Porphyrin lipid-perfluorocarbon nano preparation and preparation method and application thereof
阅读说明：本技术 一种卟啉脂质-全氟化碳纳米制剂及其制备方法和用途 (Porphyrin lipid-perfluorocarbon nano preparation and preparation method and application thereof ) 是由 戴志飞 梁晓龙 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种卟啉脂质-全氟化碳纳米制剂及其制备方法和用途,所制备的纳米制剂为核壳结构,包括外壳的卟啉脂质体膜层和内核的全氟化碳,可以作为光敏剂或声敏剂药物和氧气递送系统,向肿瘤同步递送光敏剂/声敏剂药物和氧气,在激光或超声辐照下,显著增强光动力治疗/声动力治疗效果,并改善肿瘤的乏氧微环境,有效抑制肿瘤的复发和转移。该体系将光敏剂/声敏剂药物共价连接在脂质分子中,并通过强疏水作用力包载全氟化碳,具有高稳定性、高载药量、高携氧量、不易泄漏等特点,在肿瘤诊疗方面具有很好的临床应用前景。(The invention discloses a porphyrin lipid-perfluorocarbon nano preparation and a preparation method and application thereof, the prepared nano preparation is of a core-shell structure, comprises a porphyrin liposome membrane layer of a shell and perfluorocarbon of an inner core, can be used as a photosensitizer or a sonosensitizer drug and oxygen delivery system, synchronously delivers the photosensitizer/sonosensitizer drug and oxygen to tumors, obviously enhances the photodynamic therapy/sonodynamic therapy effect under laser or ultrasonic irradiation, improves the hypoxic microenvironment of the tumors, and effectively inhibits the recurrence and metastasis of the tumors. The system connects photosensitizer/sonosensitizer medicine in lipid molecules in a covalent way, and carries perfluorocarbon through strong hydrophobic acting force, has the characteristics of high stability, high medicine carrying capacity, high oxygen carrying capacity, difficult leakage and the like, and has good clinical application prospect in the aspect of tumor diagnosis and treatment.)
1. A porphyrin lipid-perfluorocarbon nano-preparation is characterized in that: the nano preparation is a nano particle with a core-shell structure, and the structure of the nano preparation comprises a porphyrin liposome membrane layer of a shell and perfluorocarbon of an inner core; the porphyrin liposome membrane layer consists of porphyrin lipid or porphyrin lipid and phospholipid components, wherein the mole percentage of the porphyrin lipid is 5-100%, and the mole percentage of the phospholipid components is 0-95%.
2. The porphyrin lipid-perfluorocarbon nanoformulation according to claim 1, wherein: the perfluorocarbon is selected from one or more of perfluoropropane, perfluoropentane, perfluorohexane, perfluorooctane bromide and perfluorocrown ether.
3. The porphyrin lipid-perfluorocarbon nanoformulation according to claim 1, wherein: the porphyrin lipid has a structure shown as the following formula I, and comprises a hydrophilic head, a hydrophobic carbon chain and a porphyrin group;
wherein R is1And R2Selected from C6-C18 alkyl; r3Is hydroxyl or polyethylene glycol; a is 2 or 3, b is 2 or 3; x is NH or O.
4. The porphyrin lipid-perfluorocarbon nanoformulation according to claim 3, wherein: the porphyrin group is a photosensitive functional group and is selected from tetraphenylporphyrin, hematoporphyrin, protoporphyrin, pyropheophorbide-a, purpurin-18, verteporfin and chlorin e 6.
5. The porphyrin lipid-perfluorocarbon nanoformulation according to claim 1, wherein: the phospholipid component is selected from any one or more of the following compounds: distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, distearoylphosphatidylethanolamine-modified maleimide, distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
6. The porphyrin lipid-perfluorocarbon nanoformulation according to claim 1, wherein: the surface of the nano preparation is modified with tumor targeting molecules, and the tumor targeting molecules are selected from any one of antibodies, polypeptides, aptamers and folic acid capable of targeting tumors.
7. The porphyrin lipid-perfluorocarbon nanoformulation according to claim 1, wherein: the perfluorocarbon encapsulated by the nano-formulation carries oxygen.
8. The method for preparing porphyrin lipid-perfluorocarbon nano-formulation as defined in any one of claims 1 to 7, comprising the steps of:
1) dissolving porphyrin lipid or porphyrin lipid and phospholipid in mixed solvent of chloroform and methanol, vacuum drying to form film, and vacuum drying overnight;
2) adding ultrapure water, hydrating in water bath at 30-60 deg.C for 10-30min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) dropwise adding perfluorocarbon into the solution obtained in the step 2) under an ice bath condition, and simultaneously performing ultrasonic dispersion for 5-15min by using a probe to obtain the uniformly dispersed porphyrin lipid-perfluorocarbon nano preparation.
9. The method of claim 8, wherein: step 3) further removing the non-entrapped perfluorocarbon, porphyrin lipid or phospholipid by using a centrifugal method.
10. The use of the porphyrin lipid-perfluorocarbon nano-formulation of any one of claims 1-7 in the preparation of a medicament for alleviating tumor hypoxia and/or enhancing the photodynamic/sonodynamic therapeutic effect of tumors.
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a porphyrin lipid-perfluorocarbon nano preparation as well as a preparation method and application thereof.
Background
Photodynamic/sonodynamic therapy has attracted increasing attention in the research of clinical malignancy treatment over the last decades. Usually, the light/sound dynamic therapy can transfer energy to O under the combined action of light/sound sensitive agent and laser/ultrasonic irradiation2And further generates Reactive Oxygen Species (ROS) with cytotoxicity, thereby irreversibly destroying proteins or DNA of the tumor cells and inducing apoptosis of the tumor cells. Compared with conventional operations, chemotherapy and radiotherapy, the photodynamic therapy has the advantages of non-invasiveness, higher controllability, reduced side effects, negligible drug resistance, accurate treatment function, tumor intervention reservation, tumor ablation replacement and the like.
However, to date, the clinical use of photodynamic/photothermal therapy has been affected by several uncontrollable and unavoidable factors. First, due to the extreme hypoxic nature of solid tumor tissue (pO)22.5 mmHg. ltoreq.), highly dependent on O2Limiting the development of photodynamic/sonodynamic therapy. More seriously, during the photodynamic/photodynamic therapy O2The depletion of (a) may further worsen the hypoxic environment at the tumor site, reduce the efficiency of photodynamic/sonodynamic therapy, lead to further tumor growth, and may induce tumor metastasis or develop therapeutic tolerance. Hypoxia inducible factor-1 (HIF-1) is a major regulator of resistance to cell death and proliferation of cancer cells by providing a growth advantage under hypoxic stress, with increased expression of HIF commonly associated with a variety of human cancersThe mortality rate of the patients is increased to show positive correlation. In experimental models, overexpression of HIF in tumor cells promotes tumor metastasis, while inactivation of HIF reduces the metastatic potential of tumor cells. Taken together, these clinical and experimental results demonstrate an important role for HIF signaling in metastatic tumor progression.
To address this problem, researchers have developed several strategies to alleviate tumor hypoxia. Hyperbaric oxygen therapy has shown effective treatment in clinical trials, but many adverse side effects such as hyperoxic seizures and barotrauma are not negligible. In recent years, many biological reaction-based O2Generating materials, e.g. hydrogen peroxide (H)2O2) Enzyme and manganese dioxide (MnO)2) Nano material and the like through the reaction with H in tumor tissue2O2Acting to increase local O of tumor2And (4) horizontal. However, their O2The production ability is influenced by intracellular H2O2The limit of low concentration. In contrast, Perfluorocarbons (PFCs) have high biocompatibility and good oxygen solubility and are expected as artificial blood for delivering oxygen to alleviate tumor hypoxia, but the perfluorocarbons in conventional delivery systems are typically low in carrying capacity and therefore require external stimulation to trigger oxygen release. On the other hand, most of the traditional light/sound sensitive agents have the problems of insufficient drug loading capacity, serious self aggregation of the embedded light/sound sensitive drugs, early drug leakage in the circulation process and the like, so that the curative effect is limited. Thus, there is a need for more effective light/sound sensitive drug and oxygen delivery systems with high efficiency and biosafety to improve the light/sound dynamic therapeutic effect, alleviate tumor hypoxia, and inhibit tumor recurrence and metastasis.
Disclosure of Invention
Aiming at the problems in the prior art, the invention designs a novel porphyrin lipid-perfluorocarbon nano preparation so as to develop an oxygen self-supply photodynamic/sonodynamic therapy system with a fluorescence/CT imaging function. In the system, the covalent attachment of porphyrin groups to lipids can achieve high drug loading, effectively avoiding the leakage of porphyrin light/sound sensitizers during systemic circulation. The system can effectively deliver oxygen to tumor tissues after oxygen adsorption saturation and gradually release the oxygen, so that the local hypoxic condition of the tumor is obviously improved without any external stimulation. The oxygen self-supply system can improve the tumor light/sound power efficiency, and can inhibit tumor recurrence and prevent tumor cell metastasis.
It is an object of the present invention to provide an efficient oxygen delivery system for alleviating the hypoxic microenvironment of tumor tissue.
The other purpose of the invention is to provide an efficient optical/acoustic dynamic treatment system, which can realize high drug-loading rate of optical/acoustic sensitive drugs and oxygen, realize synchronous delivery of tumor tissues, realize oxygen self-supply of optical/acoustic dynamic treatment and obviously improve the optical/acoustic dynamic curative effect.
The invention also aims to provide a minimally invasive treatment method, which can effectively avoid relapse and metastasis while eliminating in-situ tumors.
The purpose of the invention is realized by the following technical scheme:
a porphyrin lipid-perfluorocarbon nano-preparation is characterized in that: the nano preparation is a nano particle with a core-shell structure, and the structure of the nano preparation comprises a porphyrin liposome membrane layer of a shell and perfluorocarbon of an inner core; the porphyrin liposome membrane layer is composed of porphyrin lipid or porphyrin lipid and phospholipid components, wherein the mole percentage of the porphyrin lipid can be 5% -100%, and the mole percentage of the phospholipid components can be 0-95%.
Further, the perfluorocarbon includes, but is not limited to, any one or combination of perfluoropropane, perfluoropentane, perfluorohexane, perfluorooctane bromide and perfluorocrown ether.
Further, the porphyrin lipid has a structure shown as the following formula I, and comprises three parts, namely a hydrophilic head part, a hydrophobic carbon chain and a porphyrin group. Wherein R is1、R2Is C6-C18 alkyl; r3Is hydroxyl or polyethylene glycol (PEG chain), wherein the PEG chain can be connected to the porphyrin lipid molecule through an amide bond or an ester bond; a, b ═ 2 or 3; x is NH or O, namely the connection mode of the photosensitizer and the lipid is an ester bond or an amido bond.
Further, porphyrin groups with photosensitizing functions include, but are not limited to, tetraphenylporphyrin, hematoporphyrin, protoporphyrin, pyropheophorbide-a, purpurin-18, verteporfin, chlorin e 6.
The synthetic procedure of porphyrin lipid refers to Chinese patent (ZL.201010222238.0), and porphyrin lipids obtained by connecting different porphyrin groups are referred to as porphyrin lipid A (tetraphenylporphyrin), porphyrin lipid B (hematoporphyrin), porphyrin lipid C (protoporphyrin), porphyrin lipid D (pyropheophorbide-a), porphyrin lipid E (purpurin-18), porphyrin lipid F (verteporfin) and porphyrin lipid G (dihydroporphin E6).
Further, the phospholipid component may include any one or more of: DSPE-mPEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) and DSPE-mPEG5000 (distearoylphosphatidylethanolamine-polyethylene glycol 5000) which can prolong the blood circulation time, DSPE-Maleimide (distearoylphosphatidylethanolamine-modified Maleimide), DSPE-mPEG2000-Maleimide (distearoylphosphatidylethanolamine-polyethylene glycol 2000-Maleimide), Dipalmitoylphosphatidylcholine (DPPC) and Distearoylphosphatidylcholine (DSPC) which can be used to modify targeting molecules.
Furthermore, the surface of the nano preparation can be modified with tumor targeting molecules, and the tumor targeting molecules comprise any one of antibodies, polypeptides, aptamers and folic acid which can target tumors.
Further, the perfluorocarbon encapsulated by the nano-formulation may carry oxygen, and the capability of the nano-formulation to carry oxygen is achieved by the dissolution of oxygen by the perfluorocarbon. In an environment with a temperature of 15-40 ℃ and an oxygen partial pressure of 0-101325 Pa, 0-2 ml of oxygen can be dissolved in per ml of perfluorocarbon.
The porphyrin lipid-perfluorocarbon nano preparation is prepared by the following steps:
1) dissolving porphyrin lipid or porphyrin lipid and phospholipid in mixed solvent of chloroform and methanol, vacuum drying to form film, and vacuum drying overnight;
2) adding ultrapure water, hydrating in water bath at 30-60 deg.C for 10-30min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) dropwise adding perfluorocarbon into the solution obtained in the step 2) under an ice bath condition, and simultaneously performing ultrasonic dispersion for 5-15min by using a probe to obtain a uniformly dispersed porphyrin lipid-perfluorocarbon nano preparation, wherein the non-entrapped perfluorocarbon and porphyrin lipid or phospholipid can be further removed by using a centrifugal method.
The porphyrin lipid-perfluorocarbon nano preparation can be used for relieving tumor hypoxia and enhancing the light/sound dynamic treatment effect of tumors.
The invention has the beneficial effects that:
the invention has the advantages that the photo/sound sensitive drugs in the porphyrin lipid are connected by covalent bonds, the perfluorocarbons and porphyrin and hydrophobic lipid carbon chains are entrapped by hydrophobic acting force, and the obtained carrier system has high drug loading capacity and high stability, can effectively avoid or reduce the early leakage of the drugs in blood circulation, and reduces the toxic and side effects.
The invention has the second advantage that the porphyrin lipid-perfluorocarbon nano preparation can simultaneously deliver the light/sound sensitive medicine and oxygen to the tumor site in a targeted way. The targeted delivery of the light/sound sensitive medicine can increase the light/sound dynamic curative effect, the targeted delivery of the oxygen can obviously improve tumor hypoxia, and the combination of the light/sound sensitive medicine and the oxygen can effectively inhibit the tumor growth and prevent and treat tumor metastasis.
The invention has the third advantage that the porphyrin lipid-perfluorocarbon nano preparation has the function of enhancing fluorescence/CT imaging and can realize the photodynamic therapy under the guidance of images.
Drawings
FIG. 1 is a schematic structural diagram of a porphyrin lipid-perfluorocarbon nano-preparation according to the present invention.
Fig. 2 is a transmission electron microscope image of the porphyrin lipid-perfluorooctyl bromide nano-preparation prepared in example 1, wherein the image shows that the nano-preparation has a spherical structure.
FIG. 3-different concentrations of O in example 62The ability of the @ PFOB @ PGL nanoparticles to release oxygen in deoxygenated water was compared.
FIG. 4 PGL nanoparticles, O in example 72The ability comparison of the @ PFOB @ PGL nanoparticles and PBS to generate active oxygen under different illumination times.
FIG. 5 detection of PGL nanoparticles, O by calcein AM/PI co-staining in example 82Fluorescence microscopy pictures of @ PFOB @ PGL nanoparticles and PBS under light and no light.
FIG. 6 HT-29 tumor in PGL nanoparticles, O in example 92Compared with the killing effect of photodynamic MTT of PBS under illumination and non-illumination.
FIG. 7 is a comparison of the in vitro detection of hypoxia in tumor regions of each group during photodynamic therapy in example 10.
FIG. 8 is a graph of tumor volume over time for each group in photodynamic therapy in example 11.
Detailed Description
The examples given in the description of the present invention are only for illustrating the present invention and do not limit the content of the present invention.
Example 1
The preparation method of the porphyrin lipid-perfluorooctyl bromide nano preparation comprises the following steps:
1) porphyrin lipid A (wherein R is a group containing a tetraphenylporphyrin photo/sonosensitive group1And R2Are both C16 alkyl; r3Is a hydroxyl group; a is 2, b is 2; x is NH) (10mmol, PGL lipid) is dissolved in a mixed solvent of chloroform and methanol (volume ratio 9:1), the liquid is dried by spinning under reduced pressure to form a film, and the film is dried in vacuum overnight;
2) adding ultrapure water, hydrating in water bath at 60 deg.C for 30min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) dropwise adding perfluorooctyl bromide (350 mu L, PFOB) into the solution in the step 2) under an ice bath condition, and ultrasonically dispersing for 5min by using a probe to obtain the uniformly dispersed porphyrin lipid-perfluorooctyl bromide nano preparation, wherein the non-entrapped perfluorooctyl bromide and porphyrin lipid can be further removed by using a centrifugal method (1000 rpm).
As a result, as shown in FIG. 2, the obtained nanoparticles were uniform in size, about 20 to 40nm in size.
Example 2
The preparation method of the porphyrin lipid-perfluorohexane nano preparation comprises the following steps:
1) porphyrin lipid B (wherein R) containing hematoporphyrin light/sound sensitive groups1And R2Are both C12 alkyl; r3Is polyethylene glycol; a is 2, b is 2; x is O) (9.5mmol) and DSPE-mPEG2000(0.5mmol) are dissolved in a mixed solvent of chloroform and methanol (volume ratio 9:1), the liquid is dried under reduced pressure to form a film, and the film is dried in vacuum overnight;
2) adding ultrapure water, hydrating in water bath at 45 deg.C for 10min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) dropwise adding perfluorohexane (330 mu L) into the solution obtained in the step 2) under an ice bath condition, and simultaneously performing ultrasonic dispersion for 10min by using a probe to obtain the uniformly dispersed porphyrin lipid-perfluorohexane nano preparation, wherein the non-entrapped perfluorohexane, porphyrin lipid and phospholipid can be further removed by using a centrifugal method (1500 rpm).
Example 3
The preparation method of the porphyrin lipid-perfluorohexane nano preparation comprises the following steps:
1) porphyrin lipid C (wherein R) containing protoporphyrin light/sound sensitive group1And R2Are both C18 alkyl; r3Is polyethylene glycol; a is 3, b is 3; x is O) (0.5mmol), DPPC (9.0mmol) and DSPE-Maleimide (0.5mmol) are dissolved in a mixed solvent of chloroform and methanol (volume ratio 9:1), the liquid is dried under reduced pressure to form a film, and the film is dried under vacuum overnight;
2) adding ultrapure water, hydrating in 50 deg.C water bath for 20min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) dropwise adding perfluorohexane (352 mu L) into the solution obtained in the step 2) under an ice bath condition, and simultaneously performing ultrasonic dispersion for 10min by using a probe to obtain the uniformly dispersed porphyrin lipid-perfluorohexane nano preparation, wherein the non-entrapped perfluorohexane, porphyrin lipid and phospholipid can be further removed by using a centrifugal method (1500 rpm).
4) 0.6mmol of tumor targeting molecule thiolated PEG folic acid is added into the nano preparation, and stirred for 24 hours, so that the thiolated PEG folic acid is coupled with Maleimide.
5) To remove thiolated PEG folate that is not attached to the surface of the carrier, exclusion chromatography column Sephadex G-50 is used to remove free thiolated PEG folate.
Example 4
The preparation method of the porphyrin lipid-perfluoropentane nano preparation comprises the following steps:
1) porphyrin lipid D (wherein R is pyrochlore-a) containing light/sound sensitive groups1And R2Are both C8 alkyl; r3Is a hydroxyl group; a is 3, b is 3; dissolving X is O) (5.0mmol), DSPC (4.5mmol) and DSPE-mPEG5000(0.5mmol) in a mixed solvent of chloroform and methanol (volume ratio 9:1), spin-drying the liquid under reduced pressure to form a film, and vacuum-drying overnight;
2) adding ultrapure water, hydrating in water bath at 40 deg.C for 15min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) under the ice-bath condition, perfluoropentane (340 mu L) is dropwise added into the solution in the step 2), and the solution is ultrasonically dispersed for 5min by using a probe, so that the uniformly dispersed porphyrin lipid-perfluoropentane nano preparation can be obtained, and the non-entrapped perfluoropentane, porphyrin lipid and phospholipid can be further removed by using a centrifugal method (1500 rpm).
Example 5
The preparation method of the porphyrin lipid-perfluorinated crown ether nano preparation comprises the following steps:
1) porphyrin lipid G (wherein R is dihydroporphin e6 light/sound sensitive group-containing porphyrin lipid G1And R2Are both C6 alkyl; r3Is a hydroxyl group; a is 2, b is 2; x is O) (7.0mmol), DSPC (2.5mmol) and DSPE-mPEG2000-Maleimide (0.5mmol) are dissolved in chloroform and methyl chlorideIn a mixed solvent of alcohol (volume ratio 9:1), carrying out vacuum spin-drying on the liquid to form a film, and carrying out vacuum drying overnight;
2) adding ultrapure water, hydrating in water bath at 60 deg.C for 25min, and performing ultrasonic treatment with ultrasonic instrument under ice bath condition to uniformly disperse lipid into water solution;
3) dropwise adding perfluorocrown ether (360 mu L) into the solution obtained in the step 2) under an ice bath condition, and simultaneously performing ultrasonic dispersion for 15min by using a probe to obtain a uniformly dispersed porphyrin lipid-perfluorocrown ether nano preparation, wherein the non-entrapped perfluorocrown ether, porphyrin lipid and phospholipid can be further removed by using a centrifugal method (2500 rpm).
4) Adding 0.6mmol tumor targeting molecule RGD polypeptide into the nano preparation, and stirring for 24 hours to couple the sulfhydryl group of the RGD polypeptide with Maleimide.
5) To remove RGD polypeptides not attached to the surface of the carrier, free RGD polypeptides were removed using exclusion chromatography column Sephadex G-50.
Example 6
Oxygen carrying and oxygen release studies were performed with the porphyrin lipid-perfluorooctylbromide nanosystem (PFOB @ PGL) obtained in example 1.
Placing PFOB @ PGL in a sterile oxygen chamber, O2Flow rate 5L/min for 15 minutes to get the perfluorooctyl bromide in PFOB @ PGL to reach oxygen saturation, named O2@ PFOB @ PGL. To measure oxygen release, a portable dissolved oxygen meter was placed in 15mL of deoxygenated water, followed by the addition of 5mL of O2@ PFOB @ PGL solution, and detecting the oxygen concentration in the solution in real time. Three replicates (n-3) were performed for each concentration. The oxygen loading capacity per 1mL of PFOB in PFOB @ PGL was calculated as follows: delta O2(enhanced oxygen concentration) x volume of final solution/volume of PFOB.
The results are shown in FIG. 3 when O2The @ PFOB @ PGL is added into deoxygenated water, so that the obvious increase of the oxygen content in the deoxygenated water can be clearly detected, and correspondingly higher oxygen release capacity is shown along with the increase of the PFOB content, which indicates that the PFOB @ PGL nano particles have excellent oxygen load and gradual oxygen release capacity in a hypoxic environment.
Example 7
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for in vitro singlet oxygen detection.
To study O2The singlet oxygen-producing ability of @ PFOB @ PGL using SOSG as singlet oxygen ((PFOB))1O2) The probe of (1). First, 0.342mL of sample and 0.38mL of 100. mu.M SOSG were mixed in a black 96-well plate (Costar) (the height of the liquid level just coincided with the upper edge of the 96-well plate), illuminated (650nm, 200mW) for an appropriate time (0-3 min), and the oxidized SOSG fluorescence was quantified by measuring the fluorescence intensity (excitation at 504nm and measurement at 525 nm) using a multifunctional microplate reader (Synergy HT, Bio-Tek). All manipulations were performed in the dark and experiments for each group were performed in triplicate.
The experimental result is shown in FIG. 4, the probe SOSG can be in1O2Is oxidized in the presence of oxygen, resulting in an increase in fluorescence intensity. The graph shows the change of the fluorescence intensity of SOSG at 520nm as a function of irradiation time, and it can be seen that the SOSG fluorescence gradually increases with the irradiation time over 3 minutes. At O2The sharp increase in SOSG fluorescence intensity with irradiation time in the case of @ PFOB @ PGL confirms O after oxygen loading2@ PFOB @ PGL may yield more1O2. In contrast, the SOSG fluorescence intensity increased much more slowly in the presence of PGL nanoparticles alone. In sharp contrast, the SOSG in PBS showed little change under light, further confirming that the oxidation of SOSG was caused by singlet oxygen rather than light.
Example 8
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for qualitative assessment of the effect of cellular photodynamic therapy.
To assess cell viability after various treatments, Calcein-AM (Calcein-AM) and PI (propidium iodide) co-staining was performed to detect the effect of photodynamic therapy on cancer cells. HT-29 cells (1X 10)5One hole) in a 6-hole cell culture box for 24h, and then using PGL nano-particles or O2@ PFOB @ PGL nanoparticles(containing an equivalent amount of 4. mu.M PGL) was added to the culture for 12 hours. Laser irradiation (650nm, 0.2 w/cm)2) After 180s, incubation was continued for 24h, adding Calcein-AM/PI stain to each well, incubating for 30min, and then washing 3 times with PBS. Finally, the cells were observed under a fluorescent microscope and images of calcein and propidium iodide were recorded in the green and red channels, respectively.
The results of the experiment are shown in FIG. 5. Only with PGL + light or O2Cell death (red fluorescence emission due to PI passage through dead cells) was observed in the @ PFOB @ PGL + light-treated group; o in comparison with PFOB-free PGL + light group2@ PFOB @ PGL + illumination shows more obvious cell death, which further proves that PFOB carries oxygen and supplies oxygen in the process of photodynamic therapy to obviously enhance the photodynamic therapy effect; while light treatment alone or incubation of PGL or O alone2No cell death was observed with @ PFOB @ PGL treatment.
Example 9
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for quantitative assessment of cellular photodynamic therapy effects.
HT-29 cells were seeded in 96-well plates (4X 10)4One/hole). After 24 hours of culture, PGL nanoparticles or O were added at different concentrations2@ PFOB @ PGL nanoparticles. To assess cytotoxicity in an oxygen deficient environment, cells were maintained in a triple gas cell incubator at 5% O2、90％N2And 5% CO2. After about 4h, each well was illuminated for 10min (650nm, 0.2 w/cm)2). Then washed with fresh medium and incubated for an additional 24 hours. MTT solution was added to each well at 37 ℃ and 5% CO2Incubate for 2 h. Finally, the absorbance at 490nm was measured with a microplate reader to evaluate the cell activity.
The results of the experiment are shown in FIG. 6. For no addition of PGL or O2@ PFOB @ PGL (concentration 0. mu.M), no decrease in cell activity was observed in the different treatments. Followed by light irradiation with PGL or O2An increase in the concentration of @ PFOB @ PGL, the activity of HT-29 cells was gradually decreased. In addition, when the PGL concentration is less than 0.25. mu.MWhen is, O2The @ PFOB @ PGL group showed higher photodynamic killing effect than PGL, since oxygen carried in PFOB effectively enhanced1O2The results are consistent with the results of calcein-AM/PI staining in example 8.
Example 10
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for the assessment of the tumor hypoxia level in vivo.
Using 4-5 weeks old BALB/c mice, HT-29 subcutaneous tumor model was constructed in the right hind leg of the mice until tumor volume increased to 100mm3At the left and right, mice were injected with 200. mu.L PBS, PGL nanoparticles (2mg/mL), O via tail vein2@ PFOB @ PGL nanoparticles (2 mg/mL). 20h after administration, the tumor tissue was irradiated with light for 3min, followed by intraperitoneal injection of pimonidazole hydrochloride (dissolved in PBS) at 60mg/kg into the mouse cavity. After 30 minutes of intraperitoneal injection, mice were sacrificed, tumors were removed, immersed in o.c.t. embedding medium, rapidly frozen in liquid nitrogen, sliced into 4 μm slices using a cryomicrotome, and Hypoxyprobe was usedTMImmunofluorescent staining of the frozen sections with a-1 plus hypoxic detection kit, staining of the nuclei for 15 minutes with 10. mu.g/mL DAPI, 0.1% PBST rinsing, mounting with an anti-fluorescence-attenuating mounting agent, visualization and taking of fluorescence images using a confocal laser microscope.
The experimental results are shown in FIG. 7, in which the PBS group and the PGL nanoparticle group showed significant tumor hypoxia, while O showed significant tumor hypoxia2The @ PFOB @ PGL group can effectively relieve hypoxia in tumors. After photodynamic therapy, the tumor hypoxia degree of the PGL nanoparticle + illumination group was more severe than that of the PBS group. In contrast, O2@ PFOB @ PGL + illumination tumor tissue had little hypoxic zone after treatment, further confirming O2The @ PFOB @ PGL system delivers oxygen and alleviates tumor hypoxia.
Example 11
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for animal photodynamic therapy effect investigation.
Mice bearing HT-29 tumors were randomized into 6 groups: PBS, PBS +Illumination, PGL, PGL + illumination, O2@ PFOB @ PGL and O2@ PFOB @ PGL + light. 24 hours after intravenous injection, different groups were treated with 650nm laser irradiation (200mW) for 20 minutes.
Experimental results as shown in fig. 8, the laser irradiation treatment alone showed little antitumor effect, indicating the safety of the laser dose used. Meanwhile, PGL and O without laser irradiation2Neither @ PFOB @ PGL showed tumor inhibition, indicating that these nanoparticles are not cytotoxic to tumor cells. In sharp contrast, there is PGL + illumination and O due to the enhanced photodynamic therapy effect under laser irradiation2The @ PFOB @ PGL + light group showed significant tumor suppression. More importantly, O2The @ PFOB @ PGL + illumination group showed better tumor treatment effect than the PGL + illumination group, which further confirmed that O2@ PFOB @ PGL nano system capable of delivering O2Slow tumor hypoxia and enhance photodynamic therapy effect to completely eliminate tumor.
Example 12
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for qualitative assessment of the effect of cellular sonodynamic therapy.
To assess cell viability after various treatments, Calcein-AM (Calcein-AM) and PI (propidium iodide) co-staining was performed to examine the effect of sonodynamic therapy on cancer cells. HT-29 cells (1X 10)5One hole) in a 6-hole cell culture box for 24h, and then using PGL nano-particles or O2@ PFOB @ PGL nanoparticle (containing equal amount of 4. mu.M PGL) was added and cultured for 12 h. Ultrasonic irradiation (frequency: 1MHz, intensity: 1.5W/cm)2Duty ratio: 50%) for 300s, incubation is continued for 24h, adding Calcein-AM/PI stain to each well, incubation for 30min, then washing 3 times with PBS. Finally, the cells were observed under a fluorescent microscope and images of calcein and propidium iodide were recorded in the green and red channels, respectively.
The results show that only with PGL + sonication or O2Cell death was observed in the @ PFOB @ PGL + sonicated group (red fluorescence was emitted because PI can permeate dead cells); and do notPFOB-containing PGL + sonophoresis group phase, O2@ PFOB @ PGL + ultrasonic irradiation shows more obvious cell death, which further proves that PFOB carries oxygen and supplies oxygen in the process of sonodynamic therapy to obviously enhance the sonodynamic therapy effect; and sonication alone or incubation of PGL or O alone2No cell death was observed with @ PFOB @ PGL treatment.
Example 13
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for quantitative assessment of cellular sonodynamic therapeutic effect.
HT-29 cells were seeded in 96-well plates (4X 10)4One/hole). After 24 hours of culture, PGL nanoparticles or O were added at different concentrations2@ PFOB @ PGL nanoparticles. To assess cytotoxicity in an oxygen deficient environment, cells were maintained in a triple gas cell incubator at 5% O2、90％N2And 5% CO2. After about 4 hours, each well was illuminated for 300s (frequency: 1MHz, intensity: 1.5W/cm)2Duty ratio: 50%). Then washed with fresh medium and incubated for an additional 24 hours. MTT solution was added to each well at 37 ℃ and 5% CO2Incubate for 2 h. Finally, the absorbance at 490nm was measured with a microplate reader to evaluate the cell activity.
The results show that for no addition of PGL or O2@ PFOB @ PGL (concentration 0. mu.M), no decrease in cell activity was observed in the different treatments. Followed by ultrasonic irradiation with PGL or O2An increase in the concentration of @ PFOB @ PGL, the activity of HT-29 cells was gradually decreased. Furthermore, O when the PGL concentration is less than 0.5. mu.M2The @ PFOB @ PGL group shows a higher sonodynamic killing effect than PGL, since oxygen carried in PFOB effectively enhances1O2The production capacity of (c).
Example 14
The oxygen-carrying porphyrin lipid-perfluorooctyl bromide nano system (O) obtained in the example 6 is used2@ PFOB @ PGL) for animal sonodynamic treatment effect investigation.
Mice bearing HT-29 tumors were randomized into 6 groups: PBS, PBS +Ultrasonic irradiation, PGL, PGL + ultrasonic irradiation, O2@ PFOB @ PGL and O2@ PFOB @ PGL + ultrasonic irradiation. After 24 hours of intravenous injection, ultrasound was applied to different groups (frequency: 1MHz, intensity: 1.5W/cm)2Duty ratio: 50%) and further examined for tumor volume changes in each group, as in example 11.
The results show that the ultrasonic irradiation treatment alone showed little antitumor effect, indicating the safety of the ultrasonic irradiation dose used. At the same time, PGL and O without ultrasonic irradiation2Neither @ PFOB @ PGL showed tumor inhibition, indicating that these nanoparticles are not cytotoxic to tumor cells. In sharp contrast thereto, PGL + ultrasound irradiation and O are due to the enhanced sonodynamic therapeutic effect under ultrasound irradiation2The @ PFOB @ PGL + ultrasonic irradiation group showed significant tumor inhibition effect. More importantly, O2The @ PFOB @ PGL + ultrasonic irradiation group showed better tumor treatment effect than the PGL + ultrasonic irradiation group, which further confirmed that O2@ PFOB @ PGL nano system capable of delivering O2Slow tumor hypoxia and enhance the sonodynamic therapeutic effect to completely eliminate tumors.