CN113559064A

CN113559064A - Novel self-oxygen-supply liposome nanoparticle and preparation method and application thereof

Info

Publication number: CN113559064A
Application number: CN202110853423.8A
Authority: CN
Inventors: 肖红俊; 谭松巍; 吴楠; 凃雅琴; 宗世民; 王维; 栗浩
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2021-10-29
Anticipated expiration: 2041-07-27
Also published as: CN113559064B

Abstract

The invention discloses a novel self-oxygen-supply liposome nanoparticle and a preparation method and application thereof. The novel self-oxygen-supply liposome nanoparticle is a core-shell structure compound and comprises an inner core containing peroxide and a lipid bilayer shell coating the inner core; the lipid bilayer shell is loaded with indocyanine green fluorescent dye; the self-oxygen-supplying liposome nanoparticle prepared by the invention has a typical shell-core spherical structure, is uniform in size and has good stability, and the stability is improved after ICG is inserted into a liposome lipid layer; meanwhile, the liposome can realize pH and temperature responsive release, improve local tumor hypoxia, combine photodynamic and photothermal effects and inhibit the growth of tumor cells in vivo; in addition, the compound has the effect of passively targeting tumor parts, can improve the distribution of the medicine, prolong the half-life period of the medicine in vivo, improve the bioavailability of the medicine and has good biological safety.

Description

Novel self-oxygen-supply liposome nanoparticle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials and nanomedicine, and particularly relates to novel self-oxygen-supply liposome nanoparticles as well as a preparation method and application thereof.

Background

Nasopharyngeal carcinoma (NPC) is one of typical malignant tumors, and seriously affects the survival and health of human beings. The current treatment modalities for nasopharyngeal carcinoma are mainly limited to traditional chemotherapy, radiation therapy, surgical therapy and some optional complementary approaches. Radiotherapy and chemotherapy are the first choice for the treatment of nasopharyngeal carcinoma, but the occurrence part of nasopharyngeal carcinoma is hidden, and early distant metastasis is the main reason of the failure of radiotherapy and chemotherapy; in addition, after the traditional chemotherapy intravenous administration, because of the lack of tumor targeting, the toxic and side effects on the whole body are great. The first-line chemotherapy medicament for treating nasopharyngeal carcinoma comprises: cisplatin, paclitaxel, 5-fluorouracil and the like have great bone marrow suppression, digestive tract reaction and auditory nerve toxic and side effects. Therefore, the development of a targeted drug delivery system by combining with the traditional chemotherapeutic drugs or the direct development of new targeted drugs becomes a hot spot of the current tumor treatment research.

Liposomes have long been approved as the first generation of nanoscale drug delivery systems for the treatment of cancer and fungal infections. It is a spherical structure formed by wrapping a hydrophilic core by double-layer lipid (mainly phospholipid) at the periphery. Depending on the water solubility of the drug, it may be encapsulated in the hydrophilic core of the liposome or in the surrounding phospholipid bilayer. Hydrophobic drugs are incorporated into the lipid membrane, while hydrophilic drugs are encapsulated within the central aqueous core. Liposomes have good biocompatibility, low toxicity and low immunogenicity, adjustable physicochemical and biophysical properties, and a unique ability to carry lipophilic and hydrophilic drugs, which makes them very promising for drug delivery and gene therapy.

Indocyanine green (ICG) is an amphiphilic fluorescent dye, the only clinically used near-infrared imaging agent approved by the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA), and has been used clinically in a variety of biomedical applications, including measurement of cardiac output, monitoring of liver function, and ophthalmic angiography. Its maximum absorption in aqueous solution is around 780nm, and in lipid environment is slightly more than 800 nm. The ICG fluorescence emission peak is around 810-830nm, and the fluorescence interference from blood and tissue (500-600nm) is extremely small at this wavelength. In addition, ICG is also a photosensitizer and a photothermal conversion agent, and can generate active oxygen and heat energy under irradiation of near-infrared laser light. And ICG molecules are very toxic at therapeutic concentrations. However, free ICG is rapidly degraded in aqueous solution due to oxidation of the double bond, not only poorly targeted, but also highly bound to plasma proteins (98%) in blood and rapidly cleared by the liver (t1/2 ≈ 2-4 min). It tends to aggregate at high concentrations and to undergo fluorescence quenching due to protein binding, thermal degradation or photobleaching, which severely limits the clinical utility of ICG.

Hypoxia within the Tumor Microenvironment (TME) is an intrinsic property of all solid malignancies. Hypoxic TME is widely recognized as an independent prognostic indicator, which has been associated with poor survival rates for various cancer types, including head and neck cancer, breast cancer, non-small cell lung cancer, cervical cancer and ovarian cancer. Hypoxia is also a major obstacle for various therapies against tumors, including photodynamic therapy (PDT) and photothermal therapy (PTT), because photosensitizers transfer near-infrared laser energy using molecular oxygen to generate highly reactive singlet oxygen and other Reactive Oxygen Species (ROS) that are induced directly or indirectly and release heat. Under laser irradiation, photosensitizers convert oxygen into ROS, which exacerbates tumor hypoxia. In addition, vessel closure due to PDT will also lead to severe hypoxia. Low oxygen levels in turn can severely inhibit ROS production in PDT, thereby limiting its therapeutic effectiveness. Therefore, the key to improving the PDT and PTT curative effect is to relieve the tumor cell hypoxia on the premise of high biological safety.

Therefore, there is a need to develop a liposome nanoparticle which uses liposome as a carrier, combines ICG and other peroxides to prepare the liposome nanoparticle, realizes pH and temperature responsive release of peroxides, can improve local tumor hypoxia, and combines photodynamic and photothermal effects to inhibit the growth of tumor cells in vivo.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides novel self-oxygen-supply liposome nanoparticles as well as a preparation method and application thereof. The liposome nanoparticle is prepared by taking liposome as a carrier and combining ICG and other peroxides, realizes pH and temperature responsive release of the peroxides, can improve local tumor hypoxia, and combines photodynamic and photothermal effects to inhibit the growth of tumor cells in vivo.

One object of the present invention is to provide a novel self-oxygen-supplying liposome nanoparticle.

A novel self-oxygen-supply liposome nanoparticle is a core-shell structure compound and comprises a kernel containing peroxide and a lipid bilayer shell coating the kernel; the lipid bilayer shell is loaded with indocyanine green fluorescent dye.

Further, the peroxide is selected from one of zinc peroxide, magnesium peroxide, barium peroxide and calcium peroxide.

Further, the lipid bilayer shell is also coated with polyethylene glycol.

Further, the polyethylene glycol is distearoylphosphatidylethanolamine-polyethylene glycol 2000, and the distearoylphosphatidylethanolamine-polyethylene glycol 2000 is obtained by reacting polyethylene glycol 2000 and distearoylphosphatidylethanolamine.

Further, the lipid bilayer includes a phospholipid and cholesterol, the cholesterol being embedded within the interior of the phospholipid.

Still further, the phospholipid is selected from one of soybean lecithin, egg yolk lecithin, glycerophospholipid, ethanolamine phospholipid.

In the invention, the PEG has strong water solubility because a large amount of ethoxy exists in the molecule, and the ethoxy can form hydrogen bonds with water molecules, so that the PEG can form a hydrated film on the surface of the liposome, and in addition, the PEG covers the surface of the liposome and prevents plasma components from approaching the liposome, thereby effectively helping the liposome avoid the recognition and phagocytosis of reticuloendothelial system (RES), and obviously prolonging the half-life period of the liposome in blood circulation. ICG as near infrared photosensitizer and photothermal converter, ZnO₂As an oxygen supply body responsive to the tumor microenvironment, loaded into the lipid membrane and the internal aqueous phase of the liposome, respectively. Upon irradiation with near infrared light (808nm), the ICG in the outer lipid layer gains sufficient energy upon photochemical conversion to generate heat to raise the local temperature and detonate the liposomes, thereby releasing ZnO₂。ZnO₂In the tumor microenvironment (H-rich)⁺) Rapid generation of O₂Relieving tumor cell hypoxia, and simultaneously ZnO₂Produced O₂Further used by photosensitizer ICG, and can produce singlet oxygen continuously under the irradiation of near infrared light to kill tumor cells so as to raise the PDT therapeutic effect. ZnO obtained finally₂@ Lip-ICG has physiological stability, long cycle time and tumor targeting ability, it can effectively penetrate into tumor tissue, accumulate in tumor tissue by enhancing permeability and retention Effect (ERP), simultaneously deliver oxygen and photosensitizer to hypoxic tumor, and respond to near-infrared laser irradiation to generate ROS and raise local temperature, thereby synergistically killing tumor cells.

The invention also aims to provide a preparation method of the novel self-oxygen-supply liposome nanoparticle.

A preparation method of novel self-oxygen-supplying liposome nanoparticles comprises the following steps:

s1, synthesizing peroxide nanoparticles by a one-pot precipitation method;

s2, synthesizing the self-oxygen-supplying liposome nanoparticles by using a thin film hydration method and taking phospholipid, cholesterol, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, indocyanine green and the peroxide nanoparticles described in the step S1 as materials.

Further, the specific synthesis method of step S2 is as follows: dissolving phospholipid, cholesterol, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and indocyanine green in methanol, adding the methanol into the peroxide nanoparticles, and uniformly dispersing by ultrasonic; then, uniformly mixing the solution, and performing rotary evaporation and drying to obtain a film; adding PBS buffer solution into the film, and performing ultrasonic oscillation until the film is completely hydrated to obtain mixed solution; and then centrifuging the mixed solution for 1-2 min under the condition of 450-500 r/min, discarding the bottom precipitate, and filtering by using a 0.45-micrometer filter membrane to obtain the self-oxygenation liposome nanoparticle.

Further, the addition amounts of the phospholipid, the cholesterol, the distearoylphosphatidylethanolamine-polyethylene glycol 2000, the indocyanine green and the peroxide are 4: 1: 1: 0.1: 1.

the last purpose of the invention is to provide the application of the novel self-oxygen-supply liposome nanoparticle.

The novel self-oxygen-supplying liposome nanoparticle is applied to the preparation of photodynamic therapeutic agents and/or drug carriers.

Compared with the prior art, the invention has the following advantages:

1) ZnO prepared by the invention₂@ Lip-ICG liposome, the particle size is 200.5nm, the zeta potential is about-22 mV; the ICG has a typical shell-core spherical structure, uniform size and good stability, and the stability is improved after the ICG is inserted into a liposome lipid layer;

2) ZnO prepared by the invention₂The @ Lip-ICG liposome can realize pH and temperature responsive release, improve local tumor hypoxia, combine photodynamic and photothermal effects and inhibit the growth of tumor cells in vivo.

3) ZnO prepared by the invention₂The @ Lip-ICG liposome has the effect of passively targeting a tumor part, and can improve the distribution of the medicament, prolong the half-life period of the medicament in vivo and improve the bioavailability of the medicament.

4) ZnO prepared by the invention₂The @ Lip-ICG liposome has good biological safety.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows ZnO in example 1 of the present invention₂The infrared spectrum characterization map of the @ Lip-ICG liposome;

FIG. 2 shows ZnO in example 1 of the present invention₂The distribution graph of the particle size of the @ Lip-ICG liposome (FIG. a) and the transmission electron micrograph (FIG. b);

FIG. 3 shows the results of the change of absorbance with time in different groups in example 1 of the present invention, where in FIG. a is the Lip group, in FIG. b is the free ICG group, in FIG. c is the ICG @ Lip group, and in FIG. d is ZnO₂@ Lip-ICG group, panel e free ICG group, ICG @ Lip group and ZnO₂Curve plot of ICG characteristic absorption peak decline rate in @ Lip-ICG group;

FIG. 4 shows dual pH and NIR stimulation on ZnO in example 2 of the present invention₂Graph of the effect of @ Lip-ICG release behaviour;

FIG. 5 shows the results of testing the singlet oxygen generating capacity in vitro of different groups in example 3 of the present invention, wherein FIG. a shows the DPBF group, FIG. b shows the free ICG group, FIG. c shows the ICG @ Lip group, and FIG. d shows the ZnO group₂The @ Lip-ICG group, graph e, contains free ICG, ICG @ Lip and ZnO when laser irradiation is performed for 5min₂A rate profile of DPBF solution degradation over time of @ Lip-ICG;

FIG. 6 shows the 808nm laser (1W/cm) of various solutions in example 4 of the present invention²) Under irradiation, a) thermal imaging at different times; b) graph of temperature versus time; c) ZnO of different concentrations₂@ Lip-ICG laser (1W/cm) at 808nm²) Under irradiation, the temperature changes with time; d) 2.50. mu.g/ml ZnO₂The graph of the change of temperature with time of @ Lip-ICG under the irradiation of laser with different power densities and 808 nm;

FIG. 7 shows the cellular uptake of ZnO in example 5 of the present invention₂The results of the @ Lip-ICG liposome are shown in the figure, a is CNE-2 cell to ZnO under the fluorescence microscope₂The uptake pattern of @ Lip-ICG, panel b for CNE-2 cells on ZnO by flow cytometry₂A histogram of the intake of @ Lip-ICG;

FIG. 8 shows ZnO in example 6 of the present invention₂A detection result graph of the toxicity effect of the @ Lip-ICG liposome on cells;

FIG. 9 shows ZnO in example 7 of the present invention₂Results plot of intracellular ROS production by the @ Lip-ICG liposomes;

FIG. 10 shows ZnO in example 8 of the present invention₂The result of the pharmacokinetics research of the @ Lip-ICG liposome in the SD rat is shown in a graph a, which is a fluorescence imaging graph of rat blood at different time points; panel b is the mean fluorescence intensity values in vivo in rats at different times;

FIG. 11 shows ZnO in example 9 of the present invention₂The distribution of the @ Lip-ICG liposome in the mouse is researched, and a figure is in vivo imaging; panel b is ex vivo imaging;

FIG. 12 shows ZnO in example 9 of the present invention₂The study result of the in vivo photothermal effect of the @ Lip-ICG liposome in mice is shown in the drawing, and a is a thermal imaging graph at different times; FIG. b is a graph showing the results of temperature changes in different processes;

FIG. 13 shows ZnO in example 9 of the present invention₂A staining result graph of the effect of the @ Lip-ICG liposome on the local tumor hypoxia state in the mouse;

FIG. 14 shows ZnO in example 9 of the present invention₂The results of the inhibition of the growth of CNE-2 subcutaneous tumors in the @ Lip-ICG liposome mice are shown in the following graphs, where a is the photograph of the tumor after the treatment is finished, b is the graph of the change in tumor volume, c is the column of the change in tumor weight, d is the graph of the change in weight of the nude mice in each group, and e is the graph of the change in tumor tissue H in each group&E, a staining result graph, and f is a TUNEL immunofluorescence staining result graph of each group of tumor tissues;

FIG. 15 shows ZnO in example 9 of the present invention₂The safety of the @ Lip-ICG liposome in the mouse is evaluated to obtain a staining result graph;

FIG. 16 shows ZnO in example 9 of the present invention₂The test result of the safety evaluation immunohistochemistry of the @ Lip-ICG liposome in mice is shown in the figure, wherein the figure a is the weight result of the main organs of the tumor-bearing mice, the figure b is the AST content bar chart of the blood of each group of mice, and the figure c is the figure of each group of miceA bar graph of ALT content in blood of mice, a bar graph d of BUN content in blood of each group of mice, and a bar graph e of CRE content in blood of each group of mice; wherein: AST: glutamic-oxalacetic transaminase; ALT: glutamic-pyruvic transaminase; BUN: urea nitrogen; and (3) CRE: creatinine.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.

Reagents, other materials and equipment used in the present invention are commercially available unless otherwise specified.

EXAMPLE 1ZnO₂Synthesis and performance characterization of @ Lip-ICG liposome

In the invention, zinc peroxide is selected to synthesize the liposome, and the other peroxide synthesis principles are similar to those of the zinc peroxide.

The invention needs soybean lecithin to synthesize liposome, and other phospholipids such as yolk lecithin, glycerophospholipid, ethanolamine phospholipid and the like can be used for synthesizing liposome.

1.1ZnO₂Synthesis of @ Lip-ICG liposomes

S1, synthesizing zinc peroxide nanoparticles by a one-pot precipitation method, wherein the specific synthesis method comprises the following steps: accurately weighing 2.98g Zn (NO)₃)₂·6H₂Dissolving O in 10ml of methanol, stirring vigorously, adding 10ml of NaOH methanol solution with the molar concentration of 2M into the solution, and continuously stirring for 20-30 min; 1.5ml of H are added further₂O₂Stirring for 4-5 h, centrifuging to obtain a precipitate, and drying the precipitate to obtain the zinc peroxide nanoparticles;

s2, dissolving 20mg of soybean lecithin, 5mg of cholesterol, 5mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and 0.5mg of indocyanine green in 8ml of methanol, adding 2ml of methanol into the zinc peroxide nanoparticles, and uniformly dispersing by ultrasonicHomogenizing; then, uniformly mixing the solution, and performing rotary evaporation and drying to obtain a film; adding 5ml of PBS buffer solution into the film, and performing ultrasonic oscillation until the film is completely hydrated to obtain a mixed solution; then centrifuging the mixed solution for 1-2 min under the condition of 450-500 r/min, discarding the bottom precipitate, and filtering with a 0.45-micrometer filter membrane to obtain ZnO₂@Lip-ICG。

1.2ZnO₂Performance characterization of the @ Lip-ICG liposomes

FT-IR: weighing 200mg of potassium bromide and about 10mg of samples, grinding and mixing the samples by using a mortar, preparing the mixture into semitransparent small round pieces by using a film pressing machine, drying the small round pieces in a drying box, and detecting the small round pieces by using a machine. (for ZnO)₂@ Lip-ICG, ICG @ Lip, freeze-drying a freshly prepared sample by using a negative pressure freeze dryer, grinding, tabletting and detecting; for free ICG, the sample was dropped directly onto a blank potassium bromide press, dried and tested. )

As a result, as shown in FIG. 1, it can be seen that 667cm^-1Is a characteristic absorption peak of the indole ring, 1385cm^-1Is ZnO₂Characteristic absorption peak of peroxy bond at 2852cm^-1And 2933cm^-1Characteristic absorption peaks for methyl and methylene groups on phospholipids; from the results, ZnO₂The occurrence of all three peaks on @ Lip-ICG indicates the formation of the complex.

And (3) particle size measurement: taking newly prepared ZnO₂Diluting the @ Lip-ICG liposome with PBS, adding 2mL of diluted liposome into a sample cell, measuring the particle size of the liposome by using a laser scattering particle size measuring instrument, wherein the sample equilibrium time is 120s, the 20 circulation times are measured, and the measuring temperature is set to be 25 ℃; the final measurement of the sample is the average of 20 measurements. The polydispersity indicates the degree of uniformity of particle size, and the smaller the polydispersity the more uniform the particles.

The results are shown in FIG. 2a, ZnO₂The average particle size of the @ Lip-ICG liposome was 200.5 nm.

Transmission Electron Microscope (TEM) observation: preparing new ZnO₂The @ Lip-ICG liposome was diluted with PBS, 10. mu.L of the diluted liposome was pipetted, slowly dropped onto a copper mesh, dried overnight at room temperature, and tested on a computer.

The results are shown in FIG. 2b, ZnO₂@LThe ip-ICG liposome is of a uniform shell-core spherical structure, the shell is a lipid bilayer, and the black compact core is ZnO₂。

Measurement of Zeta potential: adding ZnO₂The @ Lip-ICG liposome was transferred to a potential cup (the solution should submerge the conductive copper plate), the Zeta potential measurement conditions were set to equilibrium time 120s, minimum 20 cycle times were measured each time, the measurement temperature was set to 25 ℃, and the final measurement result of the sample was the average of 20 measurement results.

The test results show that ZnO₂The average zeta potential of the @ Lip-ICG liposome was-22.22 mV.

Stability test: taking freshly prepared Lip, ICG @ Lip and ZnO₂@ 3ml each of Lip-ICG and free ICG (0.5mg/ml), and the changes in absorbance were measured with an ultraviolet spectrophotometer at room temperature and in the dark for 0, 1, 2, 8, 24, and 48 hours.

The results are shown in fig. 3, which is a Lip experimental group in fig. 3a, because there is no ICG characteristic absorption peak, the absorbance at different time points is almost a coincident line; the UV characteristic absorption peak of free ICG (FIG. 3b) decreased by nearly 70% within 48h, while ICG @ Lip (FIG. 3c) and ZnO₂The UV characteristic absorption peak of the @ Lip-ICG (FIG. 3d) group remained around 80% of the initial value after 48h, indicating that binding to liposomes can effectively reduce the ICG degradation rate.

Example 2 Dual stimulation of pH and NIR on ZnO₂Effect of @ Lip-ICG Release behavior

Setting three pH conditions of 7.4, 6.5 and 5.4 respectively; two temperature conditions, namely 37 ℃ (physiological temperature) and 50 ℃ (simulating the temperature of a tumor part after near infrared light irradiation); each group accurately sucks 0.5ml of freshly prepared ZnO₂@ Lip-ICG in MWCO 3000 dialysis bag, and adding 1.5ml phosphoric acid buffer solution with corresponding pH value;

1) tying the dialysis bag, placing the dialysis bag in a blue-mouth bottle, and quickly adding 198ml of phosphoric acid buffer solution with corresponding pH value;

2) the blue-mouth bottle is placed in a constant temperature shaker at 37 ℃ or 50 ℃ for shaking dialysis at 100 rpm.

3) Sampling at set time points of 0, 15, 45, 180, 480, 1440 and 2880min, taking 5ml of dialysis external phase each time, and adding 5ml of buffer solution with corresponding pH value;

4) the sample was analyzed using an inductively coupled plasma mass spectrometer.

The results are shown in FIG. 4, from which it can be seen that Zn is present at pH 7.4 and temperature 37 ℃ within 48 hours²⁺The cumulative release rate of (a) was about 22.59%, about 42.62% at pH 6.5, and about 54.73% at pH 5.4. It is clear that when the pH is adjusted from 7.4 to 5.4, the ZnO₂The @ Lip-ICG nanoparticles exhibited a more complete release, increasing stepwise from 22.59% to 54.73%, indicating ZnO₂The release behavior of the @ Lip-ICG nanoparticles follows a pH-dependent pattern. Furthermore, Zn is present under all pH conditions²⁺The cumulative release rates were all significantly higher than 37 ℃ at 50 ℃, suggesting that temperature plays a key role in promoting release of molecules. The results show that pH and NIR can be used as a remote switch, and ZnO can be effectively and reasonably controlled₂From ZnO₂Release in the @ Lip-ICG liposomes, thereby optimizing the anti-tumor effect in vivo and in vitro.

Example 3ZnO₂Test of in vitro singlet oxygen-generating Capacity of @ Lip-ICG Liposome

DPBF is 1, 3-diphenylisobenzofuran and is capable of reacting with singlet oxygen, decreasing absorbance.

Preparing a DPBF working solution: accurately weighing 16.2mg DPBF powder in 100ml 50% ethanol solution under dark condition, ultrasonic vibrating to dissolve it sufficiently, and adjusting pH to 5.4 with 1M hydrochloric acid to obtain 6 × 10^-5DPBF working fluid of M.

The experiment was divided into four groups:

pure DPBF group: taking 18ml of prepared DPBF working solution, adding 2ml of 50% ethanol solution, and uniformly mixing for later use.

Free ICG group: taking 18ml of prepared DPBF working solution, adding 0.077mg of ICG powder and 2ml of 50% ethanol solution, and uniformly mixing for later use.

ICG @ Lip group: taking 18ml of prepared DPBF working solution, adding 0.77ml of freshly prepared ICG @ Lip solution and 1.23ml of 50% ethanol solution, and uniformly mixing for later use.

ZnO₂@ Lip-ICG group: get18ml of prepared DPBF working solution, 0.77ml of freshly prepared ZnO are added₂@ Lip-ICG solution and 1.23ml of 50% ethanol solution were mixed well for use.

The mixed solution is subpackaged into EP tubes, each tube contains 2ml, and 10 tubes are prepared by using 808nm near infrared laser system at a speed of 1W/cm²The irradiation was performed at power for 0, 10, 20, 30, 40, 50, 60, 120, 180, 300 seconds, and then absorbance of each tube was measured using an ultraviolet spectrophotometer.

The above operations were carried out under dark conditions.

As shown in FIG. 5, the absorbance of the DPBF solution alone (FIG. 5a) was measured by using a laser beam at 808nm (1W/cm)²) The absorbance decrease was negligible and was stable during 5min of continuous irradiation, indicating that DPBF did not affect the ROS generating ability. Under the same laser irradiation, the ICG (figure 5b), the ICG @ Lip (figure 5c) and the ZnO are dissociated₂The three groups of @ Lip-ICG (FIG. 5d) all produced singlet oxygen because of their significant decrease in the DPBF UV characteristic absorption peak, where ZnO₂The rate of decrease of the @ Lip-ICG group was significantly faster than the free ICG and ICG @ Lip groups, and after 30s of continuous irradiation, it was already decreased by about 80%, indicating that ZnO₂The @ Lip-ICG group produced singlet oxygen most efficiently in an acidic environment and after laser irradiation at 808 nm. This phenomenon occurs as a result of in vitro release prior to bonding because of ZnO under acidic conditions and after laser irradiation₂@ Lip-ICG can release ZnO₂And is decomposed into Zn²⁺And H₂O₂Further generates oxygen to provide raw material for generating singlet oxygen by ICG irradiated by laser, ZnO₂O produced by decomposition₂Self-feeding to enhance PDT effect. These results further indicate that ZnO₂The @ Lip-ICG nanosystem can increase ROS and show excellent PDT effect.

Example 4 photo-thermal Properties of different groups of nanoparticles

1) Investigation of the same concentration (ICG concentration 2.5. mu.g/ml) of free ICG, ICG @ Lip, ZnO₂@ Lip and ZnO₂@ Lip-ICG near infrared laser (power 1W/cm) at 808nm²) Temperature change under irradiation;

2) exploring different concentrations(ICG concentration: 0.625, 1.25, 2.5, 5.0, 10. mu.g/ml) of ZnO₂@ Lip-ICG near infrared laser (power 1W/cm) at 808nm²) Temperature change under irradiation;

3) investigation (ICG concentration: 2.5. mu.g/ml) ZnO₂@ Lip-ICG near infrared laser at 808nm with different powers (0.5, 1, 1.5, 2W/cm)²) Temperature change under irradiation;

and monitoring the temperature change of each group of mixed solution by using a FLUKE infrared thermal imager and carrying out photothermal imaging.

As shown in FIG. 6, it can be seen from FIG. 6a that the laser beam is emitted at 808nm (1W/cm)²) Under irradiation, ZnO₂The temperature of @ Lip-ICG (ICG concentration: 2.50. mu.g/ml) increased by approximately 30 ℃ in 5 minutes, and the temperature of ICG @ Lip increased by approximately 22 ℃; in contrast, under the same treatment conditions, in physiological saline solution and ZnO₂Very slight temperature rise is observed in @ Lip, and the free ICG is only raised by about 10 ℃, which indicates that ICG has NIR laser-induced photothermal performance, but the free ICG is unstable and has poor photothermal conversion efficiency, and shows better photothermal conversion efficiency after being loaded into liposome. As can be seen from FIGS. 6c and 6d, ZnO₂@ Lip-ICG shows a positive correlation pattern of concentration and irradiation duration and intensity with laser power density from 0.5W/cm²Increase to 2W/cm²Or ZnO₂The ICG concentration of @ Lip-ICG increased from 0.63. mu.g/ml to 10.00. mu.g/ml, ZnO₂The temperature of @ Lip-ICG rapidly increased within 5 minutes at 2W/cm²Irradiation of ZnO₂Temperature of @ Lip-ICG (ICG concentration: 2.50. mu.g/ml) and 0.5W/cm²Compared with ZnO of which the temperature is increased by nearly 20 ℃, and the temperature is increased by 10.00 mu g/ml₂@ Lip-ICG also in ratio of 0.63. mu.g/ml ZnO₂@Lip-ICG(1W/cm²) The temperature rise of (a) increases by nearly 20 ℃. These results show that ZnO₂@ Lip-ICG has a high efficiency in converting laser energy to thermal energy.

EXAMPLE 5 cellular uptake of ZnO₂Study of conditions of the @ Lip-ICG liposomes

And (3) observing the uptake of the drug by the cells through a fluorescence microscope:

1) when the growth rate of CNE-2 is 80%, it is digested with trypsinAnd cell pellet was collected and diluted to 1X 10 with DMEM medium containing 10% FBS and 1% double antibody⁵The cells are inoculated in a 12-well plate (1ml), slightly shaken to evenly spread the cells at the bottom of the well, and cultured in a cell culture box overnight;

2) when the cells were fully adherent, at a predetermined time, the original medium was aspirated, washed once with PBS, and added with 2.50. mu.g/ml ZnO₂@ Lip-ICG serum-free DMEM medium, placed in a cell incubator for incubation;

3) after incubation was complete, the supernatant was aspirated and washed 2 times with PBS to remove liposomes not taken up by the cells;

4) adding 300 μ l of 4% paraformaldehyde fixing solution into each well, and fixing at room temperature for 10 min;

5) after fixation is finished, removing paraformaldehyde fixing solution, and washing for 2 times by using PBS;

6) adding DAPI, and dyeing at room temperature for 5 min;

7) after dyeing, absorbing and discarding DAPI dye solution in the hole, and washing for 2 times by PBS;

8) observed with a fluorescence microscope.

Flow cytometry for detecting drug uptake by cells

1) When the human nasopharyngeal carcinoma cell CNE-2 grows to 80%, trypsinizing and collecting cell mass, and diluting to 1 × 10 with DMEM medium containing 10% FBS and 1% double antibody⁵The cells are inoculated in a 6-well plate (2ml), and the cells are evenly spread at the bottom of the well by slight shaking and cultured overnight;

3) after incubation was complete, the supernatant was aspirated and washed 2 times with PBS;

4) after digestion with EDTA-containing trypsin, the cell pellet was collected by centrifugation (2000rpm, 5 min).

5) Adding 200 mul PBS, blowing off cell mass, shaking and mixing uniformly;

6) sending to a flow cytometry for detection.

As a result, as shown in FIG. 7, the fluorescence microscopic observation result (FIG. 7a) showed that ZnO was present in the sample₂@ Lip-ICG successfully entered CNE-2 cells, ZnO₂The ICG fluorescence (red color) of @ Lip-ICG NPs is mainly distributed in cytoplasm, and the red fluorescence is increased in a time-dependent manner; the flow cytometry results (FIG. 7b) further show that ZnO₂@ Lip-ICG successfully entered CNE-2 cells and fluorescence increased time-dependently.

EXAMPLE 6ZnO₂Detection of toxic Effect of @ Lip-ICG liposomes on cells

Detecting the growth condition of CNE-2 of human nasopharyngeal carcinoma cells by CCK-8 method, adding corresponding amount of blank culture medium and CCK-8 solution, using the wells without cells as blank control group, using the wells with cells and blank culture medium as negative control group, and using ICG @ Lip group and ZnO group₂The @ Lip-ICG group was the experimental group.

1) CNE-2 cells were grown to 80%, trypsinized and cell masses were collected, and then diluted to 5X 10 in DMEM medium containing 10% FBS and 1% double antibody⁴The cells were inoculated in a 96-well plate (100. mu.l well) with 6 wells per well and cultured overnight;

2) after the cells are completely attached to the wall, the original culture medium is sucked out, 100 mu l of DMEM culture medium without FBS and double antibody is added into a blank control group and a negative control group, and pre-prepared experiments containing ICG @ Lip and ZnO with different concentrations are respectively added into the experiment groups₂@ Lip-ICG 100 μ l FBS-and diabody-free DMEM medium at concentrations of 0.31, 0.63, 1.25, 2.50, 5.00, 10.00, 20.00 μ g/ml ICG, respectively; (for the pH 6.5 group, the ordinary FBS-free and double antibody-free DMEM medium was changed to the FBS-free and double antibody-free DMEM medium in which the pH was previously adjusted to 6.5.)

3) The laser-free group was incubated in a cell incubator for 24 hours, the laser-irradiated group was incubated in a cell incubator for 4 hours, and then a near infrared laser (1W/cm) of 808nm was applied to each well²) Irradiating for 5 minutes, and continuously incubating in a cell culture box for 20 hours;

4) absorbing the culture medium in all the wells, rinsing twice by PBS, adding 100 mul of DMEM culture medium without FBS and double antibody and 10 mul of CCK-8 solution into each well, slightly shaking and uniformly mixing, and continuously incubating for 2 hours in a cell culture box;

5) absorbance at 450nm was measured using a microplate reader.

The results are shown in FIG. 8, from which it can be seen that ZnO is present under acidic conditions₂The @ Lip-ICG liposome can generate strong cell poisoning effect after being irradiated by 808nm laser, and compared with the ICG @ Lip liposome, ZnO is added₂ZnO of (2)₂The @ Lip-ICG has stronger cytotoxicity, and further shows that ZnO₂Has the functions of oxygen supply and PDT killing.

Example 7ZnO₂Study of intracellular ROS production of @ Lip-ICG liposomes

And (3) detecting active oxygen in cells by using a fluorescent probe DCFH-DA. The specific operation is as follows:

1) CNE-2 cells were grown to 80%, trypsinized and cell masses were collected, and diluted to 1X 10 in DMEM medium containing 10% FBS and 1% double antibody⁵The cells are inoculated into a 12-hole plate (1ml) with a thin glass sheet laid at the bottom, slightly shaken to evenly spread the cells at the bottom of the hole, and cultured in a cell culture box overnight;

2) when the cells are completely attached to the wall, the original culture medium is sucked out, washed twice by PBS, and the experiment components are respectively added with the pre-prepared ZnO containing 2.50 mu g/ml₂1ml of serum-free DMEM medium of @ Lip-ICG and ICG @ Lip, adding the same amount of serum-free DMEM medium without liposome into a control group, and putting the control group into a cell culture box for incubation for 12 hours; (for the pH 6.5 group, the ordinary FBS-free and double antibody-free DMEM medium was changed to the pre-prepared FBS-free and double antibody-free DMEM medium with pH 6.5.)

3) After incubation, the supernatant was discarded and washed 2 times with warm Hank's buffer;

4) add 500. mu.l DCFH-DA working solution (10. mu.M) diluted with warm Hank's buffer solution into each well, incubate for 30 min;

5) after incubation, washing with warm Hank's buffer 2 times;

6) the laser irradiation group used a 808nm laser (1W/cm)²) Irradiating each well for 5min without shockThe light irradiation group is not processed;

7) adding 300 μ l of 4% paraformaldehyde fixing solution into each well, and fixing at room temperature for 10 min;

8) after fixation is finished, removing paraformaldehyde fixing solution, and washing for 2 times by using PBS;

9) adding DAPI staining solution, and staining for 5min at room temperature;

10) after dyeing, the remaining DAPI dye solution was aspirated away and washed 2 times with PBS;

11) taking out the cell slide at the bottom of the 12-hole plate, and inversely buckling the cell slide on a glass slide on which the anti-fluorescence quencher is dripped;

12) sealing with transparent nail polish, and storing in dark place;

13) observed with a confocal laser microscope.

The results are shown in fig. 9, the control group exhibited weak green fluorescence in both light and no light, and for the ICG @ Lip group, the green fluorescence was very weak in the absence of light, almost equivalent to the control group, and after laser irradiation, moderate intensity green fluorescence was observed, with almost no difference between pH 7.4 and pH 6.5; for ZnO₂The @ Lip-ICG group, in which the green fluorescence at pH 7.4 was weak and the green fluorescence at pH 6.5 was slightly enhanced without laser irradiation; when the laser is irradiated, the green fluorescence is stronger, and when the pH is 6.5, the green fluorescence is strongest; the results show that, under acidic conditions, ZnO₂@ Lip-ICG capable of lasing at 808nm (1W/cm)²) Under irradiation, a large amount of active oxygen is generated, and then CNE-2 cells are killed.

EXAMPLE 8ZnO₂Study of pharmacokinetics of @ Lip-ICG liposome in mice

Taking 6 healthy 5-week-old female BABL/C nude mice SD rats, randomly dividing the rats into 2 groups, and 3 rats in each group; are respectively ZnO₂The @ Lip-ICG group and the Free ICG group; tail vein injection was carried out at a dose of 5mg ICG/kg. After injection, 0.15ml of blood was taken at 0.5, 1, 2, 4, 8, 24, 48 hour time points, respectively, and added to heparinized black opaque 96-well plates, and the mean fluorescence intensity value per well was measured using a small animal in vivo imaging system.

The results are shown in FIG. 10, from which FIG. 10b it can be seen that Zn is compared to the free ICG groupO₂Significant prolongation of blood circulation time of @ Lip-ICG NPs; free ICG is unstable in aqueous solution and at high concentrations aggregates and quenches, being rapidly cleared from the plasma. The stability of the liposome can be enhanced by loading the liposome in a lipid bilayer, PEG modification is carried out on the surface of the liposome, and cholesterol is also added into the liposome formula, so that ZnO can be added into the liposome₂The @ Lip-ICG can escape the recognition and phagocytosis of RES in vivo, the circulation time in vivo is greatly prolonged, and ZnO₂The Clearance (CL) of @ Lip-ICG is significantly lower than that of Free ICG group, ZnO₂The area under the @ Lip-ICG curve (AUC) is obviously higher than that of Free ICG group, and the result shows that ZnO₂@ Lip-ICG significantly prolongs drug blood circulation time.

Example 9ZnO₂Application experiment of @ Lip-ICG liposome in mouse

9.1 construction of CNE-2 subcutaneous tumor model in mice

Collecting CNE-2 cells of nasopharyngeal carcinoma growing in logarithmic phase, digesting, centrifuging, collecting cells, washing with PBS twice, removing residual FBS and culture medium, and diluting with normal saline to cell density of 1.0 × 10⁷Each/ml, put on ice for use. After the right axilla of a 5-week-old female BABL/C nude mouse is wiped and disinfected by 75% alcohol, 150 mu l of cell suspension is injected subcutaneously under the right anterior axilla, subcutaneous tumor grows out after about one week, and the tumor volume is 50-200mm³And then subsequent experiments can be carried out.

9.2ZnO₂Study on distribution of @ Lip-ICG liposomes in mice

In vivo imaging: the tumor volume is about 200mm³9 CNE-2 tumor-bearing nude mice are divided into 3 groups on average; equal amounts of Free ICG and ZnO were injected from the tail vein₂@ Lip-ICG; after injection, the nude mice are kept in a dark place, and are anesthetized by a small animal anesthesia machine after 0, 1, 2, 4, 8, 12, 24, 36 and 48 hours, and fluorescence images are collected by a small animal living body imaging system after anesthesia is finished.

In vitro imaging: the tumor volume is about 200mm³9 CNE-2 tumor-bearing nude mice are divided into 3 groups on average; equal amounts of Free ICG and ZnO were injected from the tail vein₂@ Lip-ICG; after the injection is finished, raising the seedlings for 24 hours in a dark place; sacrifice ofAnd collecting heart, liver, spleen, lung, kidney and tumor, and collecting fluorescence image with small animal living body imaging system.

The results are shown in FIG. 11, and it can be seen from FIG. 11a that the tumor-bearing nude mice were injected with ZnO in tail vein₂After the @ Lip-ICG nano-particle is used for 1h, the fluorescence intensity of a tumor part is gradually increased and reaches the highest value within 8h, then the fluorescence intensity is slowly reduced, and the existence of the tumor part nano-particle can still be observed within 48 h; the Free ICG groups are different, under the same ICG dosage, the fluorescence intensity of the liver of the nude mice is stronger, the transient fluorescence intensity of the tumor part is slightly increased along with the increase of the whole body fluorescence intensity and is rapidly reduced, and the specific aggregation phenomenon is not shown; as can be seen in FIG. 11b, ZnO compared to Free ICG 24h after IV injection₂The @ Lip-ICG has higher aggregation at the tumor site, and as a whole, the Free ICG group has significantly weaker fluorescence than ZnO₂The @ Lip-ICG group, indicates that the Free ICG moiety is metabolized or quenched.

9.3ZnO₂Research on photothermal effect of @ Lip-ICG liposome in mice

Taking 15 tumor-bearing mice, randomly dividing into 5 groups, each group comprising 3 mice, Saline group, Free ICG group, and ZnO group₂@ Lip group, ICG @ Lip group and ZnO₂The @ Lip-ICG group; each group was administered with 100. mu.l of the corresponding drug via tail vein, and after keeping dark for 8 hours, the cells were treated with 808nm laser (1W/cm)²) The tumor site was irradiated and the temperature was monitored with a thermal infrared imager.

As a result, as shown in FIG. 12, it can be seen from FIGS. 12a and 12b that ZnO is present in comparison with the other groups₂The @ Lip-ICG group showed a large temperature rise in a short time, indicating that ZnO₂The @ Lip-ICG nanoparticle has good photo-thermal conversion efficiency, and can exert the PTT effect to kill tumor cells.

9.4ZnO₂Experiment on influence of @ Lip-ICG liposome on local tumor hypoxia state in mice

Taking 15 tumor-bearing mice, randomly dividing into 5 groups, each group comprising 3 mice, Saline group, Free ICG group, and ZnO group₂@ Lip group, ICG @ Lip group and ZnO₂The @ Lip-ICG group; each group is injected with 100 μ l of corresponding medicine via tail veinAfter 24h of light-feeding, tumor tissues from each group were collected, fixed with 4% paraformaldehyde, and used for subsequent tissue sectioning and HIF-1 α staining.

The results are shown in FIG. 13, from which it can be seen that in the control group, a clear brown specific staining was observed, indicating that the tumor tissue was in a hypoxic state; the Free ICG group and the ICG @ Lip group show the same results as the control group, HIF-1 alpha is highly expressed, and the tumor part still shows the anoxic state; in contrast, ZnO₂@ Lip group and ZnO₂The brown specific staining is obviously reduced in the @ Lip-ICG group, the expression of HIF-1 alpha is obviously reduced, and the difference between the two groups is small; the above results show that ZnO₂Can surely make ZnO₂The @ Lip-ICG improves the local hypoxia of the tumor and enhances the curative effect of photodynamic.

9.5ZnO₂Experiment for inhibiting growth of CNE-2 subcutaneous tumor in mouse by @ Lip-ICG liposome and evaluating safety in vivo

50 tumors were taken and the volume was about 100mm³The tumor-bearing nude mice are randomly divided into 10 groups, and each group comprises 5 mice; the grouping is as follows: 1) saline, 2) Free ICG, 3) ZnO₂@Lip，4)ICG@Lip，5)ZnO₂@Lip-ICG，6)Saline+Laser，7)Free ICG+Laser，8)ZnO₂@Lip+Laser，9)ICG@Lip+Laser，10)ZnO₂@ Lip-ICG + Laser; the medicine is administered every other day at a dose of 5mg/kg (ICG) or 30mg/kg (lipid), and the laser irradiation is performed daily at 808nm (1W/cm)²) Irradiating the tumor part for 5min, administering the medicine for three times totally, recording the tumor volume every other day and monitoring the weight change of the nude mice; after completion of the administration, tumor tissues were collected on day 7 after the start of the administration for H&E staining and TUNEL immunofluorescence staining;

the results are shown in FIG. 14, and it can be seen from FIGS. 14a to 14c that ZnO₂The @ Lip-ICG + Laser group can obviously inhibit the tumor growth, and the ICG @ Lip + Laser group can also inhibit the tumor growth to a certain extent, but the effect is obviously inferior to that of ZnO₂A @ Lip-ICG + Laser group; the Free ICG + Laser group was less effective than the ICG @ Lip + Laser group because of its lack of targeting and high circulating clearance, insufficient drug concentration at the tumor site, and difficulty in subsequent Laser irradiation. Other not provided with laserThe irradiated groups all showed no tumor growth inhibition;

as can be seen from FIG. 14d, the body weight of each group of nude mice did not change significantly after different treatments, which illustrates to some extent the ZnO₂The @ Lip-ICG is irradiated by near infrared laser, so that the biocompatibility and the biosafety are good;

as can be seen from FIG. 14e, ZnO was compared with the control group₂The @ Lip-ICG + Laser group showed extensive cellular necrosis and reduction of intercellular gaps in tumor tissue sections, indicating that ZnO₂The @ Lip-ICG and near-infrared laser combined treatment have good anti-tumor effect;

from FIG. 14f, it can be seen that ZnO compares to the other groups₂The @ Lip-ICG + Laser group shows large-area tumor cell apoptosis, and the results show that PDT/PTT and oxygen supply ZnO₂The combined treatment scheme can exert excellent antitumor effect.

After the administration is finished, taking eyeball blood on the 7 th day after the administration is started, centrifuging, separating serum, and storing in a refrigerator at the temperature of-20 ℃ for later use; after the eyeball blood is taken out, the nude mice are immediately sacrificed, the tumor, the heart, the liver, the spleen, the lung and the kidney are stripped, photographed and weighed, and then fixed by 4 percent paraformaldehyde for the subsequent hematoxylin-eosin (HE) staining and immunohistochemical detection.

The staining results are shown in FIG. 15, which shows that no obvious pathological changes are observed in the heart, liver, spleen, lung and kidney tissue sections of each group.

The immunohistochemical detection is shown in FIG. 16, and it can be seen from FIGS. 16a-16e that the weight of the organ of each experimental group of nude mice is not significantly different from the weight of the organ of the control group of nude mice; AST, ALT, BUN and CRE in blood biochemical indexes are all in a normal range, which indicates that liver and kidney injury is not caused; these results further illustrate ZnO₂@ Lip-ICG has good biosafety.

Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein.

Claims

1. A novel self-oxygen-supply liposome nanoparticle is characterized in that the novel self-oxygen-supply liposome nanoparticle is a core-shell structure compound and comprises an inner core containing peroxide and a lipid bilayer shell coating the inner core; the lipid bilayer shell is loaded with indocyanine green fluorescent dye.

2. The novel self-donating liposomal nanoparticle according to claim 1, wherein the peroxide is selected from the group consisting of zinc peroxide, magnesium peroxide, barium peroxide, and calcium peroxide.

3. The novel self-donating liposomal nanoparticle of claim 1, wherein said lipid bilayer shell is further coated with polyethylene glycol.

4. The novel self-oxygenating liposomal nanoparticle according to claim 3, wherein the polyethylene glycol is distearoylphosphatidylethanolamine-polyethylene glycol 2000, and the distearoylphosphatidylethanolamine-polyethylene glycol 2000 is prepared by reacting polyethylene glycol 2000 and distearoylphosphatidylethanolamine.

5. The novel self-ventilating liposomal nanoparticle of claim 1, wherein said lipid bilayer comprises a phospholipid and cholesterol, said cholesterol being embedded within said phospholipid.

6. The novel self-oxygenating liposomal nanoparticle according to claim 5, wherein the phospholipid is selected from one of soy lecithin, egg yolk lecithin, glycerophospholipids, and ethanolamine phospholipids.

7. A preparation method of novel self-oxygen-supplying liposome nanoparticles is characterized by comprising the following steps:

s1, synthesizing peroxide nanoparticles by a one-pot precipitation method;

8. The method for preparing novel self-oxygen-supplying liposome nanoparticles as claimed in claim 7, wherein the specific synthesis method of step S2 is as follows: dissolving phospholipid, cholesterol, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and indocyanine green in methanol, adding the methanol into the peroxide nanoparticles, and uniformly dispersing by ultrasonic; then, uniformly mixing the solution, and performing rotary evaporation and drying to obtain a film; adding PBS buffer solution into the film, and performing ultrasonic oscillation until the film is completely hydrated to obtain mixed solution; and then centrifuging the mixed solution for 1-2 min under the condition of 450-500 r/min, discarding the bottom precipitate, and filtering by using a 0.45-micrometer filter membrane to obtain the self-oxygenation liposome nanoparticle.

9. The method for preparing novel self-oxygen-supply liposome nanoparticles as claimed in claim 8, wherein the addition amounts of the phospholipid, the cholesterol, the distearoylphosphatidylethanolamine-polyethylene glycol 2000, the indocyanine green and the peroxide are 4: 1: 1: 0.1: 1.

10. use of the novel self-oxygen-supplying liposomal nanoparticles of any one of claims 1-6 in the preparation of photodynamic therapeutic agents and/or pharmaceutical carriers.