CN115970009A - VHPK polypeptide modified fluorescent nano liposome drug delivery system, preparation method and application thereof - Google Patents
VHPK polypeptide modified fluorescent nano liposome drug delivery system, preparation method and application thereof Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a VHPK polypeptide modified fluorescent nano-liposome drug delivery system, a preparation method and application thereof, wherein the drug delivery system is a nano-liposome carrying a fluorescent probe and modified with VHPK polypeptide, and the drug delivery system comprises: the fluorescent probe is a near-infrared first-region fluorescent nano probe DiR, or the fluorescent probe is a near-infrared second-region fluorescent nano probe BBT-2FT. The nano drug delivery system can specifically target vascular cell adhesion factor-1 highly expressed by atherosclerotic vascular endothelium through polypeptide VHPK, so that the high accumulation of a near infrared fluorescence imaging probe at a target position can be realized through the specific recognition of VCAM-1 highly expressed by atherosclerotic vascular endothelium tissue, the near infrared one-region and two-region optical imaging diagnosis of atherosclerosis can be realized respectively, more comprehensive and timely diagnosis information can be obtained, and the possibility can be provided for realizing early accurate screening of atherosclerosis.
Description
Technical Field
The invention relates to the field of nano biological materials, in particular to a VHPK polypeptide modified fluorescent nano liposome drug delivery system, a preparation method and application thereof.
Background
Cardiovascular disease (CVD) is a major killer threatening human health and has become one of the most prevalent diseases in the world. Recent epidemiological investigation shows that the cardiovascular disease prevalence rate in China is still in a continuously rising stage, the total number of people suffering from cardiovascular disease in China is about 3.3 hundred million at present, and the cardiovascular disease mortality rate accounts for the first cause of the overall death of people and is far higher than that of tumors and other diseases. According to speculation, the number of people suffering from cardiovascular diseases in China still rapidly increases in the next 10 years, and the life health of people is seriously threatened. A large number of studies at home and abroad prove that Atherosclerosis (AS) is the common pathological basis of ischemic cardiovascular and cerebrovascular diseases such AS cerebrovascular diseases, coronary heart diseases, peripheral arterial diseases and the like. Since the AS is characterized by the formation of plaque, the growth or rupture of the plaque can cause the blockage and thrombosis of blood vessels, which finally results in serious vascular diseases and ischemia of affected organs, and causes serious harm to the life health of patients. Although the prognosis of high-risk AS patients is extremely aggressive, AS progresses slowly, theoretically providing an important time window for diagnosis and treatment. The occurrence of AS begins at the earliest stage of puberty, but early AS patients generally do not have corresponding clinical symptoms, which hinders early screening and prevention of AS patients, and the early indexes of CVD screened at the present stage, such AS lipoprotein, blood sugar, blood pressure and weight, are just risk factors of monitoring AS, cannot accurately reflect tiny focuses of early AS, have certain hysteresis and have poor sensitivity.
Molecular imaging enables more accurate diagnosis of AS plaque by sensitive detection of vulnerable plaque components, and it also provides more comprehensive tracking and assessment of drug intervention. Near infrared region II (NIR-II, 1100-1700 nm) fluorescence imaging has become a latest hotspot in the research field of non-invasive molecular imaging probes due to the advantages of small background signal interference, high spatial resolution, high imaging sensitivity and the like.
The near infrared fluorescence imaging has high sensitivity, low radiation and low price, and has important significance for diagnosing AS. NIR-II fluorescence imaging is a research hotspot in the technical field of current molecular imaging, and the absorption and scattering of photons and autofluorescence of biological tissues are weakened along with the increase of the wavelength, so that the NIR-II area fluorescence imaging can realize deep and high-quality imaging in vivo, thereby greatly making up for the defects of visible light and NIR-I area fluorescence imaging. Therefore, the development of the molecular imaging probe with the NIR-II fluorescence imaging function is an effective way for realizing early AS molecular detection. Among a plurality of NIR-II molecular imaging probes, benzodithiadiazole (BBTD) is widely used for constructing a D-A-D type organic small molecular NIR-II fluorescent probe as a strong electron-withdrawing group, wherein BBT-2FT has the advantages of narrow band gap, strong photoacoustic signal, high photothermal conversion efficiency, small background signal interference, high imaging sensitivity and the like, and is a latest hotspot in the research field of the NIR-II molecular imaging probes.
There are numerous targets available for active targeting design of nanoparticles in the progression of AS disease. Among them, high expression of vascular cell adhesion factor-1 (VCAM-1) is a reliable target for screening early AS lesions. A number of research reports have demonstrated that VCAM-1 is a potential marker of vascular inflammation and endothelial cell dysfunction and plays an essential role in the monocyte recruitment process of early AS. Meanwhile, VCAM-1 is highly expressed in early AS inflammatory endothelial cells, so VCAM-1 is a reliable target for AS molecular imaging or drug targeted delivery. However, conventional serum detection of VCAM-1 is difficult to determine the tissue source, and biological information at the molecular level cannot be obtained, so that the purpose of early accurate screening is difficult to achieve. Therefore, effective means are found for specifically identifying and tracking VCAM-1 in vivo, the signal change is monitored from a molecular level, the abnormality is detected before the onset of the disease, and the early accurate diagnosis of AS is expected to be realized.
Compared with other nano-carriers (such as micelles, emulsions and the like), the nano-liposome has the irreplaceable advantages of better biocompatibility, higher cell affinity, simultaneous loading of water-soluble and fat-soluble drugs and the like, and is widely applied to a plurality of fields of drug control, gene carriers and the like. Therefore, the BBT-2 FT-loaded nano-liposome is an ideal molecular imaging probe with NIR-II imaging function. In order to improve the active targeting efficiency of VCAM-1, the targeting polypeptide has the advantages of high selectivity, low immunogenicity, good biocompatibility, strong penetrability, easy operation of a synthetic modification method and the like, and is widely used for constructing an active targeting drug delivery system. The VHPKQHR (VHPK) is a specific targeting polypeptide aiming at VCAM-1 screened by researchers by using phage. VHPK is used as a linear polypeptide affinity ligand and has higher sequence homology with the known ligand VLA-4 of VCAM-1 in vivo. Research reports have confirmed that the binding and internalization rate of the VHPK modified nanoparticles by endothelial cells highly expressing VCAM-1 is 20 times higher than that of general nanoparticles. The nano-particle modified by VHPK can efficiently detect the high expression of inflammatory endothelial cells VCAM-1 in a human carotid artery AS sample. Therefore, the high accumulation of the imaging agent at the AS focus part is realized by utilizing the real-time high-definition VHPK functionalized near-infrared fluorescent nano liposome, so that the real-time high-precision molecular imaging of the early AS plaque is realized, more comprehensive and effective real-time diagnosis information is obtained, and the accurate screening of the early AS is expected to be really realized, but no published and reliable scheme is found at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a VHPK polypeptide modified fluorescent nanoliposome drug delivery system, a preparation method and application thereof aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: provided is a VHPK polypeptide modified fluorescent nanoliposome drug delivery system, which comprises: the drug delivery system is a nano liposome carrying a fluorescent probe and modified with VHPK polypeptide, wherein:
the fluorescent probe is a near-infrared first-zone fluorescent nano probe DiR, and the corresponding drug delivery system is recorded as: VHPK-Lipo @ DiR, the molar ratio of each raw material for preparing the drug delivery system VHPK-Lipo @ DiR is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =1 to 4:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.001 to 0.1;
or, the fluorescent probe is a near-infrared two-region fluorescent nano probe BBT-2FT, and the corresponding drug delivery system is recorded as: VHPK-lipo @ BBT-2FT, the molar ratio of raw materials for preparing a drug delivery system VHPK-lipo @ BBT-2FT is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: BBT-2ft =0.5 to 2:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.01 to 1.
Preferably, the molar ratio of the raw materials for preparing the delivery system VHPK-lipo @ DiR is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =2:3:6:4:1:0.2:0.01;
in the drug delivery system VHPK-lipo @ DiR, the mass percent of the nano liposome modified with the VHPK polypeptide is 99.57%, and the mass percent of the fluorescent nano probe DiR is 0.43%.
Preferably, the preparation method of the drug delivery system VHPK-lipo @ DiR comprises the following steps:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method;
2) And carrying out column purification on the VHPK-Lipo @ DiR liposome crude product to obtain the delivery system VHPK-Lipo @ DiR.
Preferably, the preparation method of the drug delivery system VHPK-lipo @ DiR comprises the following steps:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and DiR in chloroform according to a molar ratio, and stirring and hydrating at 40-50 ℃ for 10-60 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7-8;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 10-60 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and reacting for 2-6 hours at room temperature or overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ DiR liposome.
2) And carrying out column purification on the VHPK-lipo @ DiR liposome crude product to obtain the delivery system VHPK-lipo @ DiR.
Preferably, the preparation method of the delivery system VHPK-lipo @ DiR comprises the following steps:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and DiR in chloroform according to a molar ratio, and stirring and hydrating at 45 ℃ for 30 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7.4;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 30 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and standing overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ DiR liposome.
2) And purifying the VHPK-lipo @ DiR liposome crude product by adopting a G-100sephadex column to obtain the delivery system VHPK-lipo @ DiR.
Preferably, the molar ratio of the starting materials for preparing the delivery system VHPK-lipo @ BBT-2FT is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: BBT-2ft =1:2:6:4:1:0.2:0.09;
in a drug delivery system VHPK-lipo @ BBT-2FT, the mass percent of the nano liposome modified with VHPK polypeptide is 99.57%, and the mass percent of the fluorescent nano probe BBT-2FT is 0.43%.
Preferably, the preparation method of the drug delivery system VHPK-lipo @ BBT-2FT comprises the following steps:
1) Preparing a VHPK-lipo @ BBT-2FT liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and BBT-2FT in chloroform according to a molar ratio, and stirring and hydrating at 40-50 ℃ for 10-60 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7-8;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 10-60 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and reacting for 2-6 hours at room temperature or overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ BBT-2FT liposome.
2) And carrying out column purification on the VHPK-lipo @ BBT-2FT liposome crude product to obtain the drug delivery system VHPK-lipo @ BBT-2FT.
Preferably, the preparation method of the drug delivery system VHPK-lipo @ BBT-2FT comprises the following steps:
1) Preparing a VHPK-lipo @ BBT-2FT liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and BBT-2FT in chloroform according to a molar ratio, and stirring and hydrating at 45 ℃ for 30 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7.4;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 30 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and standing overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ BBT-2FT liposome.
2) And purifying the VHPK-lipo @ BBT-2FT liposome crude product by adopting a G-100sephadex column to obtain the drug delivery system VHPK-lipo @ BBT-2FT.
The invention also provides application of the drug delivery system VHPK-lipo @ DiR, which is used for near-infrared one-zone fluorescence imaging at a molecular level.
The invention also provides application of the drug delivery system VHPK-lipo @ BBT-2FT, which is used for near-infrared two-zone fluorescence imaging at a molecular level.
The invention has the beneficial effects that:
the invention adopts a film dispersion method to respectively prepare VHPK polypeptide modified near-infrared first-region DiR and near-infrared second-region BBT-2FT fluorescent probe liposomes. The nano drug delivery system can specifically target the highly expressed VCAM-1 of the atherosclerotic vascular endothelium through the polypeptide VHPK. Therefore, the high accumulation of the near-infrared fluorescence imaging probe at the target position can be realized through the specific identification of the highly expressed VCAM-1 of the atherosclerotic vascular endothelial tissue, so that the near-infrared one-region and two-region optical imaging diagnosis of atherosclerosis can be realized respectively, more comprehensive and more timely diagnosis information can be obtained, and the possibility can be provided for realizing the early accurate screening of atherosclerosis.
Drawings
FIG. 1 is the result of transmission electron microscope observation of VHPK polypeptide modified near-infrared region-I fluorescent nano-liposome drug delivery system VHPK-lipo @ DiR in example 1;
FIG. 2 is a graph showing the results of surface potential of VHPK polypeptide-modified near-infrared region-fluorescent nanoliposome delivery system VHPK-lipo @ DiR in example 1;
FIG. 3 is a photograph of the VHPK polypeptide modified near infrared one-region fluorescent nanoliposome drug delivery system VHPK-lipo @ DiR in example 1 imaged in NIR-I;
FIG. 4 is the transmission electron microscope observation result of VHPK polypeptide modified near-infrared two-region fluorescent nanoliposome drug delivery system VHPK-lipo @ BBT-2FT in example 2;
FIG. 5 is a graph showing the results of surface potential of VHPK polypeptide-modified near-infrared two-domain fluorescent nanoliposome delivery system VHPK-lipo @ BBT-2FT in example 2;
FIG. 6 is a photograph of NIR-II imaging of VHPK polypeptide modified near infrared two-region fluorescent nanoliposome delivery system VHPK-lipo @ BBT-2FT in example 2.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The test methods used in the following examples are all conventional methods unless otherwise specified. The material reagents and the like used in the following examples are commercially available unless otherwise specified. The following examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a VHPK polypeptide modified fluorescent nanoliposome drug delivery system, which comprises: the drug delivery system is a nano liposome carrying a fluorescent probe and modified with VHPK polypeptide, wherein:
the fluorescent probe is a near-infrared first-zone fluorescent nano probe DiR, and the corresponding drug delivery system is recorded as: VHPK-lipo @ DiR, the molar ratio of each raw material for preparing a drug delivery system VHPK-lipo @ DiR is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =1 to 4:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.001 to 0.1;
or, the fluorescent probe is a near-infrared two-region fluorescent nano probe BBT-2FT, and the corresponding drug delivery system is recorded as: VHPK-lipo @ BBT-2FT, the molar ratio of raw materials for preparing a drug delivery system VHPK-lipo @ BBT-2FT is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: BBT-2ft =0.5 to 2:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.01 to 1.
The VHPKQHR is a specific targeting polypeptide for accurately positioning atherosclerotic plaques, and the binding and internalization rate of the atherosclerotic plaques to the VHPKQHR modified nanoparticles is 20 times higher than that of general nanoparticles. TCEP is a very effective thiol reducing agent, is widely used as a quantitative reducing agent of disulfide bonds in protein chemistry and proteomics research, and can well protect the disulfide bonds in VHPKQHR to exert specific targeting characteristics of the TCEP.
Wherein DPPC, cholesterol and DSPE-PEG2k are common formulation components for forming liposome phospholipid bilayers. DPPC is a synthetic phospholipid which is widely applied in the field of pharmaceutical preparations, and can be used for liposome or temperature-sensitive liposome, and pharmaceutical preparations such as special lipid microspheres, pharmaceutical composites and the like. Cholesterol is an important component of liposome preparation, plays an important role in maintaining liposome structure, and can affect membrane permeability, fluidity and interactions between phospholipids, and the cholesterol content is related to the close packing of phospholipids, the degree of cooperation between hydrophobic space and phospholipids, which may result in structural changes. DSPE-PEG2k also has the effect of stabilizing liposomes and, in addition, prolongs blood circulation time as it reduces the time for the liposomes to be cleared by the reticuloendothelial system. DSPE-PEG2K-MAL is one of reactive phospholipid PEG reagents, and can react with sulfydryl (thiol, -SH) of VHPKQHR, so that the polypeptide and the liposome are stably linked and modified.
Among them, diR, as a type of environmentally sensitive fluorescent dye, is mainly used for labeling cell membranes and other lipid-soluble biological structures, and once entering cells, they gradually diffuse in the plasma membranes of the cells, and can uniformly stain the whole cells under the condition of optimal concentration. The DiR can be used for biomedical imaging of a near infrared first region and has a structural formula shown as the following formula 1:
among them, biomedical imaging of BBT-2FT in the near infrared second region (NIR-II, 1000-1700 nm) can fully improve the spatial-temporal resolution (20 ms and 25 mm) and the penetration depth (3 cm) because it has the advantages of less scattering, negligible tissue absorption and minimized auto-fluorescence effect, thus obtaining better image quality.
In a structural system of a fluorescent nano liposome drug delivery system, a phospholipid bilayer structure of a liposome similar to a cell membrane is uniformly wrapped on the outer side of the nano liposome, a fat-soluble DiR or BBT-2FT probe is embedded between hydrophobic phospholipid bilayers, and hydrophilic cavities are formed inside nanoparticles wrapped by the phospholipid bilayers, so that hydrophilic drugs can be loaded.
VHPKQHR: TCEP: and (2) DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =1 to 4:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.001 to 0.1;
the invention also provides a preparation method of the drug delivery system VHPK-lipo @ DiR, which comprises the following steps:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method:
1-1) mixing DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and DiR (3-9: 2 to 6:0.5 to 2:0.1 to 1:0.001 to 0.1) is dissolved in 1 to 6mL of trichloromethane, and is stirred and hydrated for 10 to 60 minutes at the temperature of between 40 and 50 ℃ to obtain a liposome solution;
1-2) during liposome hydration, TCEP solution (obtained by dissolving TCEP in deionized water) is prepared and NaHCO is used 3 Adjusting the pH value to 7-8;
1-3) the VHPK polypeptide was dissolved in PBS solution and then the molar ratio (VHPKQHR: TCEP =1 to 4: 1-5) mixing the obtained VHPK polypeptide solution with a TCEP solution, and reacting at room temperature for 10-60 minutes to reduce the disulfide bond of the VHPK polypeptide to obtain an activated VHPK polypeptide solution;
among them, the mechanism of reducing disulfide bond by TCEP is that the lone electron pair carried by its central atom "P" can form coordinate covalent bond with oxygen atom and has reducibility, and the resulting product has almost no side reaction with other amino acids, and is the best accepted reducing agent for disulfide bond;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), reacting for 2-6 hours at room temperature or overnight at 4 ℃ to obtain a crude product of the VHPK-lipo @ DiR liposome, wherein the reaction principle is that maleimide in a liposome component DSPE-PEG2K-MAL can react with sulfydryl (thiol, -SH) of VHPKQHR to form stable sulfydryl ether bond, so that the polypeptide and the liposome are stably linked and modified.
2) Purifying the VHPK-lipo @ DiR liposome crude product by adopting a G-100sephadex column, removing the components such as unbound VHPK polypeptide, liposome or DiR and the like, and finally obtaining the drug delivery system VHPK-lipo @ DiR.
The invention also provides a preparation method of the drug delivery system VHPK-lipo @ BBT-2FT, which specifically comprises the following steps:
1) Preparing a VHPK-lipo @ BBT-2FT liposome crude product by adopting a film dispersion method:
1-1) mixing DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and BBT-2FT (3-9: 2 to 6:0.5 to 2:0.1 to 1:0.001 to 0.1) is dissolved in 1 to 5mL of trichloromethane, and is stirred and hydrated for 10 to 60 minutes at the temperature of between 40 and 50 ℃ to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7-8;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 10-60 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and reacting for 2-6 hours at room temperature or overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ BBT-2FT liposome.
2) Purifying the VHPK-lipo @ BBT-2FT liposome crude product by adopting a G-100sephadex column, removing unbound VHPK polypeptide, liposome or BBT-2FT and other components, and finally obtaining the drug delivery system VHPK-lipo @ BBT-2FT.
The invention also provides application of the drug delivery system VHPK-lipo @ DiR, which is used for near-infrared one-zone fluorescence imaging at a molecular level, and the NIR-I fluorescence imaging of atherosclerosis at the molecular level can be realized through specific binding of the surface modified VHPK and the VCAM-1 protein highly expressed in the vascular endothelium of atherosclerosis.
The invention also provides application of the drug delivery system VHPK-lipo @ BBT-2FT, which is used for near-infrared two-region fluorescence imaging at a molecular level, and NIR-II fluorescence imaging of atherosclerosis at the molecular level is realized through specific binding of the surface modified VHPK and the VCAM-1 protein highly expressed in the vascular endothelium of atherosclerosis.
The invention adopts a film dispersion method to respectively prepare VHPK polypeptide modified near-infrared first-region DiR and near-infrared second-region BBT-2FT fluorescent probe liposomes. The nano drug delivery system can specifically target the highly expressed VCAM-1 of the atherosclerosis vascular endothelium through the polypeptide VHPK. Therefore, the high accumulation of the near-infrared fluorescence imaging probe at the target part can be realized through the specific recognition of the VCAM-1 with high expression of the atherosclerotic vascular endothelial tissue, so that the near-infrared first-region and second-region optical imaging diagnosis of atherosclerosis can be respectively realized, more comprehensive and more timely diagnosis information can be obtained, and the possibility can be provided for realizing the early accurate screening of atherosclerosis.
The foregoing is a general idea of the present invention, and detailed examples are provided below to further explain the present invention.
Example one delivery System VHPK-Lipo @ DiR
In the drug delivery system VHPK-lipo @ DiR, the mass percent of the nano liposome modified with the VHPK polypeptide is 99.57 percent, and the mass percent of the fluorescent nano probe DiR is 0.43 percent; the molar ratio of each raw material for preparing a drug delivery system VHPK-lipo @ DiR is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =2:3:6:4:1:0.2:0.01;
the preparation method comprises the following steps:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method:
1-1) mixing DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and DiR (6: 4:1:0.2:0.01 Dissolving in 3mL of chloroform at normal temperature, and stirring and hydrating at 45 ℃ for 30 minutes to obtain a liposome solution;
1-2) during liposome hydration, TCEP is dissolved in deionized water to prepare TCEP solution, and NaHCO is used 3 Adjusting the pH value to 7.4;
1-3) dissolving the VHPK polypeptide in PBS solution, and then mixing the resulting VHPK polypeptide solution with TCEP solution in molar ratio (VHPKQHR: TCEP =2: 3) Reacting at room temperature for 30 minutes to destroy disulfide bonds to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1) and standing overnight at 4 ℃ to obtain a crude product of the VHPK-Lipo @ DiR liposome.
2) Purifying the VHPK-lipo @ DiR liposome crude product by adopting a G-100sephadex column to obtain the drug delivery system VHPK-lipo @ DiR.
Preparing a control group Lipo @ DiR (namely the nano liposome which does not modify VHPK but carries the fluorescent probe) by adopting a thin film dispersion method: DPPC, cholesterol, DSPE-PEG2K-MAL and DiR (molar ratio 6; finally, G-100sephadex column is adopted for purification to obtain a control group lipo @ DiR.
The relevant test experiments were performed as follows:
1. adopting a transmission electron microscope to observe the appearance of the VHPK-Lipo @ DiR nanoparticle:
weighing a certain amount of nanoparticles, dispersing the nanoparticles in trichloromethane to obtain a nanoparticle solution with the concentration of 0.1mg/mL, dripping a sample on a copper net covered with a carbon film, airing at room temperature, and observing that the shape of the nanoparticles is regular and round through a transmission electron microscope (80 kV) and the particle size is about 175nm. As shown in fig. 1.
2. The Zeta potential of the VHPK-lipo @ DiR Nano drug delivery system at normal temperature is measured by a Nano-ZS90 type laser Nano particle analyzer, and the average value of each sample is obtained by repeating the measurement for 3 times. VHPK-Lipo @ DiR (VHPK) and Lipo @ DiR (Ctrl) nanoparticles were determined to have surface potentials of about-0.5 mV and-2.9 mV. As shown in fig. 2.
3. The in vivo NIR-I targeting exploration of VHPK-lipo @ DiR aiming at atherosclerosis mainly comprises the following four steps:
firstly, constructing an early AS animal model, adopting a 6-week-old male SPF-grade ApoE-/-gene knockout mouse and a C57 mouse, and feeding high-fat feed (containing 1% of cholesterol, 15% of lard, 0.25% of sodium cholate, 5% of egg yolk powder, 2.5% of sugar and basic diet) for 8 weeks to construct the early AS animal model.
And secondly, respectively setting two groups of VHPK-lipo @ DiR and lipo @ DiR, respectively injecting 0.1mL of the nano preparation into each group by adopting a tail vein injection method, and carrying out NIR-I optical imaging at different time points (0 and 8 h). Utilizing near infrared fluorescence spectrometerDefining the coincidence degree of the vascular autofluorescence signal and NIR-I fluorescence imaging signal, and observing the early diagnosis efficiency of VHPK-lipo @ DiR on AS with ApoE -/- Constructing an AS model animal by using a knockout mouse, taking nano liposome Lipo @ DiR (Ctrl) of unmodified VHPK AS a control, and researching the tissue distribution condition of the VHPK-Lipo @ DiR nanoparticles in vivo by using an NIR-I fluorescence imaging instrument, wherein the research result shows that: the fluorescence intensity of the VHPK-Lipo @ DiR (VHPK) nanoparticles at the AS part is time-dependent, the more the nanoparticles accumulate at the AS part along with the prolonging of time, the maximum fluorescence signal is shown at the AS part after 8h of tail vein injection (figure 3A), and the fluorescence intensity can be maintained to 24h. Compared with the control group Lipo @ DiR (Ctrl), the VHPK-Lipo @ DiR (VHPK) nanoparticle has a longer blood circulation period, is lower in nonspecific phagocytosis consumption rate by the immune system of the body, and can actively target and accumulate at the AS part more, which indicates that VHPK-Lipo @ DiR (VHPK) has obvious AS targeting (figure 3B).
Thirdly, obtaining the angiocarpy by adopting an aorta in vitro experiment method, taking a model mouse at each time point (0 and 8 h), and injecting 3% sodium pentobarbital (60 mg kg) into the abdominal cavity -1 ). After the mouse is anesthetized, the mouse is placed on an ice bag and the thoracic cavity is dissected, 0.8-1 mL of blood is rapidly obtained from the left ventricle, the mouse is rapidly transferred into an EP tube and is centrifuged for 15min at 4 ℃ and 3500 Xg, and the blood plasma is separated and stored in a refrigerator at 4 ℃ for later use. After fully perfusing with pre-cooled PBS buffer and 4% paraformaldehyde (flow rate 2 mL. Min.) -1 ) The thoracic aorta was removed (starting from the heart root to the bilateral iliac branches) with the aid of a dissecting microscope, microsurgical scissors and forceps. Whole body perfusion and aortic dissection of mice were performed on ice.
And fourthly, assessing the specific position of the atherosclerotic plaque in the blood vessel by adopting an aorta in vitro gross oil red staining method, mixing an Oil Red O (ORO) staining reagent and distilled water according to the proportion of 3:2, and filtering by using a slow filter membrane for later use. The aortic arch to iliac vessels were dissected longitudinally with the intima facing outward, and the specimens were stained with ORO solution for 30 minutes and destained in 80% isopropanol at room temperature for 30 seconds, and finally washed with PBS for 5 minutes. The extent of the entire aortic plaque was assessed by gross ORO staining and the results of the study showed that intra-aortic AS plaques occurred predominantly in the coronary arteries (junction of heart and thoracic aorta) and at the bifurcation of the hepatoabdominal aorta (fig. 3C), which is highly consistent with lipo @ dir (VHPK) at the site of actively targeted enrichment of aortic AS plaques under NIR-i fluorescence imager observation (fig. 3B and C). The result further provides a solid experimental basis for the visual diagnosis and treatment of the VHPK polypeptide modified near-infrared region-I fluorescent nano liposome drug delivery system.
Example two drug delivery System VHPK-lipo @ BBT-2FT
In the drug delivery system VHPK-lipo @ DiR, the mass percent of the nano liposome modified with the VHPK polypeptide is 99.57 percent, and the mass percent of the fluorescent nano probe DiR is 0.43 percent; the molar ratio of the raw materials of the drug delivery system VHPK-lipo @ BBT-2FT is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: BBT-2ft =1:2:6:4:1:0.2:0.09;
the preparation method of the drug delivery system VHPK-lipo @ BBT-2FT comprises the following steps:
1) Preparing a VHPK-lipo @ BBT-2FT liposome crude product by adopting a film dispersion method:
1-1) mixing DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and BBT-2FT (6: 4:1:0.2:0.09 Dissolving in 3mL of chloroform at normal temperature, and stirring and hydrating at 45 ℃ for 30 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7.4;
1-3) dissolving the VHPK polypeptide in PBS solution, and then mixing the resulting VHPK polypeptide solution with TCEP solution in molar ratio (VHPKQHR: TCEP =1: 2) Reacting at room temperature for 30 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and standing overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ BBT-2FT liposome.
2) And purifying the VHPK-lipo @ BBT-2FT liposome crude product by adopting a G-100sephadex column to obtain the drug delivery system VHPK-lipo @ BBT-2FT.
Preparation of control group lipo @ BBT-2FT: the method adopts a thin film dispersion method to prepare lipo @ BBT-2FT. DPPC, cholesterol, DSPE-PEG2K-MAL and BBT-2FT (6; purification was performed by using G-100sephadex column to obtain control group lipo @ BBT-2FT.
The relevant test experiments were performed as follows:
1. the apparent morphology of the VHPK-lipo @ BBT-2FT nanoparticles is observed by adopting a transmission electron microscope:
weighing a certain amount of nanoparticles, dispersing the nanoparticles in trichloromethane to obtain a nanoparticle solution with the concentration of 0.1mg/mL, dripping a sample on a copper net covered with a carbon film, airing at room temperature, and observing that the nanoparticles are regular and round in shape and have the particle size of about 165nm through a transmission electron microscope (80 kV). As shown in fig. 4.
2. The Zeta potential of the VHPK-lipo @ BBT-2FT Nano drug delivery system at normal temperature is measured by adopting a Nano-ZS90 type laser Nano particle size analyzer, and the average value of each sample is obtained by repeating the measurement for 3 times. VHPK-Lipo @ BBT-2FT (VHPK) and Lipo @ BBT-2FT (Ctrl) nanoparticles were determined to have surface potentials of about-2.5 mV and-2.2 mV. As shown in fig. 5.
3. The NIR-II in vivo targeting exploration of the VHPK-lipo @ BBT-2FT aiming at atherosclerosis mainly comprises the following four steps:
firstly, constructing an early AS animal model by adopting 6-week-old male SPF-grade ApoE -/- The knockout mice were fed with high-fat diet (containing 1% cholesterol, 15% lard, 0.25% sodium cholate, 5% egg yolk powder, 2.5% sugar and basal diet) for 8 weeks to construct early-stage AS animal model.
And secondly, respectively setting two groups of VHPK-lipo @ BBT-2FT and lipo @ BBT-2FT, respectively injecting 0.1mL of the nano preparation into each group by adopting a tail vein injection method, and carrying out NIR-II optical imaging at different time points (0 and 8 h). The near-infrared fluorescence spectrometer is used for determining the coincidence degree of the vascular autofluorescence signal and the NIR-II fluorescence imaging signal, the early diagnosis efficiency of VHPK-lipo @ BBT-2FT on AS is mainly observed, and ApoE is used -/- An AS model animal is constructed by a knockout mouse, a nano liposome Lipo @ BBT-2FT (Ctrl) of unmodified VHPK is used AS a control, an NIR-II fluorescence imager is adopted to research the tissue distribution condition of the VHPK-Lipo @ BBT-2FT nanoparticles in vivo, and the research result shows that: VHPK-lipo @ BBT-The fluorescence intensity of 2FT (VHPK) nanoparticles at the AS part is time-dependent, the more nanoparticles accumulate at the AS part with the time, the maximum fluorescence signal is shown at the AS part after 8h of tail vein injection (figure 6A), and the fluorescence intensity can be maintained to 24h. Compared with a control group Lipo @ BBT-2FT (Ctrl), the VHPK-Lipo @ BBT-2FT (VHPK) nanoparticle has a longer blood circulation period, is lower in nonspecific phagocytosis consumption rate by an immune system of an organism, and can actively target and accumulate at an AS part more, which indicates that the VHPK-Lipo @ BBT-2FT (VHPK) has obvious AS targeting (figure 6B).
Thirdly, obtaining the cardiovascular disease by adopting an aorta in vitro experiment method, taking a model mouse at each time point (0 and 8 h), and injecting 3% sodium pentobarbital (60 mg kg) into the abdominal cavity -1 ). After anaesthetizing, the mouse is put on an ice bag and dissected to open the thoracic cavity, 0.8-1 mL of blood is quickly taken from the left ventricle, the mouse is quickly transferred into an EP tube and is centrifuged for 15min at 4 ℃ and 3500 Xg, and the blood plasma is separated and stored in a refrigerator at 4 ℃ for later use. After fully perfusing with pre-cooled PBS buffer and 4% paraformaldehyde (flow rate 2 mL. Min.) -1 ) The thoracic aorta was removed (starting from the heart root to the bilateral iliac branches) with the aid of a dissecting microscope, microsurgical scissors and forceps. Whole body perfusion and aortic dissection of mice were performed on ice.
And fourthly, assessing the specific position of the atherosclerotic plaque in the blood vessel by adopting an aorta in vitro gross oil red staining method, mixing an Oil Red O (ORO) staining reagent and distilled water according to the proportion of 3:2, and filtering by using a slow filter membrane for later use. The aortic arch to iliac vessels were dissected longitudinally with the intima facing outward, and the specimens were stained with ORO solution for 30 minutes and destained in 80% isopropanol at room temperature for 30 seconds, and finally washed with PBS for 5 minutes. The extent of the entire aortic plaque was assessed by gross ORO staining and the results of the study showed that intra-aortic AS plaques occurred predominantly in the coronary arteries (junction of heart and thoracic aorta) and in the bifurcation of the hepatoabdominal aorta (fig. 6C), which is highly consistent with lipo @ bbt-2FT (VHPK) at the site of actively targeted enrichment of aortic AS plaques under NIR-ii fluorescence imager observation (fig. 6B and C). The result further provides a solid experimental basis for the visual diagnosis and treatment of the VHPK polypeptide modified near-infrared two-region fluorescent nano liposome drug delivery system.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the details shown in the description and the examples, which are set forth, but are fully applicable to various fields of endeavor as are suited to the particular use contemplated, and further modifications will readily occur to those skilled in the art, since the invention is not limited to the details shown and described without departing from the general concept as defined by the appended claims and their equivalents.
Claims (10)
1. A VHPK polypeptide-modified fluorescent nanoliposome drug delivery system, which is characterized by comprising: the drug delivery system is a nano liposome carrying a fluorescent probe and modified with VHPK polypeptide, wherein:
the fluorescent probe is a near-infrared one-zone fluorescent nano probe DiR, and the corresponding drug delivery system is recorded as: VHPK-lipo @ DiR, the molar ratio of each raw material for preparing a drug delivery system VHPK-lipo @ DiR is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =1 to 4:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.001 to 0.1;
or, the fluorescent probe is a near-infrared two-region fluorescent nano probe BBT-2FT, and the corresponding drug delivery system is recorded as: VHPK-lipo @ BBT-2FT, the molar ratio of raw materials for preparing a drug delivery system VHPK-lipo @ BBT-2FT is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: BBT-2ft =0.5 to 2:1 to 5:3 to 9:2 to 6:0.5 to 2:0.1 to 1:0.01 to 1.
2. The VHPK polypeptide-modified fluorescent nanoliposome delivery system according to claim 1, wherein the molar ratio of the raw materials for preparing the delivery system VHPK-lipo @ dir is VHPKQHR: TCEP: DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: diR =2:3:6:4:1:0.2:0.01;
in the drug delivery system VHPK-lipo @ DiR, the mass percent of the nano liposome modified with the VHPK polypeptide is 99.57%, and the mass percent of the fluorescent nano probe DiR is 0.43%.
3. The VHPK polypeptide modified fluorescent nanoliposome delivery system according to claim 1 or 2, wherein the method of preparation of said delivery system VHPK-lipo @ dir comprises the steps of:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method;
2) And carrying out column purification on the VHPK-Lipo @ DiR liposome crude product to obtain the delivery system VHPK-Lipo @ DiR.
4. The VHPK polypeptide modified fluorescent nanoliposome delivery system according to claim 3, wherein said delivery system VHPK-lipo @ dir is prepared by a method comprising the steps of:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and DiR in chloroform according to a molar ratio, and stirring and hydrating at 40-50 ℃ for 10-60 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7-8;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 10-60 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and reacting for 2-6 hours at room temperature or overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ DiR liposome.
2) And carrying out column purification on the VHPK-lipo @ DiR liposome crude product to obtain the delivery system VHPK-lipo @ DiR.
5. The VHPK polypeptide-modified fluorescent nanoliposome delivery system according to claim 4, wherein the preparation method of the delivery system VHPK-lipo @ DiR comprises the following steps:
1) Preparing a VHPK-lipo @ DiR liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and DiR in chloroform according to a molar ratio, and stirring and hydrating at 45 ℃ for 30 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7.4;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 30 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and standing overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ DiR liposome.
2) Purifying the VHPK-lipo @ DiR liposome crude product by adopting a G-100sephadex column to obtain the drug delivery system VHPK-lipo @ DiR.
6. The VHPK polypeptide-modified fluorescent nanoliposome delivery system according to claim 1, wherein the molar ratio of the raw materials for preparing the delivery system VHPK-lipo @ bbt-2FT is VHPKQHR: TCEP: and (2) DPPC: cholesterol: DSPE-PEG2k: DSPE-PEG2K-MAL: BBT-2ft =1:2:6:4:1:0.2:0.09;
in a drug delivery system VHPK-lipo @ BBT-2FT, the mass percent of the nano liposome modified with the VHPK polypeptide is 99.57%, and the mass percent of the fluorescent nano probe BBT-2FT is 0.43%.
7. The VHPK polypeptide modified fluorescent nanoliposome delivery system according to claim 1 or 6, wherein the method for preparing said delivery system VHPK-lipo @ bbt-2FT comprises the steps of:
1) Preparing a VHPK-lipo @ BBT-2FT liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and BBT-2FT in chloroform according to a molar ratio, and stirring and hydrating at 40-50 ℃ for 10-60 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7-8;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 10-60 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and reacting for 2-6 hours at room temperature or overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ BBT-2FT liposome.
2) And performing column purification on the VHPK-lipo @ BBT-2FT liposome crude product to obtain the delivery system VHPK-lipo @ BBT-2FT.
8. The VHPK polypeptide-modified fluorescent nanoliposome delivery system according to claim 7, wherein said delivery system VHPK-lipo @ bbt-2FT is prepared by a method comprising the steps of:
1) Preparing a VHPK-lipo @ BBT-2FT liposome crude product by adopting a film dispersion method:
1-1) dissolving DPPC, cholesterol, DSPE-PEG2K, DSPE-PEG2K-MAL and BBT-2FT in chloroform according to a molar ratio, and stirring and hydrating at 45 ℃ for 30 minutes to obtain a liposome solution;
1-2) preparation of TCEP solution with NaHCO 3 Adjusting the pH value to 7.4;
1-3) dissolving VHPK polypeptide in a PBS solution, then mixing the obtained VHPK polypeptide solution with a TCEP solution according to a molar ratio, and reacting at room temperature for 30 minutes to obtain an activated VHPK polypeptide solution;
1-4) adding the activated VHPK polypeptide solution into the liposome solution prepared in the step 1-1), and standing overnight at 4 ℃ to obtain a crude product of VHPK-lipo @ BBT-2FT liposome.
2) And purifying the VHPK-lipo @ BBT-2FT liposome crude product by adopting a G-100sephadex column to obtain the delivery system VHPK-lipo @ BBT-2FT.
9. Use of a delivery system according to any of claims 1 or 2-5, VHPK-lipo @ dir, for near infrared one-zone fluorescence imaging at the molecular level.
10. Use of a delivery system according to any one of claims 1 or 6 to 8, which is VHPK-lipo @ bbt-2FT for near infrared two-zone fluorescence imaging at the molecular level.
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