccharide composite nanoparticle according to claim 8, wherein the concentration of polyvinyl alcohol (PVA) in the aqueous solution of polyvinyl alcohol (PVA) is 1%, and the volume percentage of isopropanol in the aqueous solution of isopropanol is 2%.
10. Use of the lipopolysaccharide-loaded composite nanoparticles according to any one of claims 1 to 4 as sonosensitizers in ultrasound and in combined photoacoustic preparation of tumor drugs.
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