CN111358945A - Metalloporphyrin-loaded liposome nanoparticle, preparation method and application thereof - Google Patents

Abstract

The invention provides a metalloporphyrin-loaded liposome nanoparticle, a preparation method and application thereof, and relates to the technical field of nanomedicine. The metalloporphyrin-loaded liposome nanoparticle includes metalloporphyrin and liposome , the metalloporphyrin is encapsulated in the liposome, which alleviates the poor bioavailability and poor targeting of the existing metalloporphyrin, and limits the technical problem of its clinical application. Liposome nanoparticles encapsulate metalloporphyrins through liposomes, which not only improves the stability and biocompatibility of metalloporphyrins, but also improves the biomembrane penetration and targeting of metalloporphyrins. The sonodynamic effect is triggered under ultrasonic excitation, so that the metalloporphyrin-loaded liposome nanoparticles are easier to aggregate at the tumor cells, improve the drug utilization rate, and achieve the effect of selectively treating tumors.

Description

Metalloporphyrin-loaded liposome nanoparticle, preparation method and application thereof

technical field

The invention relates to the technical field of nano-medicine, in particular to a metalloporphyrin-loaded liposome nano-particle and a preparation method and application thereof.

Background technique

Sonodynamic therapy (SDT) excites sonosensitizers by ultrasound, and in the process of sonoluminescence-induced acoustic cracking, generates highly reactive singlet oxygen and free radicals and other reactive oxygen species (ROS) by transferring energy to oxygen molecules or water molecules. ) to kill tumor cells and achieve the purpose of treating tumors. Sonodynamic therapy utilizes ultrasonic waves to generate reactive oxygen species to treat cancer, and has become a research hotspot in the field of tumor therapy in recent years due to its advantages of deep penetration, non-invasiveness, and controllability in space and time.

At present, the research on sonosensitizers mainly focuses on the direction of organic sonosensitizers such as porphyrins. Traditional organic sonosensitizers such as porphyrin and its derivatives have insignificant sonodynamic effects due to their low accumulation in tumor sites. In recent years, various metal-centered porphyrin metal complexes have been widely reported in antibacterial and antitumor studies. As a substance in which metal ions and organic compounds are complexed by coordination bonds, metal organic complexes optimize the structure of organic raw materials and play the physiological functions of metal ions in vivo, often showing specific physiological activities.

Although metalloporphyrins can improve the sensitization effect of sonodynamic therapy to a certain extent in the sonosensitizer system, the poor bioavailability and poor targeting ability of this complex limit its clinical application.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a metalloporphyrin-loaded liposome nanoparticle, so as to alleviate the technical problems of poor bioavailability and poor targeting properties of the existing metalloporphyrins, which limit its clinical application.

The metalloporphyrin-loaded liposome nanoparticles provided by the present invention include metalloporphyrins and liposomes, and the metalloporphyrins are encapsulated in the liposomes.

Further, the metal ion in the metalloporphyrin is selected from at least one of gold, platinum, iron, cobalt, nickel, manganese, copper or zinc, preferably manganese porphyrin.

Further, the liposomes are folic acid liposomes, and the folic acid liposomes include liposomes and folic acid, and the folic acid is linked to the liposomes through chemical bonds.

Further, the particle size of the metalloporphyrin-loaded liposome nanoparticles is 50-500 nm, preferably 100-200 nm.

The second object of the present invention is to provide the preparation method of the metalloporphyrin-loaded liposome nanoparticle, comprising the following steps:

The metalloporphyrin solution and the liposome solution are mixed uniformly, and ultrasonicated to obtain metalloporphyrin-loaded liposome nanoparticles.

Preferably, the preparation method of the metalloporphyrin-loaded liposome nanoparticles comprises the following steps:

(a) mixing the metalloporphyrin solution, the lecithin solution and the phospholipid PEG substance solution uniformly, removing the solvent to obtain a metalloporphyrin-loaded liposome film;

(b) dispersing the metalloporphyrin-loaded lipid film in an aqueous solution to obtain a metalloporphyrin-loaded liposome suspension;

(c) ultrasonicating the metalloporphyrin-loaded liposome suspension to obtain metalloporphyrin-loaded liposome nanoparticles.

Further, in step (a), the mass ratio of metalloporphyrin and liposome is 1:(10-25), preferably 1:(15-20).

Further, in step (a), the phospholipid PEG substances include DSPE-PEG and derivatives thereof;

Preferably, the DSPE-PEG derivative is selected from at least one of DSPE-PEG-folate, DSPE-PEG-NH ₂ , DSPE-PEG-COOH or DSPE-PEG-NHS, preferably DSPE-PEG-folate.

Preferably, the mass ratio of the metalloporphyrin, lecithin and the DSPE-PEG-folate is 1:(5-15):(1-2), preferably 1:(8-12):(1- 2).

Further, in step (a), the organic solvent is removed by using a vacuum rotary evaporation method;

And/or, in step (b), the metalloporphyrin-loaded liposome film is dispersed in the aqueous solution by ultrasonic hydration;

And/or, in step (c), ultrasonication is performed using an ultrasonic cell disruptor.

Preferably, in step (b), the time of ultrasonic hydration is 2-15min, preferably 5-10min;

Preferably, in step (c), the ultrasonic time is 3-10 min, preferably 4-6 min.

The third object of the present invention is to provide the application of metalloporphyrin-loaded liposome nanoparticles in the preparation of drugs for treating tumors.

The fourth object of the present invention is to provide a medicine for treating tumors, including the metalloporphyrin-loaded liposome nanoparticles provided by the present invention.

The metalloporphyrin-loaded liposome nanoparticle provided by the invention not only improves the stability and biocompatibility of metalloporphyrin, but also improves the biomembrane penetration of metalloporphyrin through liposome encapsulation of metalloporphyrin At the same time, it can also trigger the sonodynamic effect under ultrasonic excitation, so that the metalloporphyrin-loaded liposome nanoparticles are easier to aggregate at the tumor cells, improve the drug utilization rate, and achieve the effect of selective tumor treatment. .

The preparation method of the metalloporphyrin-loaded liposome nanoparticle provided by the present invention encapsulates the metalloporphyrin in the liposome by ultrasound, the process is simple, the operation is convenient, and the preparation cost can be effectively reduced.

Description of drawings

Fig. 1 is a bar graph of the fluorescence intensity of the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 and the PP-loaded folic acid liposome nanoparticles provided by Comparative Example 1 at different ultrasonic times;

2 is a bar graph of cell viability after the MnPP-loaded folic acid liposome nanoparticles provided in Example 3, the PP-loaded folic acid liposome nanoparticles provided by Comparative Example 1, and a blank control group and cells;

3 is a bar graph of cell viability after the MnPP-loaded folic acid liposome nanoparticles provided in Example 3, the PP-loaded folic acid liposome nanoparticles provided by Comparative Example 1, and the blank control group and cells;

4 is a graph of ROS generated by the MnPP-loaded folic acid liposome nanoparticles provided in Example 3, the PP-loaded folic acid liposome nanoparticles provided in Comparative Example 1, and a blank control group.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

It should be noted:

In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form new technical solutions.

In the present invention, unless otherwise specified, percentage (%) or part refers to the weight percentage or weight part of the composition.

In the present invention, unless otherwise specified, the involved components or their preferred components can be combined with each other to form a new technical solution.

In the present invention, unless otherwise stated, the numerical range "a～b" represents an abbreviated representation of any combination of real numbers between a and b, wherein both a and b are real numbers. For example, the numerical range "6-22" indicates that all real numbers between "6-22" have been listed in the text, and "6-22" is just an abbreviated representation of the combination of these numerical values.

A "range" disclosed herein may be in the form of a lower limit and an upper limit, which may be one or more lower limits, and one or more upper limits, respectively.

In the present invention, unless otherwise specified, each reaction or operation step can be carried out sequentially or in sequence. Preferably, the reaction methods herein are performed sequentially.

Unless otherwise defined, professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described can also be used in the present invention.

The metal ions in metalloporphyrins attract the large π bonds of porphyrins, thereby improving the transition performance of the π electrons on the porphyrin ring, so that metalloporphyrins have the effect of killing tumors under the condition of ultrasonic excitation, so metalloporphyrins were developed. The new function of the anti-tumor function under the excitation of ultrasound. However, due to its poor biocompatibility, its application in biological research is seriously affected.

According to one aspect of the present invention, the present invention provides a metalloporphyrin-loaded liposome nanoparticle, comprising a metalloporphyrin and a liposome, and the metalloporphyrin is encapsulated in the liposome.

In the present invention, the metalloporphyrin is a metalloporphyrin complex.

The metalloporphyrin-loaded liposome nanoparticle provided by the invention not only improves the stability and biocompatibility of metalloporphyrin, but also improves the biomembrane penetration of metalloporphyrin through liposome encapsulation of metalloporphyrin At the same time, it can also trigger the sonodynamic effect under ultrasonic excitation, so that the metalloporphyrin-loaded liposome nanoparticles are more likely to aggregate at the tumor cells, improve the utilization rate of drugs, and achieve the selective treatment of tumors. Effect.

In a preferred embodiment of the present invention, the metal ions in the metalloporphyrin are selected from one or more of gold, platinum, iron, cobalt, nickel, manganese, copper or zinc, preferably manganese porphyrin.

When the metalloporphyrin is a manganese porphyrin, it has the dual functions of nuclear magnetic imaging and anti-tumor, and can realize the multi-functional diagnosis and treatment integration of acoustodynamics and nuclear magnetic imaging in tumor treatment.

In a preferred embodiment of the present invention, the liposomes are folic acid liposomes, and the folic acid liposomes include liposomes and folic acid, and the folic acid is linked to the liposomes through chemical bonds. As a water-soluble drug, folic acid can participate in various metabolic activities in the human body, and the folic acid receptor is a high-affinity folic acid-binding protein that is overexpressed on various tumor cells in the human body. Folate receptors are highly expressed in carcinoma, lung cancer, colon cancer, lung cancer, colon cancer and snuff cancer. Therefore, it is easier to achieve precise targeting of tumor cells by metalloporphyrin-loaded liposome nanoparticles after linking folic acid and liposomes through chemical bonds.

In a preferred embodiment of the present invention, the chemical bonds include ionic bonds, covalent bonds and metal bonds. Preferably, the folic acid and the liposome are connected by a covalent bond, so as to improve the stability of the connection between the folic acid and the liposome.

In a preferred embodiment of the present invention, the particle size of the metalloporphyrin-loaded liposome nanoparticles is 50-500 nm. By controlling the particle size of the metalloporphyrin-loaded liposome nanoparticles, it is convenient for them to penetrate the cell membrane and improve their targeting to tumor cells, especially when the particle size is 100-200 nm, it is more conducive to penetration It can improve the targeting of tumor cells, enhance the uptake of metalloporphyrins by tumor cells, and enhance the efficacy of sonodynamics.

Typical but non-limiting particle sizes of metalloporphyrin-loaded liposome nanoparticles are, for example, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm.

According to the second aspect of the present invention, the present invention provides the above-mentioned preparation method of metalloporphyrin-loaded liposome nanoparticles, comprising the following steps:

The metalloporphyrin solution and the liposome solution are mixed uniformly, and ultrasonicated to obtain metalloporphyrin-loaded liposome nanoparticles.

In a preferred embodiment of the present invention, the mass ratio of metalloporphyrin and liposome is 1:(10-25). By controlling the mass ratio of metalloporphyrin and liposome, the entrapment rate of metalloporphyrin in the metalloporphyrin-loaded liposome nanoparticles can be improved, and the therapeutic effect can be improved, especially when the quality of metalloporphyrin and liposome is improved When the ratio is 1:(15-20), the entrapment rate of metalloporphyrin in the metalloporphyrin-loaded liposome nanoparticles is higher, and the therapeutic effect on tumor cells is better.

Typical but non-limiting, the mass of metalloporphyrin and liposome is 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25. In a preferred embodiment of the present invention, the preparation method of metalloporphyrin-loaded liposome nanoparticles comprises the following steps:

(a) mixing the metalloporphyrin solution, the lecithin solution and the phospholipid PEG substance solution uniformly, removing the solvent to obtain a metalloporphyrin-loaded liposome film;

(b) dispersing the metalloporphyrin-loaded lipid film in an aqueous solution to obtain a metalloporphyrin-loaded liposome suspension;

(c) ultrasonicating the metalloporphyrin-loaded liposome suspension to obtain metalloporphyrin-loaded liposome nanoparticles.

In the above-mentioned preferred embodiment, in step (a), the metalloporphyrin solution, the lecithin solution and the phospholipid PEG substance solution are mixed uniformly, and then the solution is removed to load the metalloporphyrin on the lecithin and the phospholipid PEG. On the liposome film generated by the similar substances, obtain a metalloporphyrin-loaded liposome film; in step (b), disperse the metalloporphyrin-loaded liposome film in water to obtain a metalloporphyrin-loaded lipid The body suspension is facilitated in step (c), by sonication, so that the metalloporphyrin is entrapped in the liposomes.

In a preferred embodiment of the present invention, by mixing the metalloporphyrin solution and the raw materials of the liposome uniformly, then removing the solution, it is more favorable for the metalloporphyrin to be dispersed more uniformly in the liposome film.

In a preferred embodiment of the present invention, in step (a), the phospholipid PEG substances include DSPE-PEG and derivatives thereof.

In a preferred embodiment of the present invention, the phospholipid PEG substances include DSPE-PEG and its derivatives, and the DSPE-PEG derivatives are selected from DSPE-PEG-folate, DSPE-PEG-NH ₂ , DSPE-PEG-COOH or DSPE -At least one of PEG-NHS. When the phosphoesterified PEG substance is DSPE-PEG-folate, it is synergistic with lecithin, and the prepared liposome is folic acid liposome, so that the prepared metalloporphyrin-loaded liposome nanoparticles have With stronger targeting performance, it is easier to enrich at tumor cells and improve the therapeutic effect.

In a preferred embodiment of the present invention, the lecithin is soybean lecithin.

In a preferred embodiment of the present invention, the mass ratio of the metalloporphyrin, lecithin and the DSPE-PEG-folate is 1:(5-15):(1-2), preferably 1:( 8-12): (1-2). By controlling the mass ratio of metalloporphyrin, lecithin and DSPE-PEG-folate, it is beneficial to provide the encapsulation rate of metalloporphyrin in the metalloporphyrin-loaded liposome nanoparticles, especially when the mass ratio of the three is 1: (8-12): (1-2), the prepared metalloporphyrin-loaded liposome nanoparticle metalloporphyrin has a higher entrapment rate.

Typical, but non-limiting, masses of lecithin and DSPE-PEG-folate are, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1 , 13:1, 14:1 or 15:1.

In a preferred embodiment of the present invention, in step (a), the organic solvent is removed by a rotary evaporation method under reduced pressure. The process of removing the organic solvent by using the reduced pressure rotary evaporation method is simpler, more convenient and quicker.

In a preferred embodiment of the present invention, in step (b), by ultrasonic hydration, the metalloporphyrin-loaded lipid film is dispersed in an aqueous solution, so that the obtained metalloporphyrin-loaded liposome is dispersed in water It is more uniform and stable, which is more conducive to the subsequent encapsulation of metalloporphyrin in liposomes.

In a preferred embodiment of the invention, the ultrasonic hydration time is 2-15min, preferably 5-10min.

By controlling the time of ultrasonic hydration, it is favorable to disperse the metalloporphyrin-loaded lipid film uniformly in water, so as to obtain uniformly dispersed and stable metalloporphyrin-loaded liposomes.

In a preferred mode of the present invention, in step (c), an ultrasonic cell disruptor is used to perform ultrasonic waves, so as to facilitate the generation of metalloporphyrin-loaded liposome nanoparticles with uniform and stable particle size.

In a preferred embodiment of the present invention, the ultrasonic time using an ultrasonic cell disruptor is 3-10 minutes, preferably 4-6 minutes. By controlling the ultrasonic time of the ultrasonic cell disruptor, it is beneficial to encapsulate metalloporphyrin in liposomes, especially when the ultrasonic time is 4-6min, which is more conducive to reducing energy waste on the basis of ensuring the encapsulation rate.

Typically, but not limitedly, the ultrasonic time using the ultrasonic cell disruptor is 3, 4, 5, 6, 7, 8, 9 or 10 min.

According to the third aspect of the present invention, the present invention provides the application of the above-mentioned metalloporphyrin-loaded liposome nanoparticles in the preparation of drugs for treating tumors.

Such tumors include, but are not limited to, ovarian cancer, endometrial cancer, renal cancer, breast cancer, lung cancer, colon cancer and nasopharyngeal cancer.

The technical solutions provided by the present invention will be further described below in conjunction with the examples and comparative examples.

Example 1

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle, which is prepared according to the following steps:

(1) Dissolve 1 mg of MnPP in 100 uL of analytically pure dimethyl sulfoxide (DMSO) to obtain a MnPP solution with a concentration of 10 mg/mL;

(2) taking the mass ratio of soybean lecithin 10 mg and dissolving it in analytically pure chloroform, gently shaking until completely dissolved, to obtain soybean lecithin solution;

(3) Weigh 1 mg of DSPE-PEG-folate in analytically pure chloroform, shake until fully dissolved to obtain DSPE-PEG-folate solution;

(4) Mix MnPP solution, soybean lecithin solution and DSPE-PEG-folate solution evenly, and remove chloroform by rotary evaporation under reduced pressure to obtain a folic acid liposome film uniformly loaded with MnPP, and vacuum dry for 24h;

(5) adding the MnPP-loaded folic acid liposome film to a certain volume of PBS ultrasonic cleaning wave for ultrasonic hydration for 2-15 minutes to obtain the MnPP-loaded folic acid liposome suspension;

(6) The MnPP-loaded folic acid liposome suspension was subjected to intermittent ultrasonic waves for 5 minutes using an ultrasonic cell disruptor, and then filtered through a 0.22 μm filter membrane to obtain MnPP-porphyrin-loaded folic acid liposome nanoparticles.

Example 2

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle. The difference between the preparation method and the embodiment 1 is that the amount of DSPE-PEG-folate in step (3) is 2 mg.

Example 3

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle. The difference between the preparation method and the embodiment 1 is that the dosage of DSPE-PEG-folate in step (3) is 1.5 mg .

Example 4

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle, the preparation method of which is different from that of Example 3 in that the amount of soybean lecithin in step (2) is 15 mg.

Example 5

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle, the preparation method of which is different from that of Example 3 in that the amount of soybean lecithin in step (2) is 8 mg.

Example 6

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle, the preparation method of which is different from that of Embodiment 1 in that the amount of soybean lecithin in step (2) is 12 mg.

Example 7

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle, and the preparation method thereof is different from that of embodiment 1 in that the amount of soybean lecithin in step (2) is 3 mg.

Example 8

The present embodiment provides a manganese porphyrin (MnPP)-loaded folic acid liposome nanoparticle, the preparation method of which is different from that in Example 1 in that the dosage of DSPE-PEG-folate in step (3) is 0.5 mg .

Comparative Example 1

The present comparative example provides a porphyrin (PP)-loaded folic acid liposome nanoparticle, the preparation method of which is different from that of Example 1 in that PP is used instead of MnPP. Test Example 1

Take 5Ml of the MnPP-loaded folic acid liposome nanoparticle solution provided in Example 1-8, respectively, place in a 100kD ultrafiltration tube, and centrifuge at 5000rpm for 5min, discard the filtrate and add deionized water to 5mL, repeat 3 times, the concentrates were collected separately. Accurately measure an appropriate amount of the above-mentioned 8 groups of concentrated solutions, dilute to an appropriate concentration with dimethyl sulfoxide (DMSO) and analyze with an ultraviolet spectrophotometer to calculate the drug content; simultaneously accurately measure the load MnPP provided by Example 1-8. 5 mL of folic acid liposome nanoparticle solution, lyophilized, respectively weighed and recorded the weight of the nanoparticles, and calculated the drug loading rate. In addition, the MnPP-loaded folic acid liposome nanoparticle solutions provided in Examples 1-8 were diluted to an appropriate concentration, and the particle size was detected in a Malvern laser particle size analyzer. The results are shown in Table 1.

Table 1 Data table of MnPP-loaded folate liposome nanoparticles

From Table 1, it can be seen from the comparison between Examples 1-6 and 7-8 that the particle sizes of the MnPP-loaded folic acid liposome nanoparticles provided in Examples 1-6 are all less than 100 nm, and the particle size distribution is better. Uniform, the drug entrapment rate is not less than 7.5%, and it is easier to accumulate at the tumor site.

Test Example 2

The MnPP-loaded folic acid liposome nanoparticle solution provided in Example 3 and the PP-loaded folic acid liposome nanoparticle solution provided in Comparative Example 1 were respectively configured into an aqueous dispersion with an energy concentration of 50 μg/mL, and then implemented. The MnPP-loaded folic acid liposome nanoparticle aqueous dispersion provided in Example 3 and the PP-loaded folic acid liposome nanoparticle provided in Comparative Example 1 were mixed with 10 μmol/L of 2′,7′-dichlorofluorescein diacetic acid, respectively. The salt (DCFH-DA) fluorescent probe was mixed, sonicated, and sampled at different times. After the two groups of solutions after ultrasonication were diluted by the same times, the fluorescence intensity of reactive oxygen species was detected by a multi-function microplate reader, the fluorescence intensity after ultrasonication for 4 min was recorded, and a column chart was drawn.

1 is a bar graph of the fluorescence intensity of the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 at different ultrasonic times, wherein PP represents the aqueous dispersion of the PP-loaded folic acid liposome nanoparticles provided in Comparative Example 1, MnPP represents the aqueous dispersion of MnPP-loaded folic acid liposome nanoparticles provided in Example 3, and FL Intensity represents the fluorescence intensity. As shown in Figure 1, with the prolongation of the ultrasonic time, the fluorescence intensity of the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 dispersed in water was significantly enhanced, while the PP-loaded folic acid lipid provided in Comparative Example 1 With the prolongation of the ultrasonic time, the fluorescence intensity of the plastid nanoparticles increased slightly, which indicates that the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 encapsulate MnPP in the folic acid liposomes to generate reactive oxygen species. The amount of (ROS) was significantly higher than that of PP-only folate liposome nanoparticles, enabling more efficient sonodynamic therapy.

Test Example 3

Breast cancer cells 4T1 in logarithmic growth phase were seeded in a 96-well plate at 5×10 ³ cells/well, 0.2 mL of cell suspension was added to each well, and incubated in a 37°C incubator (containing 5% CO ₂ ). After 24 hours, the MnPP-loaded folic acid liposome nanoparticles and porphyrin provided in Example 3 and the PP-loaded folic acid lipid nanoparticles provided in Comparative Example 1 were diluted to 5 μg/mL, 10 μg/mL, After the concentration of 20 μg/mL, it was added to the cells respectively. At the same time, the medium was also added to the cells as a blank control group. After continuing to incubate for 24 hours, cck8 was added. The in vitro safety of nanoparticles was then calculated. The results are shown in Figure 2, where Control represents the medium blank control group, PP-LP-FA represents the PP-loaded folic acid liposome nanoparticles provided in Comparative Example 1; MnPP-LP-FA represents the MnPP-loaded folic acid lipid For bulk nanoparticles, Cell livability stands for cell viability.

It can be seen from Figure 2 that the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 have no obvious killing effect on cells, which shows that the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 has good safety. , will not cause damage to cells.

Test Example 3

Breast cancer cells 4T1 in logarithmic growth phase were seeded in a 96-well plate at 5×10 ³ cells/well, 0.2 mL of cell suspension was added to each well, and incubated in a 37°C incubator (containing 5% CO ₂ ). After 24 hours, the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 and the PP-loaded folic acid liposome nanoparticles provided in Comparative Example 1 were diluted with culture medium to a concentration of 10 μg/mL, respectively, and added to the cells. At the same time, the medium was also added to the cells as a blank control group, and the incubation was continued for 3 hours. Then, the wells in the 96-well plate that need to be sonicated were placed under a flat ultrasonic probe, and after ultrasonic excitation (2MHz, 2W) for 3 minutes, and continued incubation for 24 hours, cck8 was added, and OD (450 ) value, and then calculate the ultrasonic killing rate of nanoparticles, the results are shown in Figure 3, wherein, Contron1 represents the blank control group and does not carry out ultrasonic treatment, Control+US represents the blank control group and carries out ultrasonication, PP-LP-FA+US Representative Comparative Example 1 provides PP-loaded folic acid liposome nanoparticles and is sonicated; MnPP-LP-FA+US represents the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 and is sonicated, and Cell viability represents cells survival rate.

It can be seen from Figure 3 that the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 have significantly higher killing effect on tumor cells under the action of ultrasound than the PP-loaded nanoparticles provided in Comparative Example 1 under ultrasound. Folic acid liposome nanoparticles, this shows that the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 has a significant sonodynamic effect on breast tumor cells 4T1.

Test Example 4

Breast cancer cells 4T1 were co-incubated with the culture medium, the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 and the PP-loaded folic acid liposome nanoparticles provided in Comparative Example 1, incubated for 3h, and then subjected to ultrasonic treatment. , to detect the cell viability after sonication. The results are shown in Figure 4, where Control+US represents the medium blank control group plus ultrasonic treatment, PP-LP-FA+US represents the PP-loaded folic acid liposome nanoparticles provided in Comparative Example 1 plus ultrasonic treatment; MnPP- LP-FA represents the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 plus ultrasonic treatment, DAPI represents 4',6-diamidino-2-phenylindole, ROS represents reactive oxygen species such as free radicals, Merge represents the superposition of the two. The results show that the amount of ROS generated in the cell by the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 is significantly increased under the condition of sound dynamics (SDT), indicating that the MnPP-loaded folic acid liposome nanoparticles provided in Example 3 It has sonosensitivity and can be used as a sonosensitizer in the sonodynamic therapy of tumor therapy.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

Claims (10)

1. a metalloporphyrin-loaded liposome nanoparticle, is characterized in that, comprises metalloporphyrin and liposome, and described metalloporphyrin is encapsulated in described liposome. 2. the liposome nanoparticle of loaded metalloporphyrin according to claim 1, is characterized in that, the metal ion in described metalloporphyrin is selected from gold, platinum, iron, cobalt, nickel, manganese, copper or zinc At least one of them is preferably manganese porphyrin. 3. The metalloporphyrin-loaded liposome nanoparticle according to claim 1, wherein the liposome is a folic acid liposome, and the folic acid liposome comprises liposome and folic acid, and the Folic acid is chemically linked to the liposomes. 4. the metalloporphyrin-loaded liposome nanoparticle according to any one of claims 1-3, is characterized in that, the particle diameter of the metalloporphyrin-loaded liposome nanoparticle is 50-500nm, preferably 100-200nm. 5. the preparation method of the metalloporphyrin-loaded liposome nanoparticle according to any one of claims 1-4, is characterized in that, comprises the steps: The metalloporphyrin solution and the liposome solution are mixed uniformly, and ultrasonicated to obtain metalloporphyrin-loaded liposome nanoparticles. 6. preparation method according to claim 5, is characterized in that, comprises the steps: (a) mixing the metalloporphyrin solution, the lecithin melt and the phospholipid PEG material uniformly, removing the solvent to obtain a metalloporphyrin-loaded liposome film; (b) dispersing the metalloporphyrin-loaded lipid film in an aqueous solution to obtain a metalloporphyrin-loaded liposome suspension; (c) ultrasonicating the metalloporphyrin-loaded liposome suspension to obtain metalloporphyrin-loaded liposome nanoparticles. 7. preparation method according to claim 6, is characterized in that, in step (a), described phospholipid PEG substances comprise DSPE-PEG and derivatives thereof; Preferably, the DSPE-PEG derivative is selected from at least one of DSPE-PEG-folate, DSPE-PEG-NH ₂ , DSPE-PEG-COOH or DSPE-PEG-NHS, preferably DSPE-PEG-folate; Preferably, the mass ratio of the metalloporphyrin, lecithin and the DSPE-PEG-folate is 1:(5-15):(1-2), preferably 1:(8-12):(1- 2). 8. preparation method according to claim 6, is characterized in that, in step (a), adopts decompression rotary evaporation method to remove organic solvent; And/or, in step (b), by ultrasonic hydration, the lipid film loaded with metalloporphyrin is dispersed in the aqueous solution; And/or, in step (c), adopt ultrasonic cell disruptor to carry out sonication; Preferably, in step (b), the time of ultrasonic hydration is 2-15min, preferably 5-10min; Preferably, in step (c), the ultrasonic time is 3-10 min, preferably 4-6 min. 9. The application of the metalloporphyrin-loaded liposome nanoparticle according to any one of claims 1-4 in the preparation of a drug for treating tumors. 10. A medicament for the treatment of tumors, characterized in that it comprises the metalloporphyrin-loaded liposome nanoparticle according to any one of claims 1-4.

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