CN115671133B - Ozone therapeutic agent and preparation method and application thereof - Google Patents
Ozone therapeutic agent and preparation method and application thereof Download PDFInfo
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- CN115671133B CN115671133B CN202211109817.3A CN202211109817A CN115671133B CN 115671133 B CN115671133 B CN 115671133B CN 202211109817 A CN202211109817 A CN 202211109817A CN 115671133 B CN115671133 B CN 115671133B
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Abstract
The invention discloses an ozone therapeutic agent, a preparation method and application thereof, wherein the ozone therapeutic agent comprises perfluorinated compound nanoemulsion, ozone dissolved in the perfluorinated compound nanoemulsion and thermosensitive gel for encapsulating the perfluorinated compound nanoemulsion and the ozone. The ozone therapeutic agent obtained by filling ozone into the perfluorinated compound nanoemulsion not only can dissolve ozone in high concentration, improve the stability of ozone and prolong the half life of ozone, but also can cause obvious direct toxicity to tumor cells when used for preparing a preparation for treating tumors, and cause iron death of the tumor cells, thereby realizing postoperative anti-recurrence treatment of tumors. And when the preparation method is used for preparing the ozone therapeutic agent, the preparation is simple, the mass production can be realized, the industrialization is easy to realize, and the preparation method has good application prospect in the field of tumor treatment.
Description
Technical Field
The invention relates to the technical field of biomedical nano materials, in particular to an ozone therapeutic agent and a preparation method and application thereof.
Background
At present, the first-line tumor treatment modes such as surgical excision, radiotherapy and chemotherapy still cannot completely inhibit the tumor growth, and even after the early-stage tumor is completely surgical excised, the recurrence rate is still very high, for example, liver cancer has recurrence rate of more than 60% after surgical excision or thermal ablation. Therefore, the method has important clinical significance for postoperative recurrence treatment of tumors.
Ozone has remarkable in vitro destruction to tumor cells, and can enhance chemotherapy and radiotherapy. Ozone can induce tumor cells to produce Reactive Oxygen Species (ROS), including superoxide or hydroxyl radicals, and cause chromosome breakage. However, the tumor is inside the body and penetrates into healthy tissue, and the microenvironment is complex. In addition, ozone is highly oxidizing, has a half-life in aqueous solution of less than 8 minutes, and cannot pass through the lining layer of the lungs of a thickness exceeding 0.1 μm without reacting. Ozone is difficult to reach and penetrate into tumors by conventional methods of administration (autologous blood therapy, intraperitoneal injection, or rectal infusion).
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide an ozone therapeutic agent, a preparation method and application thereof, and aims to solve the problems of poor tumor treatment effect caused by short half-life period and poor tissue penetrability of the existing ozone.
The technical scheme of the invention is as follows:
an ozone therapeutic agent comprising: a perfluorinated compound nanoemulsion, ozone dissolved in the perfluorinated compound nanoemulsion, and a temperature sensitive gel encapsulating the perfluorinated compound nanoemulsion and the ozone.
The mass ratio of the perfluorinated compound nanoemulsion to the temperature-sensitive gel is (1-3): 100.
The ozone therapeutic agent, wherein the perfluorinated compound nanoemulsion comprises perfluorinated compounds, and cholesterol, lecithin and DSPE-PEG2000 coated on the outer layer of the perfluorinated compounds.
The ozone therapeutic agent, wherein the perfluoro compound is selected from one or more of perfluoro-n-pentane, perfluoro-hexane, perfluoro-tributylamine, perfluoro-decalin and perfluoro-15-crown-5-ether; the Wen Minning glue comprises PLA-PEG-PLA.
The ozone therapeutic agent is spherical particles with the diameter of 50-250 nm.
A method of preparing the ozone therapeutic agent comprising the steps of:
providing perfluorinated compound nanoemulsion, and carrying out ozone filling treatment on the perfluorinated compound nanoemulsion;
stirring and mixing the perfluorinated compound nanoemulsion filled with ozone with the temperature-sensitive gel to obtain the ozone therapeutic agent.
The preparation method of the ozone therapeutic agent comprises the following steps:
dissolving cholesterol, lecithin and DSPE-PEG2000 in dichloromethane, and performing rotary evaporation to remove the dichloromethane to obtain a mixture;
and dissolving the mixture in deionized water, adding a perfluorinated compound, and carrying out ultrasonic treatment on ice to obtain the perfluorinated compound nanoemulsion.
The preparation method of the ozone therapeutic agent comprises the steps of mixing the total volume of cholesterol, lecithin and DSPE-PEG2000 with the volume ratio of the perfluorinated compound to 12-18%; the power of the ultrasonic treatment on ice is 60W.
The preparation method of the ozone therapeutic agent comprises the step of filling ozone into the body at a flow rate of 4-6L/min.
Use of an ozone therapeutic agent as described above in the preparation of a formulation for the treatment of a tumour.
The beneficial effects are that: the invention provides an ozone therapeutic agent, a preparation method and application thereof, wherein the ozone therapeutic agent comprises perfluorinated compound nanoemulsion, ozone dissolved in the perfluorinated compound nanoemulsion and temperature-sensitive gel for encapsulating the perfluorinated compound nanoemulsion and the ozone. The ozone therapeutic agent obtained by filling ozone into the perfluorinated compound nanoemulsion not only can dissolve ozone in high concentration, improve the stability of ozone and prolong the half life of ozone, but also can cause obvious direct toxicity to tumor cells when used for preparing a preparation for treating tumors, and cause iron death of the tumor cells, thereby realizing postoperative anti-recurrence treatment of tumors. And when the preparation method is used for preparing the ozone therapeutic agent, the preparation is simple, the mass production can be realized, the industrialization is easy to realize, and the preparation method has good application prospect in the field of tumor treatment.
Drawings
FIG. 1 shows the synthesis of a perfluorotributylamine nanoemulsion-based ozone therapeutic agent (O) according to example 1 of the present invention 3 PFTBA@LIP);
FIG. 2 is a diagram of O in example 2 of the present invention 3 Data graph of ozone concentration measurement result in PFTBA@LIP;
FIG. 3 is a schematic diagram of embodiment 3O of the present invention 3 Quantitative analysis of HuH-7 cell migration ability after PFTBA@LIP treatment;
FIG. 4 is a graph of O in example 4 of the present invention 3 Western blotting results of HuH-7 cells after PFTBA@LIP treatment;
FIG. 5 is a graph of O in example 5 of the present invention 3 Bioluminescence image of HuH-7-Luc subcutaneously tumor bearing mice after PFTBA@LIP treatment.
Detailed Description
The invention provides an ozone therapeutic agent, a preparation method and application thereof, and aims to make the purposes, technical schemes and effects of the invention clearer and more definite, and the invention is further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the description and claims, unless the context specifically defines the terms "a," "an," "the," and "the" include plural referents. If there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature.
It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The term "and/or" as used herein includes all or any element and all combination of one or more of the associated listed items.
It will be understood by those skilled in the art that all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless defined otherwise. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The present invention provides an ozone therapeutic agent comprising: a perfluorinated compound nanoemulsion, ozone dissolved in the perfluorinated compound nanoemulsion, and a temperature sensitive gel encapsulating the perfluorinated compound nanoemulsion and the ozone.
In the invention, the ozone therapeutic agent is a compound combined with the temperature-sensitive gel after dissolving ozone by using the perfluorinated compound nanoemulsion, wherein the perfluorinated compound nanoemulsion can dissolve a large amount of ozone and prolong the half life period of the ozone, so that the ozone therapeutic agent can be used for postoperative recurrence prevention treatment of tumors; the perfluorinated compound nanoemulsion coated by the temperature-sensitive gel can efficiently penetrate through tissues and into tumors, so that the treatment effect is improved.
In some embodiments, the mass ratio of the perfluorinated nanoemulsion to the temperature sensitive gel is (1-3) 100; preferably, the mass ratio of the perfluorinated compound nanoemulsion to the temperature-sensitive gel is 1:50, so that the utilization rate of the temperature-sensitive gel carrier is improved, and a good therapeutic effect is achieved.
In some embodiments, the perfluorinated nanoemulsion comprises a perfluorinated compound and cholesterol, lecithin, and DSPE-PEG2000 (distearoyl phosphatidylethanolamine-polyethylene glycol) coated on the outer layer of the perfluorinated compound; wherein the volume ratio of the total volume of the cholesterol, the lecithin and the DSPE-PEG2000 to the perfluorinated compound is 12-18%, and the stability of the nanoemulsion can be improved under the volume ratio, so that a good therapeutic effect is achieved. In addition, the outer layer of the perfluorinated compound is coated with cholesterol, lecithin and DSPE-PEG2000, so that the perfluorinated compound nanoemulsion has good dissolving and protecting effects on ozone, and the half-life period of the ozone can be prolonged to 30 days.
In a preferred embodiment, the ratio of the total volume of cholesterol, lecithin and DSPE-PEG2000 to the volume of the perfluorinated compound is 15% to improve the stability of the nanoemulsion, thereby achieving a good therapeutic effect.
In some embodiments, the perfluorinated compound is selected from one or more of perfluoro-n-pentane, perfluoro-hexane, perfluoro-tributylamine, perfluoro-decalin, and perfluoro-15-crown-5-ether; the Wen Minning glue comprises PLA-PEG-PLA (polylactic acid-polyethylene glycol dimer); the perfluoro-n-pentane, perfluoro-hexane, perfluoro-tributylamine, perfluoro-decalin and perfluoro-15-crown-5-ether can dissolve ozone in high concentration to improve the stability of ozone, and the temperature-sensitive gel component is PLA-PEG-PLA which has temperature-sensitive phase change property, and is changed from liquid state to solid state at 32-34 ℃, so that fixed-point ozone release to the surgical wound part is realized, and postoperative anti-recurrence treatment is performed.
In some embodiments, the ozone therapeutic agent is spherical particles having a diameter of 50-250 nm; nanoparticles in this size range have high permeability and retention (EPR) effects of solid tumors.
In experimental tests, the nano emulsion of the perfluorinated compound has good dissolving and protecting effects on ozone, and can prolong the half life period to 30 days; ozone therapeutic agent obtained by filling ozone into perfluoro compound nanoemulsion, wherein the temperature sensitive gel can be changed from liquid state to solid state at tumor position, so that ozone is slowly released at operation wound position, and residual tumor cells after operation are killed.
Specifically, after the perfluorinated compound nanoemulsion is filled with ozone, the perfluorinated compound nanoemulsion is encapsulated by using the temperature-sensitive gel, so that the ozone can be dissolved at high concentration, the stability of the ozone is improved, the half life period of the ozone is prolonged, and when the perfluorinated compound nanoemulsion is used for preparing a tumor treatment preparation, obvious direct toxicity can be caused to tumor cells, iron death is caused, and postoperative anti-recurrence treatment of tumors is realized.
In addition, the invention also provides a preparation method of the ozone therapeutic agent, which comprises the following steps:
step S10: providing perfluorinated compound nanoemulsion, and carrying out ozone filling treatment on the perfluorinated compound nanoemulsion;
step S20: stirring and mixing the perfluorinated compound nanoemulsion filled with ozone with the temperature-sensitive gel to obtain the ozone therapeutic agent.
The invention combines high dissolving power of the perfluorinated compound to ozone with lipid (cholesterol, lecithin and DSPE-PEG 2000) to form perfluorinated compound nano-particles, so that the perfluorinated compound nano-particles are encapsulated by temperature-sensitive gel to form the ozone therapeutic agent; the invention utilizes the good solubility of the perfluorinated compound nanoemulsion in the temperature-sensitive gel solution, and can realize the preparation of the ozone therapeutic agent by stirring and mixing. The ozone therapeutic agent is a compound combined with the temperature-sensitive hydrogel after dissolving ozone by using the perfluorinated compound nanoemulsion, wherein the perfluorinated compound can dissolve a large amount of ozone, the half life of the perfluorinated compound is prolonged, and the ozone therapeutic agent has higher penetrating capacity in tissues, so that the therapeutic effect is better, and the ozone therapeutic agent can be used for postoperative recurrence prevention treatment of tumors. Meanwhile, the preparation method is simple, can realize mass production, is easy to realize industrialization, and has good application prospect in the field of tumor treatment.
In some embodiments, the method of preparing the perfluorocompound nanoemulsion comprises the steps of:
step S11: dissolving cholesterol, lecithin and DSPE-PEG2000 in dichloromethane, and performing rotary evaporation to remove the dichloromethane to obtain a mixture;
step S12: and dissolving the mixture in deionized water, adding a perfluorinated compound, and carrying out ultrasonic treatment on ice to obtain the perfluorinated compound nanoemulsion.
Specifically, 0.7mg of cholesterol, 61.625mg of lecithin and 9.475mg of DSPE-PEG2000 were dissolved in 12.5mL of dichloromethane, and then after removing dichloromethane by rotary evaporation at 150rpm under 34 ℃ temperature control, they were dissolved in 4.25mL of deionized water, and 0.75mL of a perfluoro compound was added, and then sonicated on ice for 10 minutes to obtain the perfluoro compound nanoemulsion; then using an oxygen tank and an ozone generator to fill ozone into the perfluorinated compound nanoemulsion, and uniformly mixing the perfluorinated compound nanoemulsion with the degradable directional thermosensitive hydrogel according to the proportion of 2% (v/v), thereby finally preparing the ozone therapeutic agent.
In some embodiments, the ratio of the total volume of cholesterol, lecithin, and DSPE-PEG2000 to the volume of the perfluorinated compound is 12-18%; the power of the ultrasonic treatment on ice is 60W. Preferably, the ratio of the total volume of cholesterol, lecithin and DSPE-PEG2000 to the volume of the perfluorinated compound is 15% to improve the stability of the nanoemulsion, thereby achieving a good therapeutic effect.
In some embodiments, the flow rate of the ozone-charging treatment is 4 to 6L/min. Preferably, the flow rate of the ozone-charging treatment is 5L/min, at which the perfluorinated compound can be caused to dissolve more ozone.
In addition, the invention also provides an application of the ozone therapeutic agent in preparing a preparation for treating tumor; the ozone therapeutic agent prepared by the preparation method can realize postoperative recurrence prevention treatment of tumors, so that the ozone therapeutic agent has good application prospect in the field of tumor treatment. Meanwhile, the preparation process is simple, convenient to operate, does not need complex and expensive equipment, and is easy to realize industrial production.
The following examples are further illustrative of the invention. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure.
Example 1: synthesis of ozone therapeutic agent (O) based on perfluorotributylamine nanoemulsion 3 /PFTBA@LIP)
61.625mg of lecithin, 10.7mg of cholesterol and 9.475mg of DSPE-PEG2000 were dissolved in 12.5mL of dichloromethane. After removal of methylene chloride by rotary evaporation at 34℃and 150rpm, it was dissolved in 4.25mL of deionized water and 0.75mL of perfluorotributylamine was added. Thereafter, the resulting solution was sonicated on ice for 10 minutes (60W) to obtain a nanoemulsion.
Ozone is filled into the nanoemulsion by using an oxygen tank and an ozone generator (5L/min), and the obtained nanoemulsion and the degradable reverse thermosensitive hydrogel are uniformly mixed according to the volume ratio of 2%, so that the ozone therapeutic agent based on the perfluorinated tributylamine nanoemulsion is synthesized.
Synthetic perfluorotributylamine nanoemulsion ozone therapeutic agent (O) 3 TEM image of/PFTBA@LIP) is shown in FIG. 1, scale bar 100nm. As can be seen from fig. 1, the synthesized ozone therapeutic agent of perfluoro tributylamine nanoemulsion has a spherical shape, and the particle size of the ozone therapeutic agent is between 50nm and 250 nm.
Example 2: perfluoro tributylamine nanoemulsion ozone therapeutic agent (O) 3 Ozone stability analysis in PFTBA@LIP)
Analysis of O preserved at 4℃by iodine reduction titration 3 Ozone content in PFTBA@LIP solution over time.
Synthetic perfluorotributylamine nanoemulsion ozone therapeutic agent (O) 3 The ozone content change of/PFTBA@LIP) is shown in FIG. 2. As can be seen from fig. 2, the ozone concentration in the aqueous solution rapidly decreases, and the ozone is completely decomposed within 48 hours. The ozone in the synthesized perfluoro-tributylamine nanoemulsion ozone therapeutic agent is kept to be decomposed relatively slowly, and more than 40% of the ozone is remained in 30 days, which proves that the perfluoro-tributylamine nanoemulsion can effectively improve the stability of the ozone.
Example 3: analysis of the Effect of ozone therapeutic Agents of perfluorotributylamine nanoemulsion on HuH-7 cell migration Capacity
The effect of ozone therapeutic agents of perfluorotributylamine nanoemulsions on HuH-7 cell migration ability was evaluated using a high content microscope.
After ozone therapeutic agent of perfluoro tributylamine nanoemulsion is filled with ozone for different time periods (0, 15,30,45,60 s), the ozone therapeutic agent is incubated with HuH-7 cells, and the displacement distance and the movement speed of the incubated HuH-7 cells are respectively measured. The results are shown in fig. 3, and the ozone therapeutic agent of the perfluoro tributylamine nanoemulsion has remarkable inhibition effect on the migration distance and the movement speed of HuH-7 cells along with the increase of the ozone concentration, which shows the influence on the migration capacity of the HuH-7 cells.
Example 4: evaluation of cellular iron death Effect of ozone therapeutic Agents of Perfluorotributylamine nanoemulsion
HuH-7 cells incubated with ozone therapeutic agent of perfluoro tributylamine nanoemulsion were collected and the protein content of GPX4, ACSL4 and GAPDH in tumor cells was determined by Western blotting. The results of fig. 4 demonstrate that ozone therapeutic agent of perfluorotributylamine nanoemulsion can cause decrease in GPX4 protein expression level and increase in ACSL4 protein expression level in HuH-7 cells, indicating that ozone treatment activates iron death pathway of tumor cells.
Example 5: evaluation of recurrence-preventing Effect of ozone therapeutic agent of Perfluorotributylamine nanoemulsion
Constructing a subcutaneous HuH-7-Luc transplanted tumor mouse model, performing surgical excision on the subcutaneous tumor on the 15 th day, and respectively spraying and loading physiological saline (physiological saline @ Gel), perfluorotributylamine nanoemulsion (PFTBA @ LIP @ Gel) and oxygenated perfluorotributylamine nanoemulsion (O) 2 PFTBA@LIP@gel), and ozone-filled gel (O) of perfluorotributylamine nanoemulsion 3 PFTBA@LIP@gel). Mice were then monitored for recurrence of subcutaneous tumors using a bioluminescence imager over 30 days. As can be seen in FIG. 5, normal saline @ Gel, PFTBA @ LIP @ Gel, and O 2 Mice in the group of/PFTBA@LIP@gel all had significant tumor recurrence in situ, whereas O 3 Mice in the group of/PFTBA@LIP@gel have no obvious tumor recurrence condition within 30 days after treatment, which indicates that the ozone therapeutic agent of the perfluorotributylamine nanoemulsion can effectively inhibit recurrence of HuH-7 subcutaneous tumor.
In summary, the present invention provides an ozone therapeutic agent, a preparation method and an application thereof, wherein the ozone therapeutic agent comprises a perfluorinated compound nanoemulsion, ozone dissolved in the perfluorinated compound nanoemulsion, and a temperature-sensitive gel encapsulating the perfluorinated compound nanoemulsion and the ozone. The ozone therapeutic agent obtained by filling ozone into the perfluorinated compound nanoemulsion not only can dissolve ozone in high concentration, improve the stability of ozone and prolong the half life of ozone, but also can cause obvious direct toxicity to tumor cells when used for preparing a preparation for treating tumors, and cause iron death of the tumor cells, thereby realizing postoperative anti-recurrence treatment of tumors. And when the preparation method is used for preparing the ozone therapeutic agent, the preparation is simple, the mass production can be realized, the industrialization is easy to realize, and the preparation method has good application prospect in the field of tumor treatment.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (8)
1. An ozone therapeutic agent, comprising: a perfluorinated compound nanoemulsion, ozone dissolved in the perfluorinated compound nanoemulsion, and a temperature-sensitive gel encapsulating the perfluorinated compound nanoemulsion and the ozone;
the perfluorinated compound nanoemulsion comprises perfluorinated compounds, cholesterol, lecithin and DSPE-PEG2000 coated on the outer layer of the perfluorinated compounds;
the preparation method of the perfluorinated compound nanoemulsion comprises the following steps:
dissolving cholesterol, lecithin and DSPE-PEG2000 in dichloromethane, and performing rotary evaporation to remove the dichloromethane to obtain a mixture;
dissolving the mixture in deionized water, adding a perfluorinated compound, and carrying out ultrasonic treatment on ice to obtain perfluorinated compound nanoemulsion;
the perfluorinated compound is perfluoro tributylamine; the Wen Minning glue comprises PLA-PEG-PLA.
2. The ozone therapeutic agent of claim 1, wherein the mass ratio of the perfluorinated nanoemulsion to the temperature sensitive gel is (1-3): 100.
3. The ozone therapeutic of claim 1, wherein the ozone therapeutic is spherical particles having a diameter of 50-250 nm.
4. A method of preparing an ozone therapeutic according to any one of claims 1 to 2, comprising the steps of:
providing perfluorinated compound nanoemulsion, and carrying out ozone filling treatment on the perfluorinated compound nanoemulsion;
stirring and mixing the perfluorinated compound nanoemulsion filled with ozone with the temperature-sensitive gel to obtain the ozone therapeutic agent.
5. The method for preparing an ozone therapeutic agent according to claim 4, wherein the method for preparing a perfluorocompound nanoemulsion comprises the steps of:
dissolving cholesterol, lecithin and DSPE-PEG2000 in dichloromethane, and performing rotary evaporation to remove the dichloromethane to obtain a mixture;
and dissolving the mixture in deionized water, adding a perfluorinated compound, and carrying out ultrasonic treatment on ice to obtain the perfluorinated compound nanoemulsion.
6. The method for producing an ozone therapeutic agent according to claim 5, wherein a ratio of a total volume of cholesterol, lecithin, and DSPE-PEG2000 to a volume of the perfluoro compound is 12 to 18%; the power of the ultrasonic treatment on ice is 60W.
7. The method for producing an ozone therapeutic agent according to claim 4, wherein the flow rate of the ozone-charging treatment is 4 to 6l/min.
8. Use of an ozone therapeutic according to any one of claims 1-2 in the preparation of a formulation for treating a tumor.
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| CN103212094A (en) * | 2013-04-28 | 2013-07-24 | 重庆医科大学附属儿童医院 | Oxygen-fluorine liposome microbubble and preparation method thereof |
| CN113101269A (en) * | 2021-04-15 | 2021-07-13 | 四川大学华西医院 | Delivery system based on nano-liposome, preparation method and application |
| CN113398257A (en) * | 2021-05-19 | 2021-09-17 | 深圳大学 | Fusion membrane-coated bionic nanoemulsion and preparation method and application thereof |
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| CN103212094A (en) * | 2013-04-28 | 2013-07-24 | 重庆医科大学附属儿童医院 | Oxygen-fluorine liposome microbubble and preparation method thereof |
| CN113101269A (en) * | 2021-04-15 | 2021-07-13 | 四川大学华西医院 | Delivery system based on nano-liposome, preparation method and application |
| CN113398257A (en) * | 2021-05-19 | 2021-09-17 | 深圳大学 | Fusion membrane-coated bionic nanoemulsion and preparation method and application thereof |
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