CN114948876A - Multifunctional microbubble and preparation method and application thereof - Google Patents

Multifunctional microbubble and preparation method and application thereof Download PDF

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CN114948876A
CN114948876A CN202210516199.8A CN202210516199A CN114948876A CN 114948876 A CN114948876 A CN 114948876A CN 202210516199 A CN202210516199 A CN 202210516199A CN 114948876 A CN114948876 A CN 114948876A
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multifunctional
microbubble
antibody
active ester
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梁晓龙
孙素会
张路路
孙利红
汤清双
徐梦红
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Peking University Third Hospital Peking University Third Clinical Medical College
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Abstract

The invention belongs to the technical field of biomedical materials, and provides a multifunctional microbubble and a preparation method and application thereof. The shell layer of the multifunctional microbubble comprises a lipid monomolecular layer and a PD-L1 antibody connected on the lipid monomolecular layer; the lipid monolayer comprises an amphiphilic drug conjugate, a phospholipid and an active ester; the active ester comprises distearoyl phosphatidyl acetamide-N-hydroxysuccinimide-polyethylene glycol 2000. The invention uses the amphiphilic drug conjugate as a film forming material to prepare the multifunctional microbubble, thereby effectively avoiding the early leakage of the drug and improving the bioavailability of the drug. The PD-L1 antibody is coupled to the surface of the multifunctional microbubble through covalent bonding, so that the delivery efficiency of the antibody is improved; the multifunctional microbubbles are broken by ultrasonic fixed-point striking to be converted into nanoparticles, and the medicine and the antibody are enriched at the tumor part at high concentration by the sonoporation effect generated in the process, so that the combination of chemotherapy and immunotherapy is realized.

Description

Multifunctional microbubble and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a multifunctional microbubble, a preparation method and application thereof.
Background
Cancer is a serious disease threatening the health of humans. Although conventional methods such as surgery, radiotherapy, chemotherapy, etc. can exhibit short-term therapeutic effects, it is urgently required to develop new therapeutic agents in order to solve the problems of many side effects such as cytotoxicity, metastasis and recurrence. In recent years, immunotherapy for tumors has attracted much attention from researchers. Immunotherapy, such as tumor vaccines, CAR-T therapy and immune checkpoint blockade therapy, show encouraging clinical outcomes in the treatment of different types of cancer, including melanoma, non-small cell lung cancer and leukemia. In the immunotherapy strategy, the blocking therapy based on the PD-1/PD-L1 immune checkpoint inhibitor brings new hopes for the treatment of cancer. However, only a few patients may benefit from immune checkpoint blockade therapy, while for the majority there is still a problem of low response rates, mainly due to poor T cell infiltration in the tumor microenvironment. Therefore, improving the response rate of immunotherapy and the infiltration level of T cells in tumor microenvironment are key issues for improving the curative effect of tumor immunotherapy.
Chemotherapy is used extensively in the treatment of tumors, particularly in patients with advanced and metastatic cancers. Clinical studies have found that chemotherapy can improve the patient's response rate to immunotherapy. This is because chemotherapy can trigger immunogenic death of cells and release molecules that make the immune system more alert. Inspired by this, researchers are trying to combine chemotherapeutic drugs with immunotherapy to develop a new combination therapy, which has been proven to be more effective in enhancing the tumor treatment effect. While PD-1/PD-L1 antibodies have been approved for the treatment of various types of tumors, such antibodies are prepared to form nanoparticles; nanoparticle-type antibodies are not able to effectively target tumor sites, and off-target and excessive use can lead to the development of side effects, such as autoimmune diseases. Therefore, how to deliver chemotherapeutic drugs and immunotherapeutic drugs with high efficiency is a key issue to be solved in cancer treatment.
Disclosure of Invention
In view of the above, the present invention aims to provide a multifunctional microbubble, a preparation method and an application thereof. The multifunctional microbubble can be converted into nano particles by fixed-point blasting at a tumor part under the action of ultrasound, and the high-concentration enrichment of tumor tissues is realized by utilizing the sonoporation effect of the ultrasound, so that the chemotherapeutic drugs in the PD-L1 antibody and the amphiphilic drug conjugate are synchronously delivered, and the combination of chemotherapy and immunotherapy is realized.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a multifunctional microbubble, which comprises a shell layer and an inner core; the shell layer comprises a lipid monomolecular layer and a PD-L1 antibody connected on the lipid monomolecular layer;
the lipid monolayer comprises an amphiphilic drug conjugate, a phospholipid and an active ester;
the active ester comprises distearoyl phosphatidyl acetamide-N-hydroxysuccinimide-polyethylene glycol 2000;
the inner core of the multifunctional microbubble is encapsulated with gas.
Preferably, the molar ratio of the amphiphilic drug conjugate to the phospholipid to the active ester is 5-20: 55-80: 5 to 30.
Preferably, the phospholipid comprises one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, and 1, 2-dipalmitoyl-SN-glycerol-3-phosphatidic acid.
Preferably, the amphiphilic drug conjugate has the structure of formula I:
Figure BDA0003639575560000021
in the formula I, A represents a hydrophobic chemotherapeutic drug, and B represents a hydrophilic chemotherapeutic drug;
x and Y represent a linking group, X and Y are the same or different;
a is 2 or 3, b is 2 or 3, and a and b are the same or different.
Preferably, said a comprises one or more of paclitaxel, camptothecin, doxorubicin, vincristine, vinblastine, etoposide, vespium, carboplatin, cisplatin, mitomycin, vinblastine amide, epirubicin, vinisovinblastine, and methotrexate; the B comprises pentafluoro-deoxyuridine and/or ifosfamide.
The invention also provides a preparation method of the multifunctional microbubble, which comprises the following steps:
dissolving and mixing the amphiphilic drug conjugate, phospholipid and active ester to obtain a first mixed system;
dropwise adding the first mixed system into a boric acid buffer solution under the condition of water bath ultrasound, and then carrying out first dialysis to obtain a first dispersed system containing nano particles;
mixing the first dispersion system containing the nano particles and the PD-L1 antibody, and sequentially performing amide reaction and second dialysis to obtain a second dispersion system containing the antibody nano particles;
and mixing the second dispersion system of the antibody-containing nanoparticles with a stabilizer, and filling an entrapped gas into the obtained mixed solution for oscillation to obtain the multifunctional microbubble.
Preferably, the pH value of the boric acid buffer solution is 8.0-8.4; the temperature of the water bath ultrasound is 30-50 ℃, and the time is 5-10 min; the intercepted molecular weight of the dialysis bag of the first dialysis is 8000-14000 Da, the temperature is 20-30 ℃, and the time is 1-2 h.
Preferably, the mass of the PD-L1 antibody is 10-60% of the mass of the nanoparticles in the first dispersion system.
Preferably, the temperature of the amide reaction is 20-30 ℃, and the time is 6-12 h; the cut-off molecular weight of the dialysis bag of the second dialysis is 150000-300000 Da, the temperature is 20-30 ℃, and the time is 2-4 h.
The invention also provides the application of the multifunctional microvesicle in the technical scheme or the multifunctional microvesicle obtained by the preparation method in the technical scheme in preparing tumor drugs.
The invention provides a multifunctional microbubble, which comprises a shell layer and an inner core; the shell layer comprises a lipid monolayer and a PD-L1 antibody linked on the lipid monolayer; the lipid monolayer comprises an amphiphilic drug conjugate, a phospholipid and an active ester; the active ester comprises distearoyl phosphatidyl acetamide-N-hydroxysuccinimide-polyethylene glycol 2000; the inner core is an encapsulated gas. The invention uses the amphiphilic drug conjugate as one of the film forming materials to prepare the multifunctional microbubble, thereby effectively avoiding the early leakage of the drug and improving the bioavailability of the drug. The PD-L1 antibody is coupled to the surface of the liposome monomolecular layer through covalent bonding to form a shell layer, so that the delivery efficiency of the antibody is improved; when the multifunctional microvesicle is applied, the multifunctional microvesicle is broken by ultrasonic fixed point to be converted into nano particles, and the medicine and the antibody are enriched at the tumor part at high concentration by the sonoporation effect generated in the process, so that the combination of chemotherapy and immunotherapy is realized.
The invention also provides a preparation method of the multifunctional microbubble, which comprises the following steps: dissolving and mixing the amphiphilic drug conjugate, phospholipid and active ester to obtain a first mixed system; dropwise adding the first mixed system into a boric acid buffer solution under the condition of water bath ultrasound, and then carrying out first dialysis to obtain a first dispersed system containing nano particles; mixing the first dispersion system containing the nano particles with a PD-L1 antibody, and sequentially carrying out an amide reaction and a second dialysis to obtain a second dispersion system containing the nano particles of the antibody; and mixing the second dispersion system of the antibody-containing nanoparticles with a stabilizer, and filling an entrapped gas into the obtained mixed solution for oscillation to obtain the multifunctional microbubble. The preparation method provided by the invention comprises the steps of firstly, synthesizing the phospholipid, the active ester and the amphiphilic drug conjugate into nano particles by an ethanol injection method; then adding a PD-L1 antibody, wherein the PD-L1 antibody forms an amido bond with active ester in the nano particle through carboxyl and is connected to the nano particle; filling the entrapped gas to prepare the multifunctional microbubble. The preparation method provided by the invention is simple to operate.
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FIG. 1 is a flow chart of the preparation of multifunctional microbubbles and the conversion of multifunctional microbubbles to nanoparticles in example 1;
FIG. 2 is a microscopic view of the multifunctional microbubbles obtained in example 1;
FIG. 3 is a graph showing a distribution of the particle size of the multifunctional microbubbles obtained in example 1;
FIG. 4 is a graph of data representing the multifunctional microbubbles obtained in example 1 after ultrasonic explosion;
FIG. 5 is a graph showing the results of in vitro imaging ability of the multifunctional microvesicles obtained in example 1;
FIG. 6 is a graph showing the results of in vivo imaging ability of the multifunctional microbubbles obtained in example 1;
FIG. 7 is a graph showing the results of cell uptake of the multifunctional microvesicles obtained in example 1;
FIG. 8 is a confocal microscope showing that the multifunctional microvesicles obtained in example 1 induce calreticulin expression in cells;
FIG. 9 is a confocal microscope showing the release of HMGB1 by the multifunctional microbubbles obtained in example 1;
FIG. 10 is a graph showing the results of tumor cell killing by the multifunctional microbubbles prepared in example 1 under the action of ultrasound;
FIG. 11 is a graph showing the in vivo NIR fluorescence imaging results of the multifunctional microvesicles obtained in example 1;
FIG. 12 is a graph showing the results of tumor suppression by the multifunctional microvesicles obtained in example 1.
Detailed Description
The invention provides a multifunctional microbubble, which comprises a shell layer and an inner core; the composition comprises a lipid monolayer and a PD-L1 antibody attached to the lipid monolayer; the lipid monolayer comprises an amphiphilic drug conjugate, a phospholipid and an active ester; the active ester comprises 1, 2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-hydroxysuccinimide-polyethylene glycol 2000;
the inner core of the multifunctional microbubble is entrapped gas.
The multifunctional microbubble provided by the invention comprises an inner core, wherein the inner core is encapsulated gas; the encapsulating gas is preferably sulfur hexafluoride or perfluoropropane, and more preferably sulfur hexafluoride.
The multifunctional microvesicles provided by the present invention comprise a shell layer. In the present invention, the shell layer includes a lipid monolayer and a PD-L1 antibody attached to the lipid monolayer.
In the present invention, the lipid monolayer comprises an amphiphilic drug conjugate, a phospholipid and an active ester; the molar ratio of the amphiphilic drug conjugate to the phospholipid to the active ester is preferably 5-20: 55-80: 5-30, more preferably 20: 70: 10.
in the present invention, the amphiphilic drug conjugate preferably has a structure represented by formula I:
Figure BDA0003639575560000051
in the formula I, A represents a hydrophobic chemotherapeutic drug, and B represents a hydrophilic chemotherapeutic drug;
x and Y represent a linking group, X and Y are the same or different;
a is 2 or 3, b is 2 or 3, and a and b are the same or different.
In the present invention, said a preferably comprises one or more of paclitaxel, camptothecin, doxorubicin, vincristine, vinblastine, etoposide, vinpocetine, carboplatin, cisplatin, mitomycin, vinblastine amide, epirubicin, vinblastine and methotrexate. In the present invention, said B preferably comprises pentafluorouridine and/or ifosfamide.
In a specific embodiment of the present invention, the amphiphilic drug conjugate preferably has a structure represented by formula I-1:
Figure BDA0003639575560000052
in the present invention, the phospholipid preferably includes one or more of Distearoylphosphatidylcholine (DSPC), Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), distearoylphosphatidylethanolamine-polyethylene glycol 5000(DSPE-PEG5000), and 1, 2-dipalmitoyl-SN-glycerol-3-phosphatidic acid (DPPA), and further preferably a phospholipid mixture of Distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000); the preferable molar ratio of distearoyl phosphatidylcholine to distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in the phospholipid mixture is 50-70: 5-10, more preferably 60: 10.
in the present invention, the PD-L1 antibody is linked to the active ester in the lipid monolayer by an amide bond.
In the present invention, the particle size of the multifunctional microbubbles is preferably 400nm to 6 μm.
The invention also provides a preparation method of the multifunctional microbubble, which comprises the following steps:
dissolving and mixing the amphiphilic drug conjugate, phospholipid and active ester to obtain a first mixed system;
dropwise adding the first mixed system into a boric acid buffer solution under the condition of water bath ultrasound, and then carrying out first dialysis to obtain a first dispersed system containing nano particles;
mixing the first dispersion system containing the nano particles with a PD-L1 antibody, and sequentially carrying out an amide reaction and a second dialysis to obtain a second dispersion system containing the nano particles of the antibody;
and mixing the second dispersion system of the antibody-containing nanoparticles with a stabilizer, and filling an entrapped gas into the obtained mixed solution for oscillation to obtain the multifunctional microbubble.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The amphiphilic drug conjugate, the phospholipid and the active ester are dissolved and mixed to obtain a first mixed system.
In the present invention, the dissolving and mixing of the amphiphilic drug conjugate, the phospholipid and the active ester preferably comprises: respectively dissolving phospholipid and active ester in ethanol to obtain a phospholipid solution and an active ester solution; dissolving the amphiphilic drug conjugate in dimethyl sulfoxide (DMSO) to obtain an amphiphilic drug conjugate solution; mixing the phospholipid solution, the active ester solution and the amphiphilic drug conjugate solution.
After the first mixed system is obtained, the first mixed system is dropwise added into a boric acid buffer solution under the condition of water bath ultrasound, and then first dialysis is carried out to obtain a first disperse system containing nano particles.
In the present invention, the volume of the solvent in the first mixed system is preferably not more than 20% of the volume of the boric acid buffer.
In the present invention, the dropping is preferably dropwise.
In the invention, the pH value of the boric acid buffer solution is preferably 8.0-8.4.
In the invention, the temperature of the water bath ultrasound is preferably 30-50 ℃; the water bath ultrasonic time is preferably 5-10 min, and timing is started after the first mixed system is dripped. According to the invention, a first mixed system is dripped into a boric acid buffer solution under the condition of water bath ultrasound, and the phospholipid, the active ester and the amphiphilic drug conjugate form nanoparticles.
In the invention, the cut-off molecular weight of the dialysis bag of the first dialysis is preferably 8000-14000 Da; the temperature of the first dialysis is preferably 20-30 ℃, and the time is preferably 1-2 h. In the present invention, the first dialysis is preferably performed in a boric acid buffer solution; the parameters of the boric acid buffer solution are preferably consistent with the technical scheme, and are not described in detail herein.
After the first dispersion system containing the nanoparticles is obtained, the first dispersion system containing the nanoparticles and the PD-L1 antibody are mixed, and then an amide reaction and a second dialysis are sequentially carried out to obtain a second dispersion system containing the nanoparticles of the antibody.
In the present invention, the mass of the PD-L1 antibody is preferably 10 to 60%, and more preferably 50% of the mass of the nanoparticles in the first dispersion system.
In the invention, the temperature of the amide reaction is preferably 20-30 ℃, and the time is preferably 6-12 h. In the present invention, the amide reaction is preferably carried out under stirring; the rotating speed of the stirring is preferably 100-300 rpm. In the invention, the carboxyl of the PD-L1 antibody and the active ester in the nanoparticle are subjected to amide reaction to form an amide bond, and the amide bond is combined on the nanoparticle.
In the invention, the cut-off molecular weight of the dialysis bag of the second dialysis is preferably 150000-300000 Da; in the invention, the temperature of the second dialysis is preferably 20-30 ℃, and the time is preferably 2-4 h. In the present invention, the second dialysis is preferably performed in a PBS buffer solution.
After the second dispersion system of the antibody-containing nanoparticles is obtained, the second dispersion system of the antibody-containing nanoparticles is mixed with a stabilizer, and the obtained mixed solution is filled with the carrier gas for oscillation to obtain the multifunctional microbubble.
In the present invention, the stabilizer preferably includes propylene glycol and glycerin. In the present invention, the volume ratio of the antibody-containing nanoparticles, propylene glycol, and glycerin in the second dispersion is preferably 10: 1: 1.
in the present invention, the volume ratio of the mixed solution to the carrier gas is preferably 1.2: 2.
in the present invention, the charging of the encapsulating gas for oscillation preferably comprises the steps of: the second dispersion is placed in a container, filled with an encapsulating gas and sealed for oscillation. In the present invention, the time of the oscillation is preferably 45 s.
The invention also provides the application of the multifunctional microvesicle in the technical scheme or the multifunctional microvesicle obtained by the preparation method in the technical scheme in preparing tumor drugs. In the present invention, the multifunctional microbubbles are used in conjunction with ultrasound.
In the invention, the frequency of the ultrasonic wave is preferably 1-3 MHz, and the intensity is preferably 0.5-2W/cm 2 The duty ratio is preferably 10-50%, and the time is preferably 1-10 min. In one embodiment of the invention, the ultrasound has a frequency of 1MHz and an intensity of 1W/cm 2 The duty ratio is preferably 20% and the time is preferably 3 min.
The multifunctional microbubbles provided by the present invention, the preparation method and the application thereof are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Respectively dissolving distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG-2000) and distearoyl phosphatidylacetamide-N-hydroxysuccinimide-polyethylene glycol 2000(DSPE-PEG2000-NHS) in absolute ethyl alcohol to obtain a distearoyl phosphatidylcholine solution, a DSPE-PEG-2000 solution and a DSPE-PEG2000-NHS solution; dissolving an amphiphilic drug Conjugate (CF) shown as a formula I in DMSO to obtain an amphiphilic drug conjugate solution; mixing stearoyl phosphatidylcholine solution, DSPE-PEG-2000 solution, DSPE-PEG2000-NHS solution and amphiphilic drug conjugate solution (wherein, the molar ratio of stearoyl phosphatidylcholine to DSPE-PEG-2000 to DSPE-PEG2000 to amphiphilic drug conjugate is 6: 1: 1: 2), dropwise adding the mixed system into 0.8mL boric acid buffer solution with the pH value of 8.4 under the condition of water bath ultrasound, and performing water bath ultrasound for 10min at 40 ℃ to form nano particles; putting the system containing the nano particles into a dialysis bag with cut-off molecular weight of 8000-14000 Da, and dialyzing in boric acid buffer solution for 1h to obtain a first dispersion system; dialyzing, placing into penicillin bottle, adding PD-L1 antibody (the mass of PD-L1 antibody is 50% of the mass of nanoparticles in the first dispersion system)) Stirring at room temperature for 12 h; dialyzing the obtained system in a PBS solution at room temperature for 2h by using a dialysis bag of 150000-300000 Da to obtain a second dispersion system; taking out, respectively adding 100 mu L of propylene glycol and 100 mu L of propylene glycol into the second dispersion system, and uniformly mixing; filling the mixed solution into a 3mL penicillin bottle, and filling sufficient sulfur hexafluoride (SF) 6 ) The gas, oscillator oscillates for 45s, resulting in multifunctional microbubbles (. alpha.PCF MBs).
Wherein the drug conjugate has a structure represented by formula I-1:
Figure BDA0003639575560000091
FIG. 1 is a flow chart of the preparation of multifunctional microvesicles and the conversion of multifunctional microvesicles into nanoparticles according to the present embodiment.
FIG. 2 is a microscope photograph of the multifunctional microbubble prepared in the present example, wherein a is a bright field image and b is a fluorescence image. As can be seen from fig. 2: the particle size of the multifunctional microbubbles is uniform and is about 2-4 microns, and the blue fluorescence of the chemotherapeutic camptothecin can be seen, so that the chemotherapeutic drug is loaded on the multifunctional microbubbles.
Fig. 3 is a distribution diagram of the particle size of the multifunctional microbubbles prepared in this example measured by the coulter technique. As can be seen from fig. 3, the particle size distribution of the obtained multifunctional microbubbles was mainly 2 to 4 μm, and the particle size distribution was concentrated and was consistent with the results observed by an optical microscope.
Fig. 4 is a representation data chart of the multifunctional microbubbles prepared in this example after ultrasonic explosion. Wherein a is a transmission electron microscope picture of the multifunctional microbubbles after ultrasonic explosion, and b is a hydration particle size distribution picture of the multifunctional microbubbles after ultrasonic explosion. As shown in FIG. 4, the multifunctional microbubbles are converted into nanoparticles after ultrasonic blasting, the nanoparticles are spherical, the particle size is 10-50 nm, and the hydrated particle size is about 100 nm.
Example 2
To observe the effect of the multifunctional microbubbles prepared in example 1 in vitro imaging, the multifunctional microbubbles prepared in example 1 (α PCF MBs) were mixed with physiological saline in a volume ratio of 1: 9 into a latex tube, placing 500mL of ultrasonically degassed water in a water tank, placing the latex tube in the middle of the liquid, using VINNO70 ultrasonic instruments, MI: 0.06 (mechanical index), probe frequency: 3-12 MHz, and observing the in vitro ultrasonic contrast effect of the multifunctional microbubble.
Fig. 5 is a result of in vitro imaging ability of the multifunctional microvesicles prepared in example 1. As can be seen from FIG. 5, the latex tube lumen exhibits an anechoic state when no multifunctional microbubbles are present. After the multifunctional microbubbles are injected, remarkable echo signal enhancement is observed in the inner cavity of the latex tube, and the time can last for more than 20min, so that the multifunctional microbubbles are proved to have good capacity of in-vitro ultrasonic development enhancement.
Example 3
To observe the effect of the multifunctional microbubbles prepared in example 1 in vivo imaging, mice inoculated with subcutaneous CT26 tumor were subjected to tumor ultrasound imaging. 200 μ L (CF concentration 5mg/kg) of multifunctional microvesicles was tail-vein injected into mice. Using VINNO70 ultrasound instrument, MI: 0.06 (mechanical index), probe frequency: 3-12 MHz, and observing the in-vivo ultrasonic contrast effect of the multifunctional microbubble.
Fig. 6 is a graph showing the results of in vivo imaging ability of the multifunctional microbubbles prepared in example 1. As can be seen from fig. 6, no ultrasound contrast effect was observed before the injection of the multifunctional microbubbles; after the multifunctional microbubble is injected for 30s, the contrast effect in the tumor is obviously enhanced, and the enhancement can last for more than 4 min.
Example 4
To trace the uptake of multifunctional microvesicles (α PCF MBs) by tumor cells, the multifunctional microvesicles of example 1 were labeled with DSPE-cy5.5. The experiment was divided into three groups: an IgG antibody-linked multifunctional microbubble group, a PD-L1 antibody-linked multifunctional microbubble group, and a PD-L1 antibody-linked multifunctional microbubble combined ultrasound group. And (3) after the multifunctional microvesicles and the cells are incubated, adding or not adding ultrasound according to grouping conditions, continuing to incubate for 4 hours, and observing by using a laser confocal microscope.
FIG. 7 is a graph showing the results of cell uptake of the multifunctional microvesicles prepared in example 1. As can be seen from fig. 7: the uptake of PD-L1 antibody-linked multifunctional microvesicles by cells was higher than that of IgG antibody-linked multifunctional microvesicles due to active targeting of PD-L1; in addition, compared with the group without ultrasonic wave, the uptake of the IgG antibody-linked multifunctional microbubbles is obviously increased after the ultrasonic wave is added, because the multifunctional microbubbles are converted into the nanoparticles in situ under the action of the ultrasonic wave, and the generated cavitation action increases the permeability of cell membranes to promote the uptake of the drugs.
Example 5
To verify that the multifunctional microvesicles can cause immunogenic death of tumor cells, calreticulin expression and HMGB1 release were observed. When immunogenic death of the cells occurs, calreticulin expression and HMGB1 release are increased. The experiments were divided into 6 groups: a control group, a free PD-L1 antibody group, a free camptothecin group, a free floxuridine group, a PD-L1 antibody linked multifunctional microbubble group, and a PD-L1 antibody linked multifunctional microbubble combined ultrasound group. For calreticulin, after different groups of drugs and cells are incubated for 4 hours, a fresh culture medium is replaced to continue incubation for 24 hours, the culture medium is removed, goat serum is sealed for 1 hour, anti-calreticulin primary antibody and anti-calreticulin secondary antibody are incubated for 30 minutes in sequence, after the cells are fixed, the cells are stained with DAPI cell nucleus staining solution, and then observed by a confocal microscope. For HMGB1, after different groups of drugs and cells are incubated for 4 hours, the fresh culture medium is replaced to continue incubation for 24 hours, the culture medium is removed, the cells are fixed, the membranes are broken and the cells are sealed after being washed by PBS, the cells are stained with fluorescence-labeled anti-HMGB 1 antibody for overnight at 4 ℃, and then the cells are stained with nuclei and observed by a confocal microscope.
FIG. 8 is a confocal microscope showing that the multifunctional microvesicles prepared in example 1 induce calreticulin expression in cells. As can be seen from fig. 8: compared with the control group and the free PD-L1 antibody group, the free camptothecin group, the free floxuridine group and the PD-L1 antibody-linked multifunctional microvesicle group cause the increasing degree of calreticulin expression, which indicates that the camptothecin and the floxuridine can cause the immunogenic death of cells and further cause the increasing of calreticulin expression, and the effect can be enhanced by combining the two medicines. When combined with ultrasound, PD-L1 antibody-linked multifunctional microvesicles can cause a higher degree of calreticulin expression, since the sonoporation generated by ultrasound promotes the uptake of chemotherapeutic drugs and thus leads to a higher degree of cell immunogenic death.
Fig. 9 is a confocal microscope photograph showing the release of HMGB1 caused by the multifunctional microbubble prepared in example 1. As can be seen from fig. 9: the free camptothecin group, the free floxuridine group, the PD-L1 antibody-linked multifunctional microvesicle group, and the PD-L1 antibody-linked multifunctional microvesicle in combination with the ultrasound group were all able to cause the release of HMGB1, indicating that the multifunctional microvesicles prepared in example 1 were able to effectively induce immunogenic death of cells.
Example 6
To assess the toxic effects of multifunctional microvesicles on cells. CT-26 cells in logarithmic growth phase were seeded in 96-well plates at a cell density of about 8X 10 per well 3 After overnight culture, cells were divided into 5 groups: a control group, an IgG antibody-linked multifunctional microbubble group, a PD-L1 antibody-linked multifunctional microbubble group, an IgG antibody-linked multifunctional microbubble combined ultrasound group, and a PD-L1 antibody-linked multifunctional microbubble combined ultrasound group. Wherein, the concentration of the camptothecin-floxuridine is 4 micromolar, and the ultrasonic parameters are as follows: frequency 1.0MHz, 20% duty cycle, intensity 1W/cm 2 1 minute. After 4 hours incubation, the cells were assayed for cell viability by changing fresh medium and incubating for an additional 24 hours with CCK-8.
FIG. 10 is a graph showing the results of the tumor cell killing by the multifunctional microvesicles prepared in example 1 under the effect of ultrasound. As can be seen from fig. 10: the cell activities generated by the control group, the multifunctional microbubble group connected with the IgG antibody, the multifunctional microbubble group connected with the PD-L1 antibody, the multifunctional microbubble combined ultrasonic group connected with the IgG antibody and the multifunctional microbubble combined ultrasonic group connected with the PD-L1 antibody are respectively as follows: 68.35 + -1.54%, 62.48 + -3.65%, 43.02 + -3.28% and 33.70 + -4.15%, indicating that the effect of ultrasound and the targeting effect of PD-L1 enhance the toxic effect of the multifunctional microvesicles on cells.
Example 7
To assess the distribution of multifunctional microvesicles in vivo, multifunctional microvesicles were labeled with DSPE-cy5.5 and subjected to in vivo near infrared fluorescence imaging. Firstly, constructing a Balb/c mouse subcutaneous CT-26 tumor model, and dividing the mouse into three groups: a multifunctional microbubble combined ultrasonic group connected with a PD-L1 antibody, a multifunctional microbubble combined ultrasonic group connected with an IgG antibody, and a multifunctional microbubble group connected with a PD-L1 antibody. Injecting the multifunctional microvesicle into a mouse body through a tail vein, adding an ultrasonic group to perform ultrasonic irradiation on a tumor part to smash the multifunctional microvesicle, then respectively performing near infrared fluorescence imaging on the mouse at 1h, 4h, 8h, 12h, 36h and 48h, dissecting the mouse after 48h, and collecting heart, liver, spleen, lung, kidney and tumor tissues to perform fluorescence imaging.
FIG. 11 is a graph of in vivo NIR fluorescence imaging of multifunctional microbubbles. Wherein a is a live body fluorescence imaging picture, and b is a fluorescence imaging picture of an isolated organ and a tumor. As can be seen from fig. 11: the tumor showed a fluorescence signal and increased gradually 1 hour after the injection of the drug, and reached a maximum at 24 hours, with the order of fluorescence enhancement: PD-L1 antibody linked multifunctional microbubble in combination with sonographer > IgG antibody linked multifunctional microbubble in combination with sonographer > PD-L1 antibody linked multifunctional microbubble group as a result of cavitation of ultrasound and PD-L1 antibody mediated targeting. In addition, the PD-L1 antibody linked multifunctional microvesicles combined with ultrasound showed the strongest fluorescence of the tumors ex vivo, which is consistent with the in vivo results.
Example 8
In-vivo chemotherapy/immunotherapy combined experiments investigate whether the prepared multifunctional microvesicles can effectively inhibit tumor growth. Mice bearing CT-26 subcutaneous tumors were randomized into 7 groups: a control group, a single ultrasonic group, a free PD-L1 antibody group, a PD-L1 antibody-linked multifunctional microbubble group, an IgG antibody-linked multifunctional microbubble combined ultrasonic combined free PD-L1 antibody group and a PD-L1 antibody-linked multifunctional microbubble combined ultrasonic group. Wherein the administration mode is tail vein injection, and the injection dosage is camptothecin-floxuridine concentration of 5 mg/kg. After treatment of each group of mice, changes in tumor volume and body weight were recorded daily (tumor volume: 1/2 × length × width) 2 )。
FIG. 12 is a graph showing the results of tumor inhibition by multifunctional microvesicles. As can be seen from fig. 12, the tumor volume growth rate of the free PD-L1 antibody group, the PD-L1 antibody-linked multifunctional microvesicle group, and the IgG antibody-linked multifunctional microvesicle in combination with the ultrasound group was significantly smaller than that of the control group and the ultrasound group alone; in addition, the multifunctional microbubble combined ultrasound group connected with the PD-L1 antibody has more remarkable curative effect than the multifunctional microbubble group connected with the PD-L1 antibody and the multifunctional microbubble combined ultrasound group connected with the IgG antibody and the free PD-L1 antibody, because the multifunctional microbubble combined ultrasound and targeted disruption technology at the tumor part and the targeted effect of the combined PD-L1 can ensure that more therapeutic agents are taken up by tumor cells and more effective therapeutic effect is achieved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (10)

1. A multifunctional microbubble, characterized in that it comprises a shell and an inner core; the shell layer comprises a lipid monomolecular layer and a PD-L1 antibody connected on the lipid monomolecular layer;
the lipid monolayer comprises an amphiphilic drug conjugate, a phospholipid and an active ester;
the active ester comprises distearoyl phosphatidyl acetamide-N-hydroxysuccinimide-polyethylene glycol 2000;
the inner core is an encapsulated gas.
2. The multifunctional microbubble of claim 1, wherein the amphiphilic drug conjugate, the phospholipid and the active ester are present in a molar ratio of 5-20: 55-80: 5 to 30.
3. The multifunctional microbubble of claim 1 or 2, wherein the phospholipid comprises one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, and 1, 2-dipalmitoyl-SN-glycerol-3-phosphatidic acid.
4. The multifunctional microbubble of claim 1 or 2, wherein the amphiphilic drug conjugate has a structure according to formula I:
Figure FDA0003639575550000011
in the formula I, A represents a hydrophobic chemotherapeutic drug, and B represents a hydrophilic chemotherapeutic drug;
x and Y represent a linking group, X and Y are the same or different;
a is 2 or 3, b is 2 or 3, and a and b are the same or different.
5. The multifunctional microbubble of claim 4 wherein A comprises one or more of paclitaxel, camptothecin, doxorubicin, vincristine, vinblastine, etoposide, vinorelbine, carboplatin, cisplatin, mitomycin, vinblastine amide, epidophyllotoxin, vinblastine, and methotrexate;
the B comprises pentafluoro-deoxyuridine and/or ifosfamide.
6. The method for preparing a multifunctional microbubble as claimed in any one of claims 1 to 5, comprising the steps of:
dissolving and mixing the amphiphilic drug conjugate, phospholipid and active ester to obtain a first mixed system;
dropwise adding the first mixed system into a boric acid buffer solution under the condition of water bath ultrasound, and then carrying out first dialysis to obtain a first dispersed system containing nano particles;
mixing the first dispersion system containing the nano particles with a PD-L1 antibody, and sequentially carrying out an amide reaction and a second dialysis to obtain a second dispersion system containing the nano particles of the antibody;
and mixing the second dispersion system of the antibody-containing nanoparticles with a stabilizer, and filling an entrapped gas into the obtained mixed solution for oscillation to obtain the multifunctional microbubble.
7. The method according to claim 6, wherein the pH of the boric acid buffer is 8.0 to 8.4; the temperature of the water bath ultrasound is 30-50 ℃, and the time is 5-10 min;
the cut-off molecular weight of the dialysis bag of the first dialysis is 8000-14000 Da, the temperature is 20-30 ℃, and the time is 1-2 h.
8. The production method according to claim 6, wherein the mass of the PD-L1 antibody is 10 to 60% of the mass of the nanoparticles in the first dispersion system.
9. The preparation method according to claim 6 or 8, wherein the temperature of the amide reaction is 20-30 ℃ and the time is 6-12 h;
the cut-off molecular weight of the dialysis bag for the second dialysis is 150000-300000 Da, the temperature is 20-30 ℃, and the time is 2-4 h.
10. Use of the multifunctional microvesicles of any one of claims 1 to 5 or the multifunctional microvesicles obtained by the preparation method of any one of claims 6 to 9 in the preparation of a tumor drug.
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