CN112546062A - Perfluorocarbon silicon plastid and preparation method and application thereof - Google Patents

Perfluorocarbon silicon plastid and preparation method and application thereof Download PDF

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CN112546062A
CN112546062A CN202011166195.9A CN202011166195A CN112546062A CN 112546062 A CN112546062 A CN 112546062A CN 202011166195 A CN202011166195 A CN 202011166195A CN 112546062 A CN112546062 A CN 112546062A
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王凡
梁晓龙
马晓途
史继云
姚美男
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Peking University Third Hospital Peking University Third Clinical Medical College
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Abstract

The invention discloses a silicon plastid-encapsulated perfluorocarbon preparation, and a preparation method and application thereof. The silicate network structure on the surface of the siliceous body ensures that the preparation has good structural stability and drug-loading stability, can reduce the early drug leakage of the preparation in a blood circulation system and reduce the toxic and side effects of the drug. The preparation can also enhance the effect of ultrasonic imaging, and realize drug delivery and drug release under image monitoring.

Description

Perfluorocarbon silicon plastid and preparation method and application thereof
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a perfluorocarbon preparation wrapped by a silica body, and a preparation method and application thereof.
Background
Tumor hypoxia is one of the most common characteristics of solid tumors. Hypoxia is a malignant feature of the tumor microenvironment, which is closely related to the resistance of tumors to various therapies, including chemotherapy, radiation therapy and photodynamic therapy. In particular, with respect to resistance to chemotherapy, hypoxia may lead to multidrug resistance (multidrug resistance) in tumors, which may ultimately lead to failure of chemotherapy. During the process of multidrug resistance, tumor hypoxia firstly increases the relative expression level of hypoxia inducible factor-1 alpha (HIF-1 alpha) of tumor cells, thereby increasing the transcription level of multidrug resistance gene 1(MDR1), and finally increasing the protein expression level of P-glycoprotein (P-gp), and P-gp is used as an 'efflux pump' of various chemotherapeutic drugs and can pump the drugs entering the tumor cells back to the outside of cells. Therefore, improving hypoxia is an effective method for enhancing the efficacy of chemotherapy. Tumor metastasis is the leading cause of death in most cancer patients. Hypoxia has been reported to promote epithelial-mesenchymal transition (epithelial-mesenchymal transition) of tumor cells, accelerating tumor metastasis. Therefore, improving hypoxia can increase the curative effect of chemotherapy and inhibit the metastasis of tumor.
Researchers have now explored a number of approaches to improve tumor hypoxia. Hyperbaric oxygen therapy can directly increase the concentration of oxygen in the systemic blood, although it has been used clinically, it lacks tumor specificity, increases the damage of radiotherapy, chemotherapy to normal tissues, and risks hyperoxicosis. There is an urgent need to develop a tumor-specific oxygen delivery system. Among the different types of oxygen delivery materials, perfluorocarbons (perfluorcarbon) have a great advantage due to their higher oxygen solubility and better biocompatibility. Perfluorocarbons have been widely used in clinical settings as contrast agents for ultrasound imaging, to prevent ischemia and reperfusion injury, and are expected to be used as artificial blood. Perfluorocarbon-based nanodroplets have been reported to alleviate tumor hypoxia and enhance the efficacy of radiation therapy and photodynamic therapy.
Although perfluorocarbons have a high oxygen solubility, their rate of oxygen release is inefficient. Unlike hemoglobin, which releases oxygen molecules rapidly under hypoxic conditions by virtue of a synergistic effect between its four subunits, perfluorocarbons release oxygen solely by virtue of the free diffusion of oxygen along a concentration gradient. Therefore, to enhance the efficiency of oxygen release from perfluorocarbons, it is necessary to rely on external stimuli to trigger the release of oxygen. Previous reports have utilized near infrared light or ultrasound to trigger the release of oxygen, but clinical use has been limited because of limited tissue penetration by near infrared light. The tissue penetrability of common ultrasound is high, but the ultrasound cannot be focused, the release of oxygen cannot be controlled at fixed points, the controlled release is not accurate enough, and normal tissues near tumors can be subjected to ultrasound treatment.
Perfluorocarbons can be used not only as carriers for oxygen delivery, but also as drug delivery systems (drug delivery systems). Small-molecule chemotherapeutic drugs are often limited in therapeutic effect and have serious side effects, and researchers have been concerned with the development of intelligent drug delivery systems for a long time to realize the synergy and attenuation of chemotherapeutic drugs. Perfluorocarbons enable drug delivery under ultrasound imaging monitoring, as well as ultrasound-triggered drug release, and hence perfluorocarbon-based drug delivery systems have unique advantages.
Good structural stability and drug loading stability are critical for drug delivery systems because it can avoid the delivery system from prematurely releasing the drug in the blood circulation system, thereby reducing the side effects of the drug. The wrapping agent of the perfluorocarbon nano material reported in the literature mainly comprises phospholipid or other surfactants, albumin, erythrocyte membranes and mesoporous nano materials, such as Bi2Se and SiO2. These encapsulates have limited drug loading stability and are complex to synthesize. There is a need to develop a perfluorocarbon drug delivery system with a simple synthetic process and high stability.
Disclosure of Invention
One of the purposes of the invention is to use the silica as a wrapping agent of the carbon fluoride, improve the structural stability and the drug-loading stability of a carbon fluoride drug delivery system, reduce the early leakage of the drug in blood circulation and reduce the side effect of the drug.
The second purpose of the invention is to trigger the carbon fluoride preparation to release oxygen and the medicine by using High Intensity Focused Ultrasound (HIFU), so as to realize the high intensity focused ultrasound stimulation response type oxygen and medicine controlled release, improve the enrichment amount of the medicine in the tumor and increase the medicine effect of the medicine.
The third purpose of the invention is to utilize the carbon fluoride preparation carried by the silica plastid to deliver oxygen to the tumor in a targeted way, improve the hypoxia of the tumor, relieve the multi-drug resistance and epithelial-mesenchymal transition of the tumor, improve the drug effect of chemotherapy and reduce the metastasis of the tumor.
The purpose of the invention is realized by the following technical scheme:
a silica body encapsulating a fluorocarbon-based compound, the silica body being a particle having a core-shell structure, the shell being a single lipid molecule layer consisting of a silica-forming lipid and a phospholipid component not containing Si, the silica-forming lipid forming a silicate network structure of the form-Si-O-Si-on an outer surface of the shell; the core contains a carbon fluoride-based compound.
Further, according to the present invention, the mole percentage of the liposome-forming lipid is 50 to 100% and the mole percentage of the phospholipid component not containing Si is 0 to 50% based on the total number of moles of the monolayer of lipid molecules. In some embodiments of the invention, the mole percentage of the liposome forming lipids is, for example, 94-95%, 60%, 50%, and the balance is a phospholipid component that is free of Si.
Further, according to the present invention, the carbon fluoride compound refers to a compound formed by replacing all or part of hydrogen bonded to carbon in an organic compound with fluorine. The organic compounds include, but are not limited to, hydrocarbons, alcohols, esters, ethers. The hydrocarbon is, for example, a saturated or unsaturated chain or cyclic hydrocarbon of C3-10. The alcohol is, for example, a C1-10 monohydric or polyhydric alcohol. The esters are, for example, C2-10 esters. The ethers are, for example, C2-30 chain or cyclic mono-or poly-ethers. In some embodiments of the invention, the fluorocarbon-based compound is selected from the group consisting of perfluoropropane, perfluoropentane, perfluorohexane, perfluorooctane bromide, perfluorocrown ether. According to the present invention, preferred are fluorocarbon-based compounds having a boiling point of 40-80 ℃ at atmospheric pressure, including but not limited to perfluorohexane, perfluoroheptane, dodecafluorohexane, monobromododecafluorohexane. The inventor of the invention finds that the carbon fluoride compound in the boiling point range can meet the requirements of targeted tumor administration, proper drug encapsulation rate and drug loading rate, oxygen carrying and drug release under the guidance of HIFU.
Further, according to the present invention, the silica liposome-forming lipid comprises an inorganic precursor, a linking group and a hydrophobic tail, which has the following structural formula: a. themCH(3-m)—CH2—Y—(CH2)p—Z—Rq，
Wherein A is: (R)1O)3Si—(CH2)3—N—C(O)—X—(CH2)n—；R1Is alkyl, preferably C1-10 alkyl, more preferably methyl, ethyl; x is-O-or a bond; n is an integer from 0 to 10, such as 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2;
m is an integer of 1 to 3; when m is 2 or 3, the 2 or 3A can be the same or different;
y is-CH2-, -O-C (O) -or-C (O) -O-;
p is an integer from 0 to 10, such as 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2;
z is-C (O) -NH (2-q) -or-CH (3-q)—；
R is alkyl with more than 5 carbon atoms, preferably linear saturated alkyl with C7-20 carbon atoms;
when Z is-C (O) -NH (2-q) -q is an integer from 1 to 2; when Z is-CH (3-q) -q is an integer from 1 to 3; when q is 2 or 3, the 2 or 3R may be the same or different.
In some embodiments of the invention, the siliceous liposome forming lipids are selected from the group consisting of complex lipids a-D as shown below. The synthesis procedure of the four complex lipids is described in the literature (chem. eur. j.2013,19, 16113-16121).
Figure BDA0002745855000000041
Further, according to the present invention, the silicate network structure on the surface of the siliceous body can be formed by in situ sol-gel reaction (in situ sol-gel reaction) by forming a siliceous lipid, for example:
-Si-OCH2CH3+H2O→-Si-OH+CH3CH2OH
2-Si-OH→-Si-O-Si-+H2O
further, according to the present invention, the Si-free phospholipid component may be selected from Si-free phospholipid components known in the art, including but not limited to: polyethylene glycol derivative DSPE-mPEG of distearoylphosphatidylethanolamine capable of prolonging blood circulation time1000～20000For example, DSPE-mPEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) and DSPE-mPEG5000 (distearoylphosphatidylethanone)Alcohol amine-polyethylene glycol 5000); maleimide-modified distearoylphosphatidylethanolamine or polyethylene glycol derivative of Maleimide-modified distearoylphosphatidylethanolamine DSPE-mPEG that can be used to link targeting molecules1000～20000For example, DSPE-Maleimide (distearoylphosphatidylethanolamine-modified Maleimide), DSPE-mPEG2000-Maleimide (distearoylphosphatidylethanolamine-polyethylene glycol 2000-Maleimide); dipalmitoylphosphatidylcholine (DPPC) and 1-myristoyl-2-stearoyl lecithin (MSPC) which can regulate the phase transition temperature of the silica body; hydrogenated soy lecithin (HSPC); distearoylphosphatidylcholine (DSPC). The non-Si containing phospholipid component may be a mixture of one or more non-Si containing phospholipids.
Further, according to the present invention, the surface of the silica body may be modified with a tumor targeting molecule selected from any one of an antibody, a polypeptide, an aptamer, folic acid or a folic acid derivative that can target a tumor. In one embodiment of the invention, the tumor targeting molecule is var7 polypeptide (ref: mol. pharmaceuticals 2014,11,2896-2905) with an amino acid sequence of ACEEQNPWARYLEWLFPTETLLLEL. In some embodiments of the invention, the tumor targeting molecule is a folate derivative, such as a sulfhydryl-polyethylene glycol2000～6000Folic acid, in particular mercapto-polyethylene glycol5000-folic acid.
Further, according to the invention, the particle size of the silica body is 10nm-1 μm.
Further, according to the present invention, the core of the silica body may encapsulate a small molecule drug. The small molecule drug is a water insoluble or hydrophobic drug, including but not limited to one or more of a chemotherapeutic drug, a protein kinase inhibitor, a photodynamic drug. In some embodiments of the invention, the small molecule drug is selected from the group consisting of doxorubicin, cisplatin, paclitaxel, irinotecan, sorafenib, gefitinib, porfimer sodium, verteporfin, indocyanine green.
Further, according to the invention, the core of the silica body may carry oxygen. The capacity of the silica body to carry oxygen is realized by dissolving oxygen by carbon fluoride compounds. When the adsorption of the silica body to oxygen reaches equilibrium, 0-2 ml of oxygen can be dissolved in each ml of carbon fluoride compound in the environment with the temperature of 15-40 ℃ and the oxygen partial pressure of 0-101325 Pa. When the partial pressure of oxygen in the environment in which the silica bodies are located increases (or decreases), the amount of oxygen adsorbed by the silica bodies increases (or decreases) accordingly. When the silica body enters a human body through intravenous injection and is transported to the area B from the area A, if the oxygen concentration of the area B is higher than that of the area A, the silica body preparation adsorbs oxygen in the area B; if the oxygen concentration in zone B is lower than that in zone a, the silica will release oxygen in zone B. The normal human body has the venous blood oxygen content of 110-180mL/L, the arterial blood oxygen content of 150-230mL/L, the venous blood oxygen concentration is lower than the arterial blood oxygen concentration, and the arterial blood oxygen concentration is higher than the tumor tissue (including tumor cells and tumor extracellular stroma) outside the tumor blood vessel. When the silica body enters the human body through intravenous injection and enters arterial blood from venous blood, the silica body adsorbs oxygen; when arterial blood enters the tumor tissue outside the tumor vessel, the silica will release oxygen. Thus, the silica can carry oxygen in arterial blood to the tumor tissue.
Further, according to the invention, the silica body has good stability: after being treated by 0-100 mu M surfactant Triton-100, the drug release rate of the silica is less than 20%. Compared with the liposome composed of common phospholipids and carrying the carbon fluoride compounds, the liposome has higher stability: after being treated by 10-100 mu M surfactant Triton-100, the drug release rate of the silica liposome is 30-90% lower than that of the liposome.
Further, according to the present invention, the silica can respond to the stimulation of the high intensity focused ultrasound to achieve the drug release and oxygen release controlled by the high intensity focused ultrasound: the silica body is processed by high-intensity focused ultrasound with the ultrasonic intensity (pulse width modulation) of 1% -10% for 0.5-5 minutes, the oxygen release rate of the silica body can reach 5% -100%, and the drug release rate of the silica body can reach 5% -100%.
Figure BDA0002745855000000051
Further, according to the present invention, the silica body also has the ability to enhance ultrasound imaging.
The invention further provides a preparation method of the silica body.
A method for preparing the aforementioned silica bodies, comprising the steps of: adding carbon fluoride compound, formed siliceous liposome and phospholipid component without Si or further containing medicine into buffer solution or water, homogenizing, and high-pressure extruding to obtain uniform particle size distribution.
Further, according to the present invention, the fluorocarbon-based compound, the liposome-forming and Si-free phospholipid component, or the further-contained drug, may be dissolved in an organic solvent prior to the addition of the buffer solution or water. The organic solvent may be alcohols (e.g., ethanol, propanol, etc.), dimethyl sulfoxide, etc.
Further, according to the present invention, the buffer solution may be a PBS buffer, a Tris-HCl buffer, or the like. The pH of the buffer solution may be 5.0-8.0.
The silica body of the invention can also be prepared by another method. The preparation method comprises the following steps: 1) hydrolyzing the formed siliceous plastid lipids in an acidic alcohol solution; 2) dissolving the phospholipid component without Si or the medicine further contained in the phospholipid component in an organic solvent, and adding the dissolved phospholipid component or the medicine further contained in the phospholipid component into the hydrolysate in the step 1) to form a mixture; 3) volatilizing the mixture of step 2) to obtain a phospholipid membrane, hydrating the membrane, and homogenizing; the fluorocarbon-based compound is added during the hydration operation or during the homogenization operation.
Further, according to the present invention, the acidic alcohol solution is prepared by dissolving alcohol in an aqueous dilute solution of acid. The alcohol may be ethanol, propanol, isopropanol, etc.; the acid may be hydrochloric acid, sulfuric acid, nitric acid, etc.
Further, according to the present invention, the organic solvent may be alcohols (e.g., ethanol, etc.), dichloromethane, chloroform, dimethylformamide, etc.
Further, according to the present invention, the hydration may use water, PBS buffer, Tris-HCl buffer, or the like.
According to the invention, the silica preparation is obtained by centrifugation after homogenization. After homogenization, the mixture may be further passed through a high-pressure extruder to obtain a silica preparation having a more uniform particle size.
According to the invention, after obtaining a preparation of silica, the preparation is modified with a tumor targeting molecule.
According to the invention, after the preparation of the silica, the material which is not encapsulated in the silica is removed, for example by washing, filtration and/or chromatography on a column.
According to the invention, if the medicine is contained, the mass ratio of the medicine to the phospholipid component (the sum of the liposome forming the silica and the phospholipid without Si) is preferably 1: 10-1: 2 when the silica is prepared, the ratio can influence the medicine loading and encapsulation rate of the silica, and the medicine loading and encapsulation rate of the silica are optimal within the preferable mass ratio range of the invention.
The invention further provides a pharmaceutical application of the silica body.
The application of the silica body in preparing oxygen delivery carriers or medicines is provided. Based on the application, the silica can be prepared into a medicament for improving tumor hypoxia.
The application of the silica body in preparing a carrier for delivering a medicament. Based on the application, the silica body can be prepared into a tumor-targeted medicament, wherein the tumor-targeted medicament is encapsulated, so that the tumor-targeted medicament can be simultaneously administered with oxygen and the medicament, the tumor hypoxia is improved, the medicament treatment effect is improved, and the tumor metastasis incidence rate is reduced. The application of the silica body in preparing tumor treatment medicines.
The application of the silica body in preparing the medicine for treating the tumor by combining with the HIFU is disclosed. The medicine can improve tumor hypoxia and reduce the incidence rate of tumor metastasis. The medicine can be used as new adjuvant chemotherapy medicine for thermotherapy. The HIFU can increase the temperature of target tissues (such as tumors) from normal body temperature (37 ℃) to target temperature (40-80 ℃) within 20 minutes and maintain the target temperature for more than 30 minutes; adjusting the power of the HIFU may change the target temperature.
In one embodiment of the invention, the tumor is a breast cancer, e.g., a triple negative breast cancer.
In one embodiment of the invention, the tumor therapy drug is doxorubicin.
The application of the silica body in preparing the ultrasonic imaging agent is provided.
The invention has the beneficial effects that:
one of the advantages of the invention is that the silica body is used as a wrapping agent of the carbon fluoride, which can improve the stability of a drug delivery system, reduce the early leakage of the drug in blood circulation and reduce the side effect of the drug. The silica body is an inorganic-organic hybrid material, the structure of the silica body is similar to that of a liposome consisting of common phospholipid, and the amphiphilic composite lipid is self-assembled to form micro or nano particles. However, compared with the traditional phospholipid liposome, the surface of the liposome is of a single-atom-layer network structure formed by organosilicate, and the liposome has higher structural stability and drug-loading stability. Compared with silicon nanoparticles, the phospholipid layer structure of the silica reduces the overall hardness and density of the nanoparticles, reduces the injection dosage of silicon element, and improves the biological safety. The carbon fluoride compound has strong hydrophobicity and very low loading content by adopting the silica body. The invention improves the microstructure of the silica body through research, skillfully solves the problem of high content entrapment of the highly hydrophobic carbon fluoride compounds by adopting a monomolecular layer silica body structure, and keeps the stability of the silica body as a delivery system.
The preparation of the carbon fluoride compound encapsulated by the silica body can respond to the stimulation of the high-intensity focused ultrasound, and the high-intensity focused ultrasound can trigger the rapid release of the medicine and the oxygen in the target area. High-intensity focused ultrasound (HIFU) is a non-invasive technique used clinically for thermal ablation therapy. HIFU can precisely control and elevate the temperature of target tissue by converting ultrasonic energy into thermal and mechanical energy. HIFU can slightly elevate the temperature of the target tissue (<43 ℃), and has been used for the heat-sensitive release of drugs without damaging the tissue. A slight increase in temperature increases tumor blood flow, so HIFU itself may also alleviate tumor hypoxia to some extent. The siliceous body can respond to the stimulation of the HIFU, so as to release the oxygen or the medicine carried in the siliceous body, realize the accurate fixed-point control of the release of the oxygen or the medicine, and better relieve the hypoxic of the tumor by combining the heating effect of the HIFU.
The third advantage of the invention is that the preparation of the carbon fluoride compound carried by the silica plastid can deliver the drug and the oxygen to the tumor site in a targeted way. The targeted delivery of the drug can increase the drug effect of the drug, and the targeted delivery of the oxygen can improve tumor hypoxia, relieve the multi-drug resistance and epithelial-mesenchymal transition of the tumor, improve the drug effect and reduce the tumor metastasis.
The preparation of the carbon fluoride compound encapsulated by the silica body has the effect of enhancing ultrasonic imaging, can realize drug delivery and drug release under image monitoring, and can estimate the treatment effect.
Drawings
FIG. 1: the invention discloses a structural schematic diagram of a silicon plastid-encapsulated carbon fluoride preparation.
FIG. 2: the cryoetching scanning electron microscope image of the silicon plastid-entrapped fluorocarbon preparation prepared in example 3 shows that the nano preparation is spherical and has an average particle size of about 100 nm.
FIG. 3: the drug loading stability of the silica-encapsulated fluorocarbon formulations prepared in example 3 and example 5, respectively, was compared to that of the common phospholipid-encapsulated fluorocarbon formulations.
FIG. 4: the controlled release effect of the drug after the high intensity focused ultrasound treatment of the silicon plastid-entrapped fluorocarbon preparation prepared in example 3 is evaluated.
FIG. 5: evaluation of oxygen controlled release effect of the silica-encapsulated fluorocarbon formulation prepared in example 3.
FIG. 6: ultrasonic imaging effect evaluation of the silica-encapsulated fluorocarbon formulations prepared in example 3.
FIG. 7: the drug-loading stability of the preparation consisting of different kinds of carbon fluoride.
FIG. 8: sonogram plots of different formulations before (a-d) and after (I-V) 1 minute of focused ultrasound treatment with M-HIFU (PWM ═ 2%): (a) and (I) D-PFP-vPCs, (b) and (II) D-PFH-vPCs, (c) and (III) D-PFOB-vPCs, (D) and (IV) D-PFCE-vPCs, (V) PBS.
FIG. 9: therapeutic effect of the preparation at cellular level.
FIG. 10: the preparation can improve the distribution of the medicine in vivo.
FIG. 11: the in vivo chemotherapy effect of the preparation.
FIG. 12: the preparation has effect in reducing tumor metastasis.
FIG. 13: the preparation has therapeutic effect as new adjuvant chemotherapy means.
Detailed Description
The present invention will be described in further detail with reference to examples. However, those skilled in the art will appreciate that the scope of the present invention is not limited to the following examples. In light of the present disclosure, those skilled in the art will recognize that many variations and modifications may be made to the embodiments described above without departing from the spirit and scope of the present invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples are all commercially available products or can be prepared by known methods; the manipulations performed are all known in the art or performed according to the user's manual of commercially available products.
Example 1 preparation of silica-Encapsulated fluorocarbon formulations
(1) Mu.mol of complex lipid A, 0.5. mu. mol of DSPE-mPEG2000, 4. mu. mol of the small molecule drug doxorubicin and 20. mu.L of perfluorohexane were dissolved in 2mL of ethanol, injected into 20mL of PBS, and during and within 15 minutes after injection, the 20mL of PBS was sonicated using a water bath sonicator (Prima, UK, model PM3-900 TL). Thereafter, sonication was continued for 10 minutes at 50% output under ice bath conditions using a probe-type sonicator (Qsonica, usa, model Q700). The liquid was repeatedly extruded through a polycarbonate filter (pore size: 100nm, Whatman, USA) 3 times by means of a high-pressure extruder (Northern Lipid, USA) to obtain a preparation having an average particle size of about 100 nm.
(2) To remove doxorubicin that was not encapsulated in the siliceous preparation, the preparation obtained in step (1) was first filtered through a 0.45 μm filter and free doxorubicin was removed using exclusion chromatography column Sephadex G-50.
(3) The formulation was tested for carbon fluoride and drug loading: taking 0.1mL of 100mg/mL of the preparation (the concentration of the preparation is calculated by taking the weight of the preparation after freeze-drying as the weight of a solute, the solute is equivalent to 10mg of the freeze-dried silica, and the freeze-dried silica does not contain perfluorohexane due to volatilization of the perfluorohexane), dissolving the silica with 500 muL of methanol, detecting the content of the perfluorohexane by a gas chromatograph, and determining that 6.24mg of the perfluorohexane is contained in each 10mg of the freeze-dried silica. Similarly, 0.1mL of 100mg/mL of the preparation was dissolved in an acidified isopropanol solution (10% of 1mol/L hydrochloric acid, 90% isopropanol) and the content of doxorubicin was determined by fluorescence spectrophotometry, whereby the doxorubicin-loaded amount was 17.5% and the encapsulation efficiency was 84.9%, based on the determination that 1.75mg of doxorubicin was contained per 10mg of the lyophilized silica.
(4) Detecting whether the outer layer of the silica plastid of the preparation is a single lipid molecular layer or not, wherein the method I comprises the following steps: the thickness of the outer shell of the preparation is observed by a cryo-electron microscope, and the result shows that the thickness of the outer shell of the preparation is about 2nm, and the thickness of the phospholipid bilayer is about 4.5nm, so that the outer shell of the preparation is a lipid monolayer. The second method comprises the following steps: dissolving 9.5 mu mol of the compound lipid A, 0.5 mu mol of DSPE-mPEG2000, 2 mu mol of micromolecular drug adriamycin and 20 mu L of perfluorohexane, 0.01 mu mol of water-soluble dye sodium calcein and 0.01 mu mol of fat-soluble dye nile red in 2mL of ethanol, and detecting the ultraviolet-visible light absorption curve of the prepared preparation by the same subsequent treatment method as the steps (1) and (2). The detection result shows that the preparation contains a characteristic absorption peak of nile red and does not have a characteristic absorption peak of calcein sodium, which indicates that the preparation does not have a water cavity, so that the outer layer is a monolayer lipid molecular layer.
Example 2 preparation of silica Encapsulated fluorocarbon formulations
(1) Mu. mol of complex lipid B was hydrolyzed in acidic ethanol at 40 ℃ for 30 minutes. The acidic ethanol solution is prepared by adding dilute hydrochloric acid into ethanol solution, and adjusting pH value to 3.0 with pH meter.
(2) Dissolving 1 mu mol of paclitaxel, a small molecule drug, 4 mu mol of phospholipid components without Si (including 3 mu mol of hydrogenated soybean lecithin (HSPC) and 1 mu mol of DSPE-mPEG2000) in 2mL of chloroform, adding the hydrolysate obtained in step (1), volatilizing the liquid to form a phospholipid film, vacuum-drying overnight, adding 5mL of ultrapure water and 20 mu L of perfluorocrown ether, shaking and hydrating for 60 minutes, and then continuously performing ultrasonic treatment for 10 minutes by using a probe type ultrasonic instrument (Qsonica, model Q700, USA) at an output power of 50% under an ice bath condition to obtain a preparation with an average particle size of about 200 nm.
(3) To remove paclitaxel that is not encapsulated in the siliceous preparation, the preparation obtained in step (2) is first filtered through a 0.45 μm filter and then freed paclitaxel is removed using exclusion chromatography column Sephadex G-50.
(4) Using the same assay as in example 1, it was determined that 5.1mg of perfluorocrown ether and 0.9mg of paclitaxel were contained per 10mg of lyophilized siliceous mass.
Example 3 preparation of silica-Encapsulated fluorocarbon formulations with tumor targeting molecules
(1) Mu.mol of the complex lipid A, 0.2. mu. mol of DSPE-mPEG2000, 4. mu. mol of the small molecule drug doxorubicin, 20. mu.L of perfluorohexane, and 0.3. mu. mol of DSPE-mPEG2000-Maleimide were dissolved in 2mL of ethanol, and injected into 20mL of PBS, and during and within 15 minutes after the injection, the 20mL of PBS was sonicated using a water-bath sonicator (Prima, UK, model PM3-900 TL). Thereafter, sonication was continued for 10 minutes at 50% output under ice bath conditions using a probe-type sonicator (Qsonica, usa, model Q700). The liquid was repeatedly extruded through a polycarbonate filter (pore size: 100nm, Whatman, USA) 3 times by means of a high-pressure extruder (Northern Lipid, USA) to obtain a preparation having an average particle size of about 100 nm.
(2) 0.6 mu Mol of tumor targeting molecule var7 polypeptide (var7 can be targeted to the tumor site by virtue of the acidic microenvironment of the tumor, the amino acid sequence is ACEEQNPWARYLEWLFPTETLLLEL, the detailed information is described in the literature: Mol Imaging Biol (2016)18:686-696), and the thiol group of var7 polypeptide is coupled with Maleimide by stirring at 4 ℃ for 24 hours.
(3) To remove doxorubicin that was not encapsulated in the siliceous preparation and var7 that was not attached to the surface of the siliceous body, the preparation obtained in step (2) was first filtered through a 0.45 μm filter and free doxorubicin and var7 were removed using exclusion chromatography column Sephadex G-50.
(4) Using the same assay method as in example 1, the assay result was 5.9mg of perfluorohexane and 1.6mg of doxorubicin per 10mg of lyophilized silica.
Example 4 preparation of silica-Encapsulated fluorocarbon formulations with tumor targeting molecules
(1) Dissolving 9.5 μmol of complex lipid B, 0.2 μmol of DSPE-mPEG5000, 2 μmol of small molecule drug porfimer sodium, 20 μ L of perfluorooctyl bromide and 0.3 μmol of DSPE-Maleimide (distearoyl phosphatidyl ethanolamine modified Maleimide) in 2mL of ethanol, injecting into 20mL of PBS, and subjecting the 20mL of PBS to ultrasonic treatment by using a water bath ultrasonic instrument (Prima corporation, UK, model PM3-900TL) during and within 15 minutes after injection. The liquid was repeatedly extruded through a polycarbonate filter (pore size: 10 μm, Whatman, USA) 3 times by means of a high-pressure extruder (northern lipid, USA) to obtain a preparation having a particle size of about 10 μm.
(2) 0.6 mu mol of tumor targeting molecule SH-PEG5000-Folate (sulfhydryl-polyethylene glycol 5000-folic acid) is added into the preparation, and the mixture is stirred for 24 hours at 4 ℃ so that the sulfhydryl of the SH-PEG5000-Folate is coupled with Maleimide.
(3) In order to remove porfimer sodium not encapsulated in the siliceous preparation and tumor targeting molecules not attached to the surface of the siliceous body, the preparation obtained in step (2) was repeatedly washed three times with 50% ethanol and stored in PBS for future use.
(4) The same test method as in example 1 was used, and the test results showed that 7.3mg of perfluorooctyl bromide and 0.7mg of porfimer sodium were contained per 10mg of the lyophilized silica.
Example 5 preparation of Liposome Encapsulated fluorocarbon formulations consisting of common Phospholipids
(1) Mu.mol Distearoylphosphatidylcholine (DSPC), 0.2. mu. mol DSPE-mPEG2000, 3.0. mu. mol cholesterol, 2.0. mu. mol small molecule drug doxorubicin, 20. mu.L perfluorohexane, 0.3. mu. mol DSPE-mPEG2000-Maleimide were dissolved in 2mL ethanol, and injected into 20mL PBS, and during and within 15 minutes after injection, 20mL PBS was sonicated using a water bath sonicator (Prima, UK, model PM3-900 TL). Thereafter, sonication was continued for 10 minutes at 50% output under ice bath conditions using a probe-type sonicator (Qsonica, usa, model Q700). The liquid was repeatedly extruded through a polycarbonate filter (pore size: 100nm, Whatman, USA) 3 times by means of a high-pressure extruder (Northern Lipid, USA) to obtain a preparation having an average particle size of about 100 nm.
(2) 0.6 mu mol of tumor targeting molecule var7 polypeptide is added into the preparation, and the mixture is stirred for 24 hours at 4 ℃ so that the sulfhydryl of var7 polypeptide is coupled with Maleimide.
(3) To remove doxorubicin that was not encapsulated in the formulation and var7 that was not attached to the surface of the liposomes, the formulation obtained in step (2) was first filtered through a 0.45 μm filter and free doxorubicin and var7 were removed using exclusion chromatography column Sephadex G-50.
(4) Using the same examination method as in example 1, it was determined that 5.3mg of perfluorohexane and 1.4mg of doxorubicin were contained per 10mg of the lyophilized liposomes.
Example 6 evaluation of drug-loaded stability of silica-encapsulated fluorocarbon formulations
(1) The surfactant Triton-100 solubilization method was used to compare the drug loading stability of different fluorocarbon formulations. The fluorocarbon formulations prepared in example 3 and example 5 were dispersed in Triton-100 solutions of different concentrations, respectively.
(2) After shaking for 10 minutes at room temperature, sampling to detect the fluorescence intensity of the adriamycin, detecting the drug amount of the adriamycin released from the preparation by a fluorescence quenching method, and calculating the percentage of the released adriamycin in the total adriamycin in the preparation, wherein the formula is as follows:
Figure BDA0002745855000000121
wherein F (t) is fluorescence intensity of doxorubicin of the Triton-100-treated sample, F0Is the fluorescence intensity of doxorubicin of the control sample not treated with Triton-100, while Fc is the mean fluorescence intensity of doxorubicin of the five samples after complete destruction by 1% Triton-100 and 1M hydrochloric acid. The fluorescence intensity of the adriamycin is detected by a fluorescence spectrophotometer, the detected excitation wavelength is 480nm, and the detected emission wavelength is 580 nm.
As shown in FIG. 3, with the increase of Triton-100 concentration, doxorubicin in the liposome-entrapped fluorocarbon preparation composed of common phospholipids was released in a large amount, but only a small amount of doxorubicin in the liposome-entrapped fluorocarbon preparation was released. The result shows that the carbon fluoride coated by the silica liposome has higher stability than the carbon fluoride coated by the liposome and can resist the solubilization of the surfactant.
Example 7 evaluation of the drug Release ability of silica-encapsulated fluorocarbon formulations under high intensity focused ultrasound control
(1) The silica-encapsulated fluorocarbon preparation prepared in example 3 was dispersed in PBS at 37 ℃ and treated with high intensity focused ultrasound for 4 minutes with a Pulse Width Modulation (PWM) of 2%. At various times during sonication, the solution was sampled and immediately cooled on ice to stop drug release.
(2) The release rate of the drug is detected by a fluorescence quenching method, and the percentage of released adriamycin in the total adriamycin in the preparation is calculated by the following formula:
Figure BDA0002745855000000122
where F (t) is the fluorescence intensity of doxorubicin for the sample after high intensity focused ultrasound treatment, cooled on ice and equilibrated to room temperature, F0 is the fluorescence intensity of doxorubicin for the control sample without high intensity focused ultrasound treatment, and Fc is the average fluorescence intensity of doxorubicin for five samples after extensive destruction by 1% Triton-100 and 1M hydrochloric acid. The fluorescence intensity of the adriamycin is detected by a fluorescence spectrophotometer, the detected excitation wavelength is 480nm, and the detected emission wavelength is 580 nm.
The detection result is shown in figure 4, and the carbon fluoride preparation encapsulated by the silica can rapidly release the adriamycin under the treatment of high-intensity focused ultrasound. The high-intensity focused ultrasound can trigger the preparation to release the drug, so that the controlled release of the drug is realized.
Example 8 evaluation of oxygen delivery and controlled Release capabilities of silica-Encapsulated fluorocarbon formulations
(1) In a sterile oxygen chamber (O)2Flow rate 5L/min), 1mL of the preparation prepared in example 3 was oxygenated for 1 minute and then rapidly injected into 4mL of PBS.
(2) The dissolved oxygen concentration of 4ml pbs was monitored before and after injection using a portable dissolved oxygen meter (Rex, model JPBJ-608, china). 1mL of oxygenated or non-oxygenated PBS containing no siliceous preparation was injected into 4mL of PBS for use as a control.
(3) The time of injection was defined as 0 minutes, and the injected solution was irradiated with high intensity focused ultrasound (PWM ═ 2%) for 2 minutes at 20, 30, 40, and 50 minutes after injection, respectively, to trigger the release of oxygen from the formulation. The oxygen concentration was recorded using a dissolved oxygen meter for a total of about 60 minutes.
As shown in FIG. 5, after 1mL of oxygenated preparation was injected into 4mL of PBS, the oxygen content of PBS increased rapidly and the preparation released oxygen rapidly each time it was treated with high intensity focused ultrasound, forming a prominent "fluctuating peak" in the oxygen concentration profile, indicating that the preparation was able to carry oxygen efficiently and that high intensity focused ultrasound triggered the release of oxygen.
Example 9 evaluation of ultrasound imaging Effect and carbon fluoride controlled Release Effect of silica-Encapsulated carbon fluoride preparations
(1) Establishing a mouse subcutaneous tumor model: 4T1 cells were administered at 5X 10 per mouse6The amount of each cell is inoculated on the subcutaneous side of the thigh of a BALB/c mouse, and the mouse is raised in an SPF level environment;
(2) tumor size up to about 100mm3In this case, 200. mu.L of the preparation prepared in example 3 was injected via the tail vein, and doxorubicin was injected at a dose of 5 mg/kg;
(3) the tumor site was treated with high intensity focused ultrasound (PWM ═ 2%) irradiation, and ultrasound imaging was performed before the high intensity focused ultrasound treatment and after different durations of continuous irradiation, respectively. Ultrasound images of the tumor site were acquired using a Resona 7 ultrasound system (Mindray, China) equipped with an L12-3E ultrasound probe (3-12MHz) with a Mechanical Index (MI) set at 0.044.
The detection result is shown in figure 6, and after the preparation is injected, the ultrasonic signal of the tumor area is not obviously changed. However, after the injection of the formulation and the treatment of the tumor with High Intensity Focused Ultrasound (HIFU), the ultrasound signal of the tumor becomes stronger with the increase of the HIFU treatment time, and finally, a significantly enhanced ultrasound signal appears in the whole tumor area, which indicates that the treatment of HIFU can convert the carbon fluoride formulation into bubbles to generate the ultrasound signal. This also suggests that HIFU treatment may facilitate the release of carbon fluoride from the formulation.
Example 10 evaluation of drug-loaded stability of formulations consisting of different kinds of fluorocarbons
Formulations consisting of perfluoropentane, perfluorohexane, perfluorooctane bromide and perfluorocrown ether, designated respectively as D-PFP-vPCs, D-PFH-vPCs, D-PFOB-vPCs, D-PFCE-vPCs, were prepared according to the method described in example 3; a preparation of perfluorohexane coated with common phospholipids, designated D-PFH-vPLs, was prepared according to the procedure described in example 5.1 mL of this formulation was dispersed in 10mL of fetal bovine serum at 37 ℃ with constant stirring (500 rpm), simulating the environment in which different formulations would be in the blood circulation system in vivo, during which time the solution was sampled at different times and the amount of doxorubicin-releasing drug from the formulation was determined using the fluorescence dequenching method described in example 6.
The results are shown in FIG. 7, where D-PFP-vPCs leaked about 50% of doxorubicin while the other three D-vPCs only leaked < 10% of doxorubicin within 48 hours, indicating that the drug loading stability of the formulation consisting of perfluoropentane was poor. The adriamycin leakage rate of the D-PFH-vPLs is far higher than that of the D-PFH-vPCs, and the adriamycin leakage rate and the D-PFH-vPLs are respectively 78.5% and 8.4%, which shows that the preparation has better drug loading stability compared with a common phospholipid-coated perfluorohexane preparation.
Example 11 evaluation of the in vitro ultrasound contrast Effect of formulations consisting of different types of carbon fluoride
Formulations consisting of different types of fluorocarbons prepared in the previous example 10 were loaded in latex tubes, immersed in water at 37 c, and examined for ultrasonic signals before and after HIFU treatment using a 2% PWM (Pulse-Width Modulation) power using a Resona 7 ultrasonic imaging system (Mindray, china) using an L12-3E ultrasonic examination probe (3-12MHz) with a Mechanical Index (MI) of 0.044.
As shown in fig. 8, the formulations of four fluorinated carbons did not have significant ultrasound signals without HIFU pretreatment. After 3 minutes of HIFU (PWM ═ 2%) pretreatment, obvious ultrasonic signals appear in D-PFP-vPCs and D-PFH-vPCs. However, the ultrasonic signals of D-PFOB-PCs and D-PFCE-vPCs are not obvious. Therefore, after HIFU pretreatment, only the formulation consisting of perfluoropentane and perfluorohexane had ultrasound contrast ability, and the formulation consisting of perfluorooctyl bromide and perfluorocrown ether had no ultrasound contrast ability.
Example 12 evaluation of the therapeutic Effect of the preparations on the cellular level
4T1 cells were seeded into 24-well plates (10 per well)4One cell) filled with 1% O2The cells are induced to enter a hypoxic state by culturing in a hypoxic incubator for 24 hours. The D-PFH-vPCs preparation prepared according to the method of example 3 was dispersed in 2 mlpmi-1640 medium (final concentration of doxorubicin 5 μ g/mL), saturated with nitrogen or oxygen, respectively (filtration of nitrogen or oxygen through a 0.22 μm gas filter), added to the cells, immediately treated with HIFU (PWM ═ 2%) for 3 minutes, incubated for 4 hours, the cells were washed three times with the medium, cultured for further 24 hours, and the survival rate of the cells was examined using CCK-8 kit (Dojindo, japan).
The results are shown in fig. 9, and the experimental group "the preparation saturated with nitrogen + HIFU treatment" has lower survival rate of tumor cells than the experimental group "the preparation saturated with nitrogen", which indicates that HIFU can promote the preparation to release adriamycin. Compared with the experimental group of 'the preparation saturated with oxygen and the HIFU treatment', the survival rate of the tumor cells is lower, which shows that the oxygen carried by the preparation can improve the hypoxia state of the tumor cells and reduce the survival rate of the tumor cells. And the anti-tumor effect of the experimental group 'oxygen-saturated preparation + HIFU treatment' is much higher than that of the experimental group 'free adriamycin'.
The present inventors further investigated the effect of the relevant treatment methods on the expression levels of the relevant genes and proteins (HIF-1. alpha., MDR1 and P-gp) for multiple drug resistance in tumor cells using PFH-vPCs without doxorubicin loading, under nitrogen or oxygen saturation conditions, with PBS as a control (the relative expression level of MDR1 gene was quantified by RT-qPCR, and the relative expression levels of HIF-1. alpha. and P-gp proteins were quantified by western blotting). As a result, it was found that: cell passage through vPCs (O)2) After + HIFU treatment, the expression levels of all three markers were significantly reduced, while vPCs (N)2) + HIFU or PBS (O)2) + HIFU had no significant effect on the expression levels of the three markers. The results show that PFH-vPCs are capable of carrying oxygen and that PFH-vPCs (O)2) The + HIFU can effectively reduce the expression level of the genes and proteins related to the multiple drug resistance of the tumor cells after relieving the tumor hypoxia.
Example 13 evaluation of the formulations for improvement in vivo drug distribution
4T1 tumors were implanted subcutaneously in the hind legs of BALB/c mice on both sides until the tumor volume increased to 100mm3In this case, the preparation (D-vPCs) prepared in example 3, the perfluorohexane preparation (D-vPLs) coated with common phospholipid prepared in example 5, Free doxorubicin (Free DOX), and doxorubicin liposome were injected intravenously, respectively
Figure BDA0002745855000000151
The dose was 5mg of doxorubicin per kg of mouse body weight. Immediately after injection, tumors on one side were sonicated with HIFU (PWM ═ 2%) for 15 minutes. After 24 hours, the mice were sacrificed, the tumors and major organs were collected, weighed, 0.5mL of PBS was added per 50mg of tissue, and after clipping, thoroughly homogenized using an electric tissue homogenizer. To 100 u L homogenate add 900mu.L of precipitation buffer (containing 800. mu.L of isopropanol, 100. mu.L of 10 vol% Triton-100 and 100. mu.L of water). After standing at 4 ℃ for 24 hours, 12000g of the mixture was centrifuged, and the fluorescence intensity of doxorubicin in the supernatant was measured by a multifunctional microplate reader. Doxorubicin was dispersed in a mouse tissue homogenate without administration treatment at a known concentration, and the fluorescence intensity of doxorubicin was measured by the same treatment as described above, to prepare a standard calibration curve for each tissue. The tissue content of doxorubicin was calculated from the corresponding standard calibration curve and the final result was expressed as the percentage of doxorubicin content per gram of tissue (ID%/g) of the total dose of doxorubicin injected.
As a result, as shown in FIG. 10, the tumors on one side treated with HIFU had higher adriamycin uptake (34.2vs 1.2% ID/g) than the tumors on the other side not treated with HIFU after the injection of D-vPCs, which indicates that D-vPCs have good HIFU responsiveness. The experimental group "D-vPCs + HIFU" had the highest tumor doxorubicin uptake, 16.3 times (34.2vs 2.1% ID/g) that of the experimental group "Free DOX", and was the experimental group
Figure BDA0002745855000000161
8.8 times higher (34.2vs 3.9% ID/g).
Myocardial damage is one of the most serious side effects of doxorubicin. Although in contrast to Free DOX,
Figure BDA0002745855000000167
the myocardial uptake of doxorubicin was somewhat reduced, however, the experimental group "D-vPCs + HIFU" had a lower myocardial uptake, which is compared to
Figure BDA0002745855000000162
Compared with the prior art, the reduction is 75.0% (0.3vs 1.2% ID/g); compared with D-vPLs, a reduction of 81.3% (0.3vs 1.6% ID/g) was achieved. With the Free DOX, the process is simple,
Figure BDA0002745855000000163
compared with D-vPLs, the doxorubicin intake in the kidney and lung of the mice treated with the D-vPCs + HIFU is also lowest.
EXAMPLE 14 evaluation of the in vivo chemotherapeutic Effect of the formulations
BALB/c mouse with 4T1 tumor is prepared, and the tumor grows to about 100mm3On the left and right, mice were randomly divided into nine groups of 5 mice each: (1) PBS; (2) vPCs (O)2)+HIFU；(3)Free DOX；(4)
Figure BDA0002745855000000164
(5)D-vPCs；(6)D-vPLs+HIFU；(7)D-vPCs+HIFU；(8)D-PCs+HIFU；(9)D-vPCs(O2) + HIFU. D-vPCs and vPCs are doxorubicin-loaded and non-doxorubicin-loaded formulations modified with tumor targeting molecule var7, respectively, prepared according to the method of example 3, D-PCs are doxorubicin-loaded, non-modified tumor targeting molecule var7, prepared according to the method of example 3, and D-vPLs are common phospholipid-loaded perfluorocarbon formulations (containing doxorubicin), prepared according to example 5. For groups (2) and (9), "(O)2) "means that the mice are given hyperoxic respiratory treatment: the mice were fitted with an oxygen breathing mask starting 5 minutes before injection and breathing of high concentration oxygen was continued for 20 minutes. All groups of the administered agents were dispersed in PBS and injected into mice via tail vein, with a volume of 200. mu.L per mouse. For the group treated with HIFU sonication, the tumor was sonicated with HIFU (PWM ═ 2%) immediately after injection to trigger the release of oxygen and DOX. For groups (1) and (2) that did not contain DOX, the drug was injected twice daily, once in the morning and once in the evening, for 10 days from day 0 to day 9; for the other doxorubicin-containing groups, injections were given once daily at 5mg/kg doxorubicin dose on days 0, 2, 4, 6, and 8, respectively. For group (9), "D-vPCs (O) were administered once daily on days 0, 2, 4, 6, 8 in the morning2) + HIFU ", administering once a night" vPCs (O)2) + HIFU "and" vPCs (O) twice daily on days 1, 3, 5, 7, 92) + HIFU "once in the morning and once in the evening, thus, it is ensured that the mice of group (9) and group (2) receive the same number of hyperoxic breaths. Tumor volumes were measured every two days. The tumor volume was calculated as V ═ L × W2) L and W are the length and width of the tumor, respectively. At the end of the treatment (day 16), the tumor growth inhibition rate was calculated and the formula was calculatedThe formula is as follows: (tumor volume in PBS group-tumor volume in experimental group)/tumor volume in PBS group.
As shown in FIG. 11, first, vPCs (O)2) The + HIFU had no significant effect on tumor growth, indicating to some extent the good biological safety of vPCs and HIFU. Free DOX and
Figure BDA0002745855000000165
with similar tumor growth inhibition rate (20.6% vs 25.77%). The tumor treatment effect of the D-vPCs + HIFU is far better than that of the D-vPCs, Free DOX,
Figure BDA0002745855000000166
The tumor growth inhibition rate is improved to 83.2 percent. D-vPCs + HIFU also showed better tumor growth inhibition than D-PCs + HIFU, D-vPLs + HIFU (P-PCs + HIFU)<0.05), because the targeting property of the tumor is increased compared with that of the D-PCs, and the D-vPCs have better drug-loading stability compared with that of the D-vPLs.
By improving tumor hypoxia, and relieving hypoxia-mediated multidrug resistance, D-vPCs (O)2) + HIFU has a higher antitumor effect than D-vPCs + HIFU. With Free DOX,
Figure BDA0002745855000000171
D-vPCs + HIFU in comparison to D-vPCs (O)2) The tumor growth inhibition rate of the + HIFU is as high as 94.6%, which is respectively improved by 3.59, 2.67 and 0.14 times (P) compared with the former three<0.0001)。
Example 15 evaluation of the Effect of the formulations on reducing tumor metastasis
4T1 cells (4T1-Luciferase) transfected with the Luciferase gene were prepared. BALB/c mice bearing 4T1-Luciferase subcutaneous tumors were prepared until the tumor volume grew to approximately 100mm3On the left and right, mice were randomly divided into 7 groups of 5 mice: (1) PBS; (2) vPCs; (3) vPCs (O)2)+HIFU；(4)Free DOX；(5)
Figure BDA0002745855000000172
(6)D-vPCs+HIFU；(7)D-vPCs(O2) + HIFU. D-vPCs and vPCs are respectively entrapping plants prepared according to the method of example 3A preparation of a modified tumor targeting molecule var7 without doxorubicin; "(O)2) "means that the mice are given hyperoxic respiratory treatment. Mice were treated by the dosing method described in example 14. The transfer and distribution of 4T1 cells in vivo can be dynamically monitored by bioluminescence imaging of luciferase. At the end of the in vivo treatment experiment (day 16), different groups of tumors were observed for lung metastasis using the IVIS Spectrum small animal in vivo imaging system (PerkinElmer, usa): mice were injected intraperitoneally with 150mg/kg of D-fluorescein potassium salt and imaged 15 minutes after injection.
Results as shown in figure 12, all five mice in the PBS group (group 1) developed spontaneous lung metastases at day 16 of the in vivo treatment experiment. By Free DOX (group 4) or
Figure BDA0002745855000000173
Five administrations (group 5) showed a significant increase in lung metastases, although the growth of orthotopic tumors was inhibited to some extent. Via vPCs (O)2) Multiple treatment with + HIFU (group 3) showed significantly lower lung metastasis levels than PBS (group 1), indicating vPCs (O)2) + HIFU can inhibit lung metastasis of the carcinoma in situ by reducing hypoxia of the carcinoma in situ. The D-vPCs + HIFU group (group 6) had significantly lower lung metastasis than the PBS group (group 1). D-vPCs (O)2) The extent of lung metastasis with + HIFU (group 7) was lower than with D-vPCs + HIFU (group 6), and no significant lung metastasis was observed in all five mice, suggesting that the formulation may improve the hypoxic extent of the carcinoma in situ, inhibiting spontaneous lung metastasis of the tumor.
Example 16 evaluation of the therapeutic efficacy of formulations as a novel adjunctive chemotherapeutic approach
BALB/c mice bearing 4T1 subcutaneous tumors were prepared until the tumor volume grew to approximately 100mm3On the left and right sides, the mice were randomly divided into four groups of 6 mice each, which were treated as follows: (group 1) PBS → HT; (group 2) Free DOX → HT; (group 3)
Figure BDA0002745855000000174
→ HT; (group 4) D-vPCs (O)2) + HIFU → HT. HT stands for hyperthermia: irradiation of the tumor using HIFU (PWM ═ 8%)Tumor for 15 minutes. HT was given on day 0 and chemotherapy was given once on days minus 4 and minus 2, respectively. Four groups of mice were given PBS, Free DOX,
Figure BDA0002745855000000181
and D-vPCs (O)2) + HIFU, the drug is injected via tail vein, the drug dose is 5mg/kg adriamycin, the injection volume is 200 μ L, wherein, D-vPCs (O)2) The + HIFU processing method is as described in embodiment 14: mice were injected with D-vPCs (prepared from example 3) via tail vein with inhalation of hyperoxia, and tumor sites were irradiated with HIFU (PWM ═ 2%) for 15 minutes.
As a result, as shown in FIG. 13, since H-HIFU-mediated hyperthermia itself failed to eradicate all tumor cells, tumors of all mice rapidly recurred through group 1 of PBS pretreatment (days-4 and-2) and hyperthermia treatment (day 0). With Free DOX or
Figure BDA0002745855000000182
In groups 2 and 3, which had been pretreated with chemotherapy (days-4 and-2) and heat treatment (day 0), the recurrence rate of the tumors decreased to 66.7% and 50.0%, respectively. By means of D-vPCs (O)2) + HIFU group 4 with chemotherapy pretreatment and hyperthermia treatment, no tumor recurrence was observed for the next 60 days. Thus, with Free DOX and
Figure BDA0002745855000000183
in contrast, D-vPCs (O)2) The + HIFU is more suitable to be used as a new auxiliary chemotherapy (chemotherapy pretreatment) means to inhibit the tumor recurrence after the thermotherapy.

Claims (10)

1. A silica body encapsulating a fluorocarbon-based compound, the silica body being a particle having a core-shell structure, the shell being a single lipid molecule layer consisting of a silica-forming lipid and a phospholipid component not containing Si, the silica-forming lipid forming a silicate network structure of the form-Si-O-Si-on an outer surface of the shell; the core contains a carbon fluoride compound;
the mole percentage of the liposome-forming lipid is 50-100% and the mole percentage of the phospholipid component without Si is 0-50% based on the total mole of the monolayer of lipid molecules;
the siliceous liposome forming lipid has the following structural formula: a. themCH(3-m)—CH2—Y—(CH2)p—Z—Rq，
Wherein A is: (R)1O)3Si—(CH2)3—N—C(O)—X—(CH2)n—；R1Is alkyl, preferably C1-10 alkyl, more preferably methyl, ethyl; x is-O-or a bond; n is an integer of 0 to 10, preferably 0, 1, 2;
m is an integer of 1 to 3; when m is 2 or 3, the 2 or 3A can be the same or different;
y is-CH2-, -O-C (O) -or-C (O) -O-;
p is an integer of 0 to 10, preferably 0, 1, 2;
z is-C (O) -NH (2-q) -or-CH (3-q)—；
R is alkyl with more than 5 carbon atoms, preferably linear saturated alkyl with C7-20 carbon atoms;
when Z is-C (O) -NH (2-q) -q is an integer from 1 to 2; when Z is-CH (3-q) -q is an integer from 1 to 3; when q is 2 or 3, the 2 or 3R can be the same or different;
preferably, the siliceous plastid forming lipid is selected from the group consisting of complex lipids a-D:
Figure FDA0002745854990000021
；
preferably, the carbon fluoride compound is a carbon fluoride compound having a boiling point of 40 to 80 ℃ at normal pressure.
2. The siliceous body of claim 1, wherein said Si-free phospholipid component is selected from the group consisting of: polyethylene glycol derivative DSPE-mPEG of distearoyl phosphatidyl ethanolamine1000～20000Distearoyl phospholipid modified by maleimideAcylethanolamide, polyethylene glycol derivative DSPE-mPEG of distearoylphosphatidylethanolamine modified by maleimide1000～20000Dipalmitoylphosphatidylcholine, 1-myristoyl-2-stearoyl lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine.
3. The silica body according to claim 1 or 2, wherein the surface of the silica body is modified with a tumor targeting molecule, preferably the tumor targeting molecule is selected from any one or more of an antibody, a polypeptide, an aptamer, folic acid or folic acid derivative for targeting tumor; preferably, the tumor targeting molecule is var7 polypeptide having an amino acid sequence of ACEEQNPWARYLEWLFPTETLLLEL; preferably the tumour targeting molecule is a folate derivative; preferably, the tumor targeting molecule is sulfhydryl-polyethylene glycol2000～6000-folic acid.
4. The siliceous body according to any one of claims 1 to 3, which has a particle size of 10nm to 1 μm.
5. The silica body of any one of claims 1-4, wherein the core of the silica body comprises a water-insoluble or hydrophobic small molecule drug; preferably, the small molecule drug is selected from one or more of chemotherapeutic drugs, protein kinase inhibitors and photodynamic drugs; preferably, the small molecule drug is selected from the group consisting of doxorubicin, cisplatin, paclitaxel, irinotecan, sorafenib, gefitinib, porfimer sodium, verteporfin, indocyanine green.
6. A method for the preparation of a silica body according to any one of claims 1 to 5, characterised in that it comprises the following steps: adding carbon fluoride compound, formed siliceous liposome and phospholipid component without Si or further contained medicine into buffer solution or water, homogenizing, and high-pressure extruding to obtain preparation with uniform particle size distribution;
preferably, the carbon fluoride-based compound, the liposome-forming lipid and the Si-free phospholipid component, or the further drug, are dissolved in an organic solvent prior to the addition of the buffer solution or water, preferably the organic solvent is selected from alcohols, dimethyl sulfoxide;
preferably, the pH of the buffer solution is 5.0 to 8.0.
7. A method for the preparation of a silica body according to any one of claims 1 to 5, characterised in that it comprises the following steps: 1) hydrolyzing the formed siliceous plastid lipids in an acidic alcohol solution; 2) dissolving the phospholipid component without Si or the medicine further contained in the phospholipid component in an organic solvent, and adding the dissolved phospholipid component or the medicine further contained in the phospholipid component into the hydrolysate in the step 1) to form a mixture; 3) volatilizing the mixture of step 2) to obtain a phospholipid membrane, hydrating the membrane, and homogenizing; adding the carbon fluoride compound during hydration operation or homogenization operation;
preferably, the acidic alcohol solution is prepared by dissolving alcohol in dilute aqueous acid solution; preferably the alcohol is ethanol, propanol or isopropanol; preferably the acid is hydrochloric acid, sulfuric acid or nitric acid;
preferably, the organic solvent is an alcohol, dichloromethane, chloroform or dimethylformamide;
preferably, the hydration uses water, PBS buffer solution, Tris-HCl buffer solution;
preferably, the homogenized mixture is further processed through a high pressure extruder to obtain a silica preparation with more uniform particle size.
8. The preparation method according to claim 6 or 7, wherein the mass ratio of the drug to the phospholipid component is 1:10 to 1: 2;
preferably, after obtaining a preparation of silica, the preparation is modified with a tumor targeting molecule.
9. Use of a silica body according to any one of claims 1 to 5 in the manufacture of a carrier or medicament for delivery of oxygen and/or a drug; preferably the medicament is a tumour therapy medicament; preferably the drug is in combination with HIFU; preferably the drug is used as a neoadjuvant chemotherapy drug for thermotherapy; preferably the medicament ameliorates tumor hypoxia; preferably the medicament reduces tumor metastasis; preferably the tumour is breast cancer.
10. Use of the silica bodies of any one of claims 1 to 5 in the preparation of an ultrasound imaging agent.
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CN113332619A (en) * 2021-05-28 2021-09-03 西安交通大学 Ultrasonic conformal activation and monitoring imaging method and system for phase-change nano-droplet drug carrier
CN114732796A (en) * 2022-02-18 2022-07-12 北京大学第三医院（北京大学第三临床医学院） Double-targeting drug-loaded microbubble and preparation method and application thereof
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CN116603101A (en) * 2023-07-20 2023-08-18 北京大学第三医院（北京大学第三临床医学院） Gold-siliceous body-polycaprolactone scaffold system and preparation method and application thereof
CN116603101B (en) * 2023-07-20 2023-10-03 北京大学第三医院（北京大学第三临床医学院） Gold-siliceous body-polycaprolactone scaffold system and preparation method and application thereof

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