CN114099718B - Collagen-targeted contrast agent with multi-mode image detection and drug loading treatment effects, and preparation method and application thereof - Google Patents
Collagen-targeted contrast agent with multi-mode image detection and drug loading treatment effects, and preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
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- A—HUMAN NECESSITIES
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- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- A61K9/5123—Organic compounds, e.g. fats, sugars
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- 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 relates to the technical field of nano drug-loaded contrast agents, in particular to a collagen-targeted contrast agent with multi-mode image detection and drug-loaded treatment effects, and a preparation method and application thereof. The contrast agent comprises a lipid shell membrane, wherein a gold nanorod, triazamidine and perfluoropentane are wrapped in the lipid shell membrane, and CNA35 is connected to the surface of the lipid shell membrane. The technical problem that a multi-mode molecular developer aiming at myocardial fibrosis is lacking at present can be solved by the scheme. The contrast agent with the effect of precisely positioning the lesion part and the functions of ultrasonic imaging and photoacoustic imaging can be applied to medical practice operation of myocardial fibrosis multi-mode development.
Description
Technical Field
The invention relates to the technical field of nano drug-loaded contrast agents, in particular to a collagen-targeted contrast agent with multi-mode image detection and drug-loaded treatment effects, a preparation method and application thereof.
Background
Myocardial fibrosis (CF) is the pathological basis for heart failure and malignant arrhythmia in various heart diseases, and the main pathological changes are excessive deposition of matrix components among cells, such as collagen fibers (I, III type is the main) and the like. The excessive deposition of matrix components between cells and the severity of myocardial fibrosis are closely related to the prognosis of patients, and early recognition and timely blocking of the progression of myocardial fibrosis are of great significance. Although imaging examinations represented by Magnetic Resonance Imaging (MRI) and drugs represented by renin-angiotensin system (RAS) inhibitors play an important role in diagnosis and treatment of myocardial fibrosis, five-year mortality in heart failure patients is still up to 50%. Therefore, it is necessary to search for new detection patterns and therapeutic approaches for myocardial fibrosis.
The detection of myocardial fibrosis mainly includes imaging examination, serum metabolite detection, and myocardial biopsy. Serological examination can be used for primarily assessing the presence or absence of myocardial fibrosis, but positioning and quantification cannot be performed; the positive rate of myocardial biopsy is related to the material sampling part, is an invasive operation, and has less clinical application. Imaging examinations are an important means of assessing myocardial fibrosis, with Cardiac Magnetic Resonance (CMR) examinations being the most common. Gadolinium delayed enhancement cardiac magnetic resonance imaging (LGE-MRI) is currently considered a gold standard for noninvasive detection of focal myocardial fibrosis. For diffuse fibrosis changes, the extracellular volume (ECV) of the myocardium can be calculated by CMR T1 imaging, indirectly assessing the extent of myocardial fibrosis. However, the CMR examination has high requirements on image acquisition and analysis and the heart rate and respiratory coordination of patients, and part of patients cannot perform the CMR examination due to claustrophobia, renal insufficiency, or metal implants in the body, so that the clinical use of the CMR examination is limited. The CT examination can also evaluate diffuse fibrosis by ECV measurement, but for focal fibrosis, the conventional CT imaging has low signal-to-noise ratio, and is difficult to distinguish normal myocardium from fibrosis myocardium. PET-CT can be used for identifying viable myocardium and necrotic myocardium, and has certain prompt significance for myocardial fibrosis. However, PET-CT images have low resolution and radioactivity, and are only applied to myocardial fibrosis with few detections. Ultrasonic speckle tracking imaging techniques, strain rate imaging, and ultrasonic tissue characterization based on ultrasonic back-scattering techniques can be quantitatively analyzed by related software to find differences in the fibrillated myocardium from normal myocardium. But lack of specificity, and the heterogeneity of the measured values of different ultrasonic instruments, limits clinical applications.
At present, the clinic still lacks a medicament for specifically treating myocardial fibrosis, and the medicament aiming at RAS can reverse myocardial fibrosis, but has side effects of depressurization and the like, and partial patients cannot tolerate the medicament, so that the sufficient use of the medicament is limited. Therefore, it is an urgent need to develop a therapeutic means or medicament capable of effectively treating myocardial fibrosis and avoiding side effects.
Research shows that molecular image diagnosis can break through the bottleneck of the conventional medical imaging technology as the forefront technology of medical images, and the pathological changes of diseases are specifically revealed on the molecular and cellular level of living bodies, so that the accuracy of diagnosis is greatly improved. The method is applied to the fields of early diagnosis of diseases such as tumors, development of accurate medicaments and the like. In recent years, molecular imaging technology has also been developed rapidly in the field of cardiovascular diseases, and is mainly focused on the identification of vulnerable plaques of atherosclerosis and the detection of myocardial viability. In the aspect of detection of myocardial fibrosis, the basic research of preparing CT, MRI and nuclear medicine related molecular developer by applying specific molecular probes is available, and the existence, degree and prognosis of myocardial fibrosis can be evaluated in a targeted specificity mode, but the method is still in the early stage of animal experiments and clinic. However, the single developing modes have certain limitations, such as high spatial resolution of CT detection, but low signal-to-noise ratio; ultrasonic detection is convenient in real time, but evaluation of the three-dimensional space structure is not good enough. Along with the development of multi-mode medical imaging technology and the proposal of a new concept of realizing the diagnosis and treatment integration of the tumor field by the multifunctional nano-particles, the preparation of the diagnosis and treatment integration multifunctional drug-loaded contrast agent becomes the front edge and the hot spot of the research in the biomedical field, targets the drug for fibrosis cardiac muscle, can reduce the side effect of the drug and is hopeful to become a new method for treating the cardiac muscle fibrosis. However, the current lack of multi-modal molecular imaging agents and related therapeutic agents for myocardial fibrosis does not meet the requirements for the overall assessment and treatment of myocardial fibrosis.
Disclosure of Invention
The invention aims to provide a collagen-targeted contrast agent with multi-modal image detection and drug loading treatment effects, so as to solve the technical problem of lack of multi-modal molecular developers and targeted therapeutic drugs aiming at myocardial fibrosis at present.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a collagen-targeted contrast agent with multi-mode image detection and drug loading treatment effects comprises a lipid shell membrane, wherein gold nanorods and perfluoropentane are wrapped in the lipid shell membrane, and CNA35 is connected to the surface of the lipid shell membrane.
The scheme also provides a preparation method of the collagen-targeted contrast agent with multi-mode image detection and drug loading treatment effects, which comprises the following steps:
s1: DPPC, DSPE-PEG2000-NH 2 Dispersing shell membrane raw materials consisting of DSPG and CH in chloroform to obtain a mixture I;
s2: adding a mixed solution containing gold nanorod solution and triazamidine or a solution containing gold nanorod into the mixture I to obtain a mixture II, and performing primary sound vibration treatment on the mixture II;
s3: performing rotary evaporation on the mixture II subjected to the first sound vibration treatment to obtain a film;
s4: dissolving the film by using a buffer solution to obtain a mixture III;
S5: adding perfluoropentane into the mixture III under ice bath condition, and performing secondary sound vibration treatment to obtain suspension;
s6: centrifuging the suspension to obtain a solid phase; resuspending the solid phase with a solution of 4-morpholinoethanesulfonic acid to obtain a resuspension;
s7: activating CNA35 using EDC and NHS to obtain a CNA35 solution;
s8: adding CNA35 solution into the heavy suspension, and incubating under ice bath and shaking conditions to obtain a connection product;
s9: and cleaning the connection product, centrifuging to obtain nanoparticles, and re-suspending the nanoparticles by using a buffer solution to obtain a nanoparticle solution.
The scheme also provides application of the collagen-targeted contrast agent with multi-mode image detection and drug loading treatment effects in preparation of the therapeutic drug for myocardial fibrosis.
The principle and the advantages of the scheme are as follows:
according to the technical scheme, I, III type collagen specific binding protein CNA35 in the development process of myocardial fibrosis is used as a molecular probe, simultaneously a gold nanorod is used as a photoacoustic development material, phospholipid with better biocompatibility is used as a coating material, and finally the myocardial fibrosis multi-modal developed contrast agent is prepared. Wherein, the phospholipid adopted by the contrast agent shell membrane has the same components as the cell membrane in the body, and has no toxic or side effect on organisms; the targeting of CNA35 is good; the perfluoropentane (PFP) can be dissolved in blood through dispersion and is discharged through expiration, so that the perfluoropentane does not participate in biochemical degradation in the body, and the stability is good; compared with gold nanoparticles, the gold nanorods have better stability and biocompatibility, the gold nanorods have larger optical cross section than the gold nanoparticles with the same mass, and are more stable in the manufacturing process and in-vivo action process. The contrast agent prepared by the scheme has an X-ray absorption coefficient higher than that of the iodine contrast agent under the same concentration, and the cytotoxicity experiment also proves the safety of the invention. The ultrasonic examination has wide application in the aspect of cardiovascular diseases, the perfluoropentane has good acoustic phase change property, the gold nanorod has good photoacoustic imaging property, the perfluoropentane and the gold nanorod are simultaneously loaded by liposome, the specific polypeptide CNA35 of target myocardial fibrosis is connected on a lipid shell membrane, and the contrast agent can meet the requirements of ultrasonic enhancement/photoacoustic multi-mode imaging under the excitation of LIFU.
In the preparation process of the contrast agent, a method different from the prior art of sound vibration treatment is used, so that the encapsulation rate of the gold nanorods is greatly improved. The traditional negative pressure rotary evaporation-double sound vibration method is used for preparing drug-loaded liposome, generally, the drug to be wrapped and the like are firstly subjected to sound vibration with liquid phase change materials PFP, PFH and the like to be wrapped, and then the obtained product and the lipid film suspension subjected to rotary evaporation are subjected to sound vibration to be wrapped for the second time, the method is commonly used when the liposome wraps water-soluble drugs, but for gold nanorods, the metal materials are far more than water-soluble small molecular drugs in volume and mass, and the method is easy to leak out the cash nanorods or display incomplete wrapping in the wrapping process, so that the encapsulation rate is obviously reduced. The method selects a method of pre-sound vibration before rotary steaming, fully and uniformly mixing gold nanorods with negative charges on the surfaces and phospholipid groups in a lipid environment, comprehensively contacting with lipid shell membranes, and adsorbing the gold nanorods with the negative charges on the surfaces of the shell membranes by utilizing the characteristics of the negative charges on the surfaces. The second sound vibration can wrap the gold nano rod while wrapping the PFP, and through two actions, the gold nano rod can be wrapped in the liposome inner capsule, and some of the gold nano rod exists in the lipid bilayer membrane, so that the encapsulation rate of the gold nano rod in the liposome is increased to 33.6%.
In-vitro multi-mode imaging experiments show that the prepared contrast agent has the characteristics of good ultrasonic enhancement imaging and photoacoustic imaging, and the imaging capability is gradually enhanced along with the increase of the concentration. In-vivo multi-mode imaging experiments indicate that the enhanced signal can be detected by ultrasound 30min after the intravenous injection of the targeted nanoparticles, the enhanced signal has a good positioning effect on pathological tissues, the photoacoustic instrument detects a relatively strong photoacoustic signal after 90min, and the size of the displayed pathological range corresponds to that of pathological sections made of corresponding tissues. In vivo multi-mode imaging shows that the prepared targeting nanoparticle can very sensitively locate a lesion site through ultrasonic imaging, and the range of photoacoustic imaging corresponds to the lesion range. Therefore, the contrast agent can be used as a contrast agent with the effect of precisely positioning the lesion part and the functions of ultrasonic imaging and photoacoustic imaging, and is applied to medical practice operation of myocardial fibrosis multi-mode development.
Further, the lipid shell membrane is also internally wrapped with triazamidine.
The scheme also provides application of the collagen-targeted contrast agent with multi-mode image detection and drug loading treatment effects in preparation of the contrast agent for myocardial fibrosis.
By adopting the technical scheme, the loaded medicine triazamidine (Diminazene aceturate, DIZE) is released to a fibrosis area in a targeted manner after the membrane is ruptured under the action of LIFU irradiation, so that the effect of targeted treatment on myocardial fibrosis is achieved. The nano particles prepared by the scheme contain DIZE, and the DIZE can prevent myocardial hypertrophy caused by pressure load, relieve myocardial injury induced by ischemia, and have multiple cardiovascular protection effects of improving myocardial fibrosis, improving diastolic function, resisting inflammation, reducing pulmonary artery pressure and the like. Recent studies have shown that the protective effect of DIZE on myocardial infarction in rats is even better than that of the ACE inhibitor enalapril. Meanwhile, the DIZE has stable property, low price and easy acquisition, and is a very promising drug for treating myocardial fibrosis. However, ACE2 agonist diameter has significant side effects through oral administration to the gastrointestinal tract, and has associated side effects at conventional therapeutic doses, limiting its clinical use. The DIZE is integrated in the nanoparticle in the scheme, and intravenous administration is carried out, so that the side effect of the DIZE is reduced to a great extent. Therefore, the nanoparticles of the present scheme can be applied as a therapeutic drug for myocardial fibrosis in the practical operation of treatment of related diseases.
Further, the lipid shell membrane comprises DPPC and DSPE-PEG2000-NH with the mass ratio of 20:7:3:4 2 DSPG and CH.
The contrast agent shell membrane of the scheme adopts phospholipid, has the same components as in-vivo cell membranes, and has no toxic or side effect on organisms. The component content of the lipid shell membrane is very important for the encapsulation efficiency of the gold nanorods, and the mass ratio of 20:7:3:4 can ensure that the stability and the fluidity of the lipid bilayer meet the requirements, reduce the leakage of the gold nanorods in the shell membrane and increase the stability of the liposome.
Further, the particle size was 311.+ -. 4.5nm, and the potential was-38.1.+ -. 0.3mV.
The particle size of the contrast agent of the scheme is about 311+/-4.5 nm, the contrast agent can pass through pathological capillary gaps (about 700 nm) and has the potential of-38.1+/-0.3 mV, and the contrast agent can ensure that the prepared nano particles are relatively stable and are not easy to precipitate too fast.
Further, in S1, DPPC, DSPE-PEG2000-NH 2 The mass ratio of DSPG to CH is 20:7:3:4; the dosage ratio of the shell membrane raw material to the chloroform is 34mg:15ml.
By adopting the technical scheme, the stability and the fluidity of the lipid bilayer can be ensured to meet the requirements by adopting the mass ratio, the leakage of the gold nanorods in the shell membrane can be reduced, and the encapsulation rate of the gold nanorods can be improved. The mass ratio of DPPC, DSPE-PEG2000-NH2, DSPG and CH is very important to improve the encapsulation efficiency of the gold nanorods, and the inventor tries other ratios once, and discovers that the encapsulation efficiency of the gold nanorods does not meet the subsequent application requirements.
Further, in S2, the volume ratio of the gold nanorod solution to chloroform is 0.5:15; the concentration of gold nanorods in the gold nanorod solution was 1.0mg/ml.
The gold nanorods with the above dosage can ensure ideal encapsulation rate, and avoid material waste caused by excessive use of the gold nanorods.
Further, in S2, the power of the first vibration treatment is 60w, the time is 1min, and the intermittent vibration method is adopted.
By adopting the technical scheme, the condition of the sound vibration treatment can ensure that gold nanorods with negative charges carried on the surfaces are fully and uniformly mixed with phospholipid groups in a lipid environment, the gold nanorods are in full contact with lipid shell membranes, and the gold nanorods are adsorbed on the surfaces of the shell membranes by utilizing the characteristics of the negative charges on the surfaces.
Further, in S5, the power of the second sound vibration treatment is 60w, the time is 3min, and a discontinuous sound vibration method is adopted; the volume ratio of perfluoropentane to chloroform is 0.2:15.
by adopting the technical scheme, the intermittent sound vibration method (and the whole-course ice bath method) is adopted, so that on one hand, the phase change of liquid fluorocarbon caused by temperature rise is avoided, and on the other hand, the influence of heat generated by sound vibration on the quality of balling is avoided. Besides, the second sound vibration can wrap the gold nanorods while wrapping the perfluoropentane, so that the encapsulation rate of the gold nanorods is improved.
Further, in S5, the power of the second sound vibration treatment is 60w, the time is 3min, and a discontinuous sound vibration method is adopted; the volume ratio of perfluoropentane to chloroform is 0.2:15; in S6, the concentration of the 4-morpholinoethanesulfonic acid solution is 0.1M, and the pH value is 8.0; in S7, the mass ratio of EDC to NHS is 3:1, and the pH value of the CNA35 solution is 5.2; in S8, the pH of the CNA35 solution was adjusted from 5.2 to 8.0, and the CNA35 solution was added to the resuspension.
By adopting the technical scheme, the mass ratio of the 4-morpholinoethanesulfonic acid solution, EDC and NHS and the pH value of the CNA35 solution can ensure the full activation of the CNA35 and improve the connection rate of the CNA35 and the lipid shell membrane. In addition, the carbodiimide method is a mature method for connecting carboxyl and amino, and can realize the connection of the high CNA35 and the lipid shell membrane more efficiently and stably.
In conclusion, the multi-mode contrast agent with the particle size of about 311+/-4.5 nm can pass through pathological capillary gaps (about 700 nm), has the potential of about-38.1+/-0.3 mV, and can ensure that the preparation of nano particles is relatively stable and is not easy to precipitate too quickly. The transmission electron microscope can see that the nano particles are black spherical, the morphology is more regular, and the particle size of the prepared nano particles is not obviously changed within 1 week under the condition of 4 degrees through detection verification. CNA35 is connected to the surface detected by flow cytometry and confocal microscopy, and a foundation is laid for subsequent targeting development. When the low-intensity focused ultrasound (LIFU) acts for 2w and 4min, the phase change of perfluoropentane liquid gas in the nanoparticles can be excited, obvious bubbles can be generated under a microscope, a basis is provided for the subsequent ultrasonic contrast imaging, the gold nanorod content in the nanoparticles measured by an ICP method is higher, and the subsequent photoacoustic imaging effect is ensured. The scheme provides the contrast agent with the effect of precisely positioning the lesion part and the functions of ultrasonic imaging and photoacoustic imaging, and can be applied to medical practice operation of myocardial fibrosis multi-mode development.
Drawings
FIG. 1 is a schematic diagram of the structure of CNA35-GDP@NPs according to example 1 of the present invention.
FIG. 2 is an appearance and transmission electron microscope image of CNA35-GP@NPs and CNA35-GDP@NPs of example 3 of the present invention.
FIG. 3 shows the results of the Malvern laser particle size/potentiometer test of example 3 of the present invention.
FIG. 4 shows the result of the CNA35-GDP@NPs targeting experiment (immunofluorescence) of example 4 of the present invention.
FIG. 5 is an image of ultrasound imaging (effect of different excitation power and time on imaging effect) of nanoparticles of example 5 of the present invention under in vitro LIFU excitation.
FIG. 6 is an image of ultrasound imaging of nanoparticles of example 5 of the present invention under in vitro LIFU excitation (effect of different nanoparticle concentrations on imaging effect).
Fig. 7 is an in vitro photoacoustic imaging image of nanoparticles of example 5 of the present invention.
FIG. 8 is an in vivo ultrasound imaging image of nanoparticles of example 5 of the present invention.
Fig. 9 is an image of in vivo photoacoustic imaging of nanoparticles of example 5 of the present invention.
Fig. 10 is an ultrasonic image of cardiac function evaluation after treatment of myocardial fibrosis and a graph showing changes in cardiac Ejection Fraction (EF) and left ventricular short axis reduction rate (FS) index before and after treatment according to example 6 of the present invention.
FIG. 11 is a graph showing the collagen occupancy ratio analysis of pathological Masson staining and myocardial fibrosis areas after treatment of myocardial fibrosis in example 6 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
Example 1:
a specific preparation method of a collagen-targeted contrast agent (CNA 35-GNR/DIZE/PFP@NPs, CNA35-GDP@NPs for short) with multi-modal image detection and drug-loaded treatment effects is as follows:
(1) DPPC (dipalmitoyl phosphorylcholine), DSPE-PEG2000-NH 2 (amino-linked polyethylene glycol 2000 modified distearoyl phosphatidylethanolamine), DSPG (1, 2-distearoyl-sn-glycerol-3-phosphorylglycerol) and CH (cholesterol) were added to a 50ml centrifuge tube at a mass ratio of 20:7:3:4 together with 34mg, and 15ml of chloroform was added for dissolution to form a mixture I.
(2) To the above organic solvent, 0.5ml of a mixed aqueous solution was added, wherein the concentration of GNR was 1.0mg/ml (gold nanorod, aspect ratio was 3, siamitraz organism) and the concentration of triazamidine was 12mg/ml. And (3) carrying out sonic vibration emulsification on the suspension in the centrifuge tube once by using a sonic vibrator, wherein the sonic vibration power is 60w and the time is 1min (5 s on;5s off), so as to form a mixture II.
(3) Transferring the sound vibration back complex emulsion to a round bottom flask, and placing the round bottom flask on a rotary steaming instrument for rotary steaming to form an adherence film, wherein parameters of the rotary steaming instrument are as follows: the temperature is 50 ℃, the rotating speed is 60rpm, and the time is 30min.
(4) After evaporation, the round bottom flask was removed, 6ml of PBS solution was added, and the flask was rinsed with shaking in an ultrasonic cleaner until the bottom film eluted, forming a suspension (mixture III) which was transferred to a 10ml EP tube.
(5) 200ul PFP (perfluoropentane) is added to the suspension obtained in the step (4) under the whole ice bath condition, and the suspension is vibrated by a vibrator for 3min (5 s on;5s off) under the power of 60 w.
(6) And (3) carrying out low-temperature centrifugal cleaning on the suspension obtained after the second sound vibration for three times, wherein the parameters of a centrifugal machine are as follows: the nanoparticle obtained after the third centrifugation is resuspended in 6ml of pH8.0 MES solution (4-morpholinoethanesulfonic acid, concentration 0.1M) at 4℃at 8000rpm for 5min and placed in a refrigerator at 4℃for further use.
(7) The carbodiimide method is used for linking targets, EDC/NHS (1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) with the mass ratio of 3:1 is weighed, 4ml of MES (0.1M) with the pH of 5.2 is used for dissolving, 400ul of CNA35 (I, III type collagen specific binding protein) solution with carboxyl at the tail end is added, and shaking incubation is carried out on a shaking table for 2 hours, so as to activate the carboxyl at the tail end of the CNA35, and the rotation speed of the shaking table is 150 revolutions per minute, thus obtaining CNA35 solution.
(8) And (3) regulating the pH of the CNA35 solution obtained in the step (7) to 8.0, then adding the heavy suspension obtained in the step (6), and incubating for 12 hours under ice bath conditions by shaking at low temperature.
(9) And (3) carrying out low-temperature centrifugal cleaning on the obtained solution for three times, and re-suspending the solution with 6ml of PBS after the third centrifugation to obtain the targeted nanoparticle CNA35-GDP@NPs solution.
The contrast agent with the multi-mode image detection and drug loading treatment effects for targeting collagen is shown in the figure 1, and comprises a lipid shell membrane, wherein gold nanorods, DIZE and PFP are wrapped in the shell membrane, and CNA35 is connected to the surface of the shell membrane.
Example 2:
based on the embodiment, the specific preparation method for preparing the collagen-targeted contrast agent with multi-mode image detection (CNA 35-GNR/PFP@NPs, CNA35-GP@NPs for short) by independently wrapping the nano gold rod is as follows:
(1) DPPC (dipalmitoyl phosphorylcholine), DSPE-PEG2000-NH 2 (amino-linked polyethylene glycol 2000 modified distearoyl phosphatidylethanolamine), DSPG (1, 2-distearoyl-sn-glycerol-3-phosphorylglycerol) and CH (cholesterol) were added to a 50ml centrifuge tube at a mass ratio of 20:7:3:4 together with 34mg, and 15ml of chloroform was added for dissolution to form a mixture I.
(2) To the organic solvent was added 0.5ml of GNR (concentration: 1.0mg/ml, solvent: water). And (3) carrying out sonic vibration emulsification on the suspension in the centrifuge tube once by using a sonic vibrator, wherein the sonic vibration power is 60w and the time is 1min (5 s on;5s off), so as to form a mixture II.
(3) Transferring the sound vibration back complex emulsion to a round bottom flask, and placing the round bottom flask on a rotary steaming instrument for rotary steaming to form an adherence film, wherein parameters of the rotary steaming instrument are as follows: the temperature is 50 ℃, the rotating speed is 60rpm, and the time is 30min.
(4) After evaporation, the round bottom flask was removed, 6ml of PBS solution was added, and the flask was rinsed with shaking in an ultrasonic cleaner until the bottom film eluted, forming a suspension (mixture III) which was transferred to a 10ml EP tube.
(5) 200ul PFP (perfluoropentane) is added to the suspension obtained in the step (4) under the whole ice bath condition, and the suspension is vibrated by a vibrator for 3min (5 s on;5s off) under the power of 60 w.
(6) And (3) carrying out low-temperature centrifugal cleaning on the suspension obtained after the second sound vibration for three times, wherein the parameters of a centrifugal machine are as follows: the nanoparticle obtained after the third centrifugation is resuspended in 6ml of pH8.0 MES solution (4-morpholinoethanesulfonic acid, concentration 0.1M) at 4℃at 8000rpm for 5min and placed in a refrigerator at 4℃for further use.
(7) The carbodiimide method is used for linking targets, EDC/NHS (1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) with the mass ratio of 3:1 is weighed, 4ml of MES (0.1M) with the pH of 5.2 is used for dissolving, 400ul of CNA35 (I, III type collagen specific binding protein) solution with carboxyl at the tail end is added, and shaking incubation is carried out on a shaking table for 2 hours, so as to activate the carboxyl at the tail end of the CNA35, and the rotation speed of the shaking table is 150 revolutions per minute, thus obtaining CNA35 solution.
(8) And (3) regulating the pH of the CNA35 solution obtained in the step (7) to 8.0, then adding the heavy suspension obtained in the step (6), and incubating for 12 hours under ice bath conditions by shaking at low temperature.
(9) And (3) carrying out low-temperature centrifugal cleaning on the obtained solution for three times, and re-suspending the solution with 6ml of PBS after the third centrifugation to obtain the targeted nanoparticle CNA35-GP@NPs solution.
Example 3:
characterization and characterization tests were performed on CNA35-GDP@NPs and CNA35-GP@NPs prepared in examples 1 and 2, and the experimental results are as follows:
(one) aiming at the nanoparticles prepared in the embodiment 1, the appearance of the nanoparticle solution subjected to PBS (phosphate buffer solution) is grey yellow, no rapid precipitation and development are carried out after the solution is stood, the shape is spherical under observation of a microscope, the distribution is uniform, the size is uniform, and a single CNA35-GDP@NPs is in a black spherical structure under a transmission electron microscope, and the shape is more regular, as shown in figure 2. Aiming at the nanoparticles prepared in the embodiment 2, the appearance of the nanoparticle solution subjected to PBS (phosphate buffer solution) resuspension is grey white, no rapid precipitation and development are carried out after the solution is stood, the observation form under a microscope is spherical, the distribution is uniform, the size is uniform, and a single CNA35-GP@NPs can be seen to be in a black spherical structure under a transmission electron microscope, and the form is more regular, as shown in figure 2.
And (II) detecting the particle size of CNA35-GDP@NPs by a Malvern laser particle size/potentiometer to be 311+/-4.5 nm, wherein the potential is-38.1+/-0.3 mV, the particle size of CNA35-GP@NPs is 299+/-3.0 nm, and the potential is-43.0+/-0.8 mV. The results of the detection of CNA35-GDP@NPs of example 1 and CNA35-GP@NPs of example 2 are shown in FIG. 3.
(III) aiming at the CNA35-GDP@NPs of example 1, dyeing a lipid shell membrane by using a DiI fluorescent dye in the process of preparing nanoparticles, and dyeing the CNA35 by using FITC, wherein the prepared nanoparticles are observed under a laser confocal microscope, and the red fluorescent shell membrane with DiI and the CNA35 with FITC green fluorescence are well fused, so that the CNA35 is well connected to the surface of the lipid shell membrane of the nanoparticles, and meanwhile, the continuous target rate is 83+/-2.1% by using a flow cytometry.
Measuring the content of gold nanorods in CNA35-GDP@NPs by using an ICP method, wherein the detection result is as follows: gold nanometerThe standard curve equation for rod GNR is y= 143861.4980 x+10024.0478, r 2 =0.9999, from which the encapsulation efficiency of GNR in nanoparticles was calculated to be 33.6%. The content of the gold nanorods in the CNA35-GP@NPs is measured by adopting the same method, and the detection result is as follows: the standard curve equation of the gold nanorod GNR is y= 170175.6244 x+18499.1582, r 2 =0.9997, from which the encapsulation efficiency of GNR in nanoparticles was calculated to be 34.3%. Indicating that the encapsulation efficiency of the gold nanorods is not greatly influenced by drug loading or not.
The traditional negative pressure rotary evaporation-double sound vibration method is used for preparing drug-loaded liposome, generally, the drug to be wrapped and the like are firstly subjected to sound vibration with liquid phase change materials PFP, PFH and the like to be wrapped, and then the obtained product and the lipid film suspension subjected to rotary evaporation are subjected to sound vibration to be wrapped for the second time, the method is commonly used when the liposome wraps water-soluble drugs, but for gold nanorods, the metal materials are far more than water-soluble small molecular drugs in volume and mass, and the phenomenon that the cash nanorods leak out or are not fully wrapped easily in the wrapping process of the method, so that the encapsulation rate is obviously reduced. Therefore, how to effectively encapsulate gold nanorods in the nanoparticles of the present solution is a technical key point for implementing the present solution, and the inventors conducted a great deal of tests on the encapsulation method of gold nanorods, with the following details:
Experiment 1: CNA35-GP@NPs were prepared by the method of example 2, except that in (1), 34mg of DPPC, DSPE-PEG2000-NH2, DSPG, CH were added to a 50ml centrifuge tube at a mass ratio of 10:4:3:3, and dissolved in 15ml of chloroform. The encapsulation efficiency (aiming at gold nanorods) of CNA35-GP@NPs obtained in the experiment is 21.6%.
Experiment 2: CNA35-GP@NPs were prepared by the method of example 2, in (1), DPPC, DSPE-PEG2000-NH 2 34mg of DSPG and CH are added into a 50ml centrifuge tube according to the mass ratio of 20:7:3:4, and 15ml of chloroform is added for dissolution. The difference is that the experiment is not carried out with the sound vibration treatment of (2), the rotary steaming step of (3) is directly carried out, the mixed solution (mixture I) obtained in (1) is put into a round bottom flask and put on a rotary steaming instrument for rotary steaming to form a wall-attached film, and the aqueous solution of GNR is directly added into the elution of (4) to obtain a suspensionIn the mixture III), hydration is carried out for 10min and then (5). The encapsulation efficiency (aiming at gold nanorods) of CNA35-GP@NPs obtained in the experiment is 6.08%.
Experiment 3: CNA35-GP@NPs were prepared by the method of reference example 2, except that in (1), DPPC, DSPE-PEG2000-NH 2 The mass ratio of DSPG and CH is 20:7:3:2, and the encapsulation rate (aiming at gold nanorods) of CNA35-GP@NPs obtained in the experiment is 27.0%.
Experiment 4:
(1) DPPC (dipalmitoyl phosphorylcholine), DSPE-PEG2000-NH 2 (amino-linked polyethylene glycol 2000 modified distearoyl phosphatidylethanolamine), DSPG (1, 2-distearoyl-sn-glycerol-3-phosphorylglycerol) and CH (cholesterol) were added to a 50ml centrifuge tube at a mass ratio of 20:7:3:4 together with 34mg, and dissolved in 15ml of chloroform.
(2) Carrying out rotary steaming treatment on the mixed solution in the step (1), and carrying out rotary steaming instrument parameters: the film is obtained at 50 ℃ and at a rotating speed of 60rpm for 30 min.
(3) Adding 6ml of PBS solution into the membrane, placing the membrane into an ultrasonic cleaner for vibration cleaning until the membrane at the bottom of the bottle is eluted, forming a membrane suspension, and transferring the membrane suspension to a 10ml EP tube.
(4) A mixed aqueous solution of 0.5ml of GNR and triazamidine was mixed with 200ul of PFP (perfluoropentane), followed by a first sonication, power of shaking 60w, time 1min (5 s on;5s off), to obtain a drug mixture.
(5) And (3) adding the drug mixed solution in the step (4) into the film suspension obtained in the step (3) under the whole-process ice bath condition, and carrying out sound vibration by using a sound vibration instrument, wherein the sound vibration power is 60w, and the time is 3min (5 s on;5s off).
(6) And (3) carrying out low-temperature centrifugal cleaning on the suspension obtained after the second sound vibration for three times, wherein the parameters of a centrifugal machine are as follows: the nanoparticle obtained after the third centrifugation is resuspended in 6ml of pH8.0 MES solution (4-morpholinoethanesulfonic acid, concentration 0.1M) at 4℃at 8000rpm for 5min and placed in a refrigerator at 4℃for further use.
(7) The carbodiimide method is used for linking targets, EDC/NHS (1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) with the mass ratio of 3:1 is weighed, 4ml of MES (0.1M) with the pH of 5.2 is used for dissolving, 400ul of CNA35 (I, III type collagen specific binding protein) solution with carboxyl at the tail end is added, and shaking incubation is carried out on a shaking table for 2 hours, so as to activate the carboxyl at the tail end of the CNA35, and the rotation speed of the shaking table is 150 revolutions per minute, thus obtaining CNA35 solution.
(8) And (3) adjusting the pH of the CNA35 solution obtained in the step (7) to 8.0, then adding the heavy suspension obtained in the step (6), and incubating overnight under ice bath conditions by shaking at low temperature.
(9) And (3) carrying out low-temperature centrifugal cleaning on the obtained solution for three times, and re-suspending the solution with 6ml of PBS after the third centrifugation to obtain the targeted nanoparticle CNA35-GDP@NPs solution.
The encapsulation efficiency (aiming at gold nanorods) of CNA35-GDP@NPs obtained in the experiment is 12.48%.
The amount of DPPC used in experiment 1 was too small, and GNR, DPPC, DSPE-PEG2000-NH were not used in experiment 2 2 Mixing DSPG and CH in advance by sound vibration, wherein the CH content in experiment 3 is too low, mixing GNR (gold nanorod) aqueous solution and PFP in advance in experiment 4, and performing sound vibration treatment to obtain nanoparticles with encapsulation rates of 21.6%, 6.08%, 27.0% and 12.48%, respectively. The encapsulation rate of the nanoparticle obtained by the scheme is greatly different from that of the nanoparticle obtained by the scheme. In conclusion, the preparation method adopting the technical scheme can greatly improve the encapsulation rate of the gold nanorods in the nanoparticles, and the inventor analyzes the reason for the phenomenon as follows: the method selects a method of pre-sound vibration before rotary steaming, fully and uniformly mixing gold nanorods with negative charges on the surfaces and phospholipid groups in a lipid environment, comprehensively contacting with lipid shell membranes, and adsorbing the gold nanorods with the negative charges on the surfaces of the shell membranes by utilizing the characteristics of the negative charges on the surfaces. The second sound vibration can wrap the gold nano rod while wrapping the PFP, and through two actions, the gold nano rod can be wrapped in the liposome inner capsule, and some gold nano rods exist on the lipid bilayer membrane, and the encapsulation rate of the gold nano rod obtained by comparing with the traditional method is 12.48 percent, so that the encapsulation rate of the gold nano rod in the liposome is increased to 33.6 percent. On the other hand, the proportion of the phospholipid component is increased, so that the lipid bilayer can be stabilized, the fluidity of the membrane is reduced, the leakage of the gold nanorods inside the shell membrane is reduced, and the stability of the liposome is improved.
And fifthly, detecting the encapsulation efficiency of the medicine DIZE (aiming at CNA35-GDP@NPs of example 1) by adopting a high performance liquid phase method, wherein the detection result is that the encapsulation efficiency of the medicine is 23.78%, and the medicine loading rate is 3.5%.
(sixth) stability of nanoparticle preparation of example 1: the particle size of the nanoparticles does not change significantly when the nanoparticles are stored at 4 ℃ for 1 week.
(seventh) optical observation of morphological changes during heating of nanoparticle CNA35-GDP@NPs of example 1:
the detection result shows that the liquid-gas phase change of the nano particles occurs along with the extension of the heating time at 45 ℃, the volume is obviously increased, when the nano particles are heated for 10min, the obviously increased phase change nano particles are observed under the mirror, and part of bubbles are broken.
(eighth) biosafety detection of CNA35-GDP@NPs of example 1:
the primary SD rat heart fibroblast is extracted for culture, and CCK8 experiment shows that the activity of the cells is not obviously reduced along with the increase of the concentration in the range of 0.74mg/ml to 5.9mg/ml of the nanoparticle concentration. Healthy SD rats are injected with CNA35-GDP@NPs nanoparticle solution through tail vein, blood is collected at different time points, the rats are sacrificed after 72 hours to take important viscera (heart, liver, spleen, lung and kidney) for pathological HE staining, and the blood sample is subjected to blood test to obtain normal liver and kidney functions and myocardial enzyme indexes, and compared with healthy rats, the blood sample is not statistically different (P < 0.01).
Example 4
The targeting ability of CNA35-GDP@NPs prepared in example 1 was examined in this example, and the specific contents are as follows:
reference methods establish models of myocardial fibrosis following myocardial infarction in SD rats (reference: ferriera JCB, campos JC, qvit N, et al A selective inhibitor of mitofusin 1-. Beta. IIPKC association improves heart failure outcome in rates. Nat Commun. 2019;10 (1): 329. Published 2019 Jan 18. Doi:10.1038/s 41467-018-08276-6). Nanoparticles were classified into targeted groups CNA35-GDP@NPs and non-targeted groups GNR/DIZE/PFP@NPs (GDP@NPs) (i.e., nanoparticles not linked to CNA 35) by in vitro and in vivo observation of in vitro targeting.
(1) In vitro targeting experimental study of CNA 35-GDP@NPs:
taking a rat myocardial pathological tissue which is successfully modeled as an instant frozen section, incubating 200ul of a target nanoparticle CNA35-GDP@NPs solution simultaneously dyed with DiI (lipid shell membrane) and FITC (CNA 35, green fluorescence) and a non-target nanoparticle GDP@NPs solution dyed with DiI (lipid shell membrane) with the frozen section for half an hour respectively, staining the cell nucleus with DAPI, and observing under a confocal microscope to observe that CNA35-GDP@NPs dyed with more red-green fluorescence are accumulated around the cell nucleus, while the GDP@NPs group (nanoparticles not connected with CNA 35) is not accumulated around the cell nucleus with red fluorescent nanoparticles.
(2) In vivo targeting experimental study of CNA 35-GDP@NPs:
the number of myocardial fibrosis model mice which are successfully modeled is selected, 1ml (5.9 mg/ml) of CNA35-GDP@NPs solution and 1ml of non-targeted GDP@NPs solution are respectively injected through tail veins, the rats are sacrificed after 30min, heart pathological tissue frozen sections are taken under the condition of light shielding, nuclei are stained by DAPI, CNA35-GDP@NPs which are more stained with red and green fluorescence are observed to be aggregated around the nuclei under a confocal microscope, and red fluorescent nanoparticles are not aggregated around the nuclei in the GDP@NPs group, as shown in figure 4.
The in vitro targeting experiment result shows that the targeting group nanoparticle can be connected to the myocardial fibrosis pathological tissue part through CNA35, which indicates that CNA35 is well connected to the surface of the nanoparticle in the preparation process, and the binding capacity with heart I, III type collagen is not affected. The in vivo targeting experiment shows that the targeting nanoparticle can actively target the collagen fiber aggregation site through the capillary wall of the heart lesion region by the EPR effect, thereby providing a good foundation for the subsequent targeting and developing of the lesion region.
Example 5
This example investigated the ultrasound/photoacoustic imaging of the multimodal contrast agent of CNA35-gdp@nps prepared in example 1, with the following specific experimental results:
1. Ultrasonic imaging under in vitro LIFU excitation
Preparing 3% agarose gel hole model, and performing the steps of the preparation method at different working time (1 min, 2min, 3min,4min, 5 min) and different LIFU excitation powers (1 w/cm 2 、2w/cm 2 、3w/cm 2 ) The imaging capability of the nanoparticle B mode and the contrast mode is evaluated under the condition, and the ultrasonic gray value under the enhancement mode is measured and analyzed by using DFY software, and the result is shown in figure 5. CNA35-GDP@NPs contrast medium with different concentrations (0.37 mg/ml, 0.74mg/ml, 1.47mg/ml, 2.95mg/ml, 5.90 mg/ml) was added to each well, and irradiated (2W/cm) with LIFU meter 2 4 min), using an Esaote MyLab90 ultrasonic diagnostic apparatus, using a B-mode and contrast mode for contrast, the results are shown in fig. 6, with increasing concentration, the intensity of the ultrasound image increases.
2. In vitro photoacoustic imaging
A3% agarose gel well model was prepared, and CNA35-GDP@NPs contrast medium of different concentrations (0.37 mg/ml, 0.74mg/ml, 1.47mg/ml, 2.95mg/ml, 5.9 mg/ml) was added to each well, and photoacoustic imaging obtained by equipping an lz250 probe with a Vevo LAZR photoacoustic imaging apparatus (visual sonic Co., canada). The excitation wavelength was 705nm and the photoacoustic signal intensity was quantified by the Vevo Zr software analysis, as shown in fig. 7, with increasing concentration, the nanoparticle photoacoustic signal intensity increased and had a good linear relationship with concentration.
3. In vivo ultrasound imaging
The myocardial fibrosis rat model after myocardial infarction which is successfully modeled is selected and divided into two groups, and the CNA35-GDP@NPs and the non-targeted GDP@NPs nanoparticle solutions are injected through tail veins, and 30min before injection, 30min after injection and 30min+LIFU (2 w/cm) 2 4 min) after excitation, ultrasound B-mode and Contrast mode imaging was performed, as shown in fig. 8, without significant enhancement of ultrasound signal before the injection of the targeted nanoparticles and before the excitation of the LIFU, and enhancement of ultrasound signal at lesions after the irradiation of LIFU.
4. In vivo photoacoustic imaging
The myocardial fibrosis rat model after myocardial infarction which is successfully modeled is selected and divided into two groups, CNA35-GDP@NPs and non-targeted GDP@NPs nanoparticle solutions are injected through tail veins, and after 90min, photoacoustic imaging is obtained by using a Vevo LAZR photoacoustic imaging device, wherein the excitation wavelength is 705nm. As shown in fig. 9, a clear photoacoustic signal was visible at the lesion 90min after the injection of the targeting nanoparticle.
Example 6
This example describes the study of the multimode contrast agent of CNA35-GDP@NPs prepared in example 1 in the local LIFU excitation treatment of SD rat myocardial fibrosis.
The experiment adopts a myocardial fibrosis model caused by the myocardial infarction of SD rats, the rats are randomly divided into 5 groups, 5 groups each, and the rats are divided into blank groups, and myocardial fibrosis treatment effects are observed after 30 days of treatment by intravenous injection of DIZE groups, GDP@NPs groups, CNA35-GDP@NPs groups and CNA35-GDP@NPs+LIFU acting groups.
(1) Therapeutic effects on myocardial fibrosis
After 30 days of treatment, the myocardial fibrosis SD rats grouped above were examined for heart morphology by cardiac ultrasound (MyLab 90, esaote, italy) and assessed for cardiac improvement, and an ultrasound image is shown in fig. 10. The cardiac Ejection Fraction (EF) and left ventricular short axis shortening (FS) index changes before and after treatment are shown in fig. 10. The result shows that the cardiac function of rats in the treatment group irradiated by the targeted CNA35-GDP@NPs+LIFU is obviously improved, and the anti-myocardial fibrosis effect is obvious.
(2) Assessment of collagen fraction as stained MASSON sections of cardiac pathological tissue
After anesthesia, the treated SD rats were stained with Masson to observe the degree of myocardial fibrosis, and myocardial fibrosis collagen was analyzed by Image J software, and the results are shown in FIG. 11. The Masson stained image of heart tissue is shown in FIG. 11, in order from left to right, as blank, venous DIZE, GDP@NPs, CNA35-GDP@NPs and CNA35-GDP@NPs+LIFU acting groups. A statistical plot of collagen area fraction is shown below in fig. 11. FIG. 11 shows that the targeted CNA35-GDP@NPs+LIFU irradiation treatment groups had smaller fibrotic staining areas than the other groups and the collagen deposition was significantly improved compared to the control group. Meanwhile, compared with the single DIZE rat myocardial fibrosis degree of the vein, the target group treatment CNA35-GDP@NPs+LIFU is obviously improved, and the collagen ratio is statistically different (P < 0.01).
In-vitro multi-mode imaging experiments show that the prepared nanoparticles have the characteristics of good ultrasonic enhanced imaging and photoacoustic imaging, and the developing capability is gradually enhanced along with the increase of the concentration. In-vivo multi-mode imaging experiments indicate that the enhanced signal can be detected by ultrasound 30min after the intravenous injection of the targeted nanoparticles, the enhanced signal has a good positioning effect on pathological tissues, the photoacoustic instrument detects a relatively strong photoacoustic signal after 90min, and the size of the displayed pathological range corresponds to that of pathological sections made of corresponding tissues. In vivo multi-mode imaging shows that the prepared targeting nanoparticle can very sensitively locate a lesion site through ultrasonic imaging, the range of photoacoustic imaging corresponds to the lesion range, and the active targeting capability of the targeting nanoparticle is proved to be a main reason for accurately locating the lesion site through non-targeting group comparison. In the aspect of targeted drug delivery accurate treatment, experiments show that the prepared drug-loaded nanoparticle has the characteristic of targeted drug delivery accurate, and under the excitation of local LIFU irradiation, the concentrated targeted release of the drug is realized, compared with the systemic drug delivery mode with the same dosage, the targeted nanoparticle drug delivery can avoid the side effect of the drug on a systemic system and organs as much as possible, and the local concentration of the drug is enriched, so that the therapeutic effect of the drug in a lesion area on myocardial fibrosis can be amplified.
The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (9)
1. A collagen-targeted contrast agent with multi-modal image detection and drug-loading therapeutic effects, characterized in that: the lipid shell membrane is internally wrapped with gold nanorods, perfluoropentane and optionally triazamidine, and the surface of the lipid shell membrane is connected with CNA35;
the lipid shell membrane comprises the components of DPPC, DSPE-PEG2000-NH2, DSPG and CH in a mass ratio of 20:7:3:4;
the preparation method comprises the following steps:
s1: dispersing shell membrane raw materials consisting of DPPC, DSPE-PEG2000-NH2, DSPG and CH in chloroform to obtain a mixture I;
S2: adding a mixed solution containing gold nanorod solution and triazamidine or a solution containing gold nanorod into the mixture I to obtain a mixture II, and performing primary sound vibration treatment on the mixture II;
s3: performing rotary evaporation on the mixture II subjected to the first sound vibration treatment to obtain a film; the power of the first sound vibration treatment is 60w, the time is 1min, and a discontinuous sound vibration method is adopted;
s4: dissolving the film by using a buffer solution to obtain a mixture III;
s5: adding perfluoropentane into the mixture III under ice bath condition, and performing secondary sound vibration treatment to obtain suspension; the power of the second sound vibration treatment is 60w, the time is 3min, and a discontinuous sound vibration method is adopted;
s6: centrifuging the suspension to obtain a solid phase; resuspending the solid phase with a solution of 4-morpholinoethanesulfonic acid to obtain a resuspension;
s7: activating CNA35 using EDC and NHS to obtain a CNA35 solution;
s8: adding CNA35 solution into the heavy suspension, and incubating under ice bath and shaking conditions to obtain a connection product;
s9: and cleaning the connection product, centrifuging to obtain nanoparticles, and re-suspending the nanoparticles by using a buffer solution to obtain a nanoparticle solution.
2. A collagen-targeted contrast agent with multimodal imaging detection and drug delivery therapy according to claim 1, wherein: the lipid shell membrane is internally wrapped with gold nanorods, perfluoropentane and triazamidine.
3. A collagen-targeted contrast agent with multimodal imaging detection and drug delivery therapy according to claim 2, wherein: the particle size is 311+ -4.5 nm, and the potential is-38.1+ -0.3 mV.
4. A preparation method of a collagen-targeted contrast agent with multi-modal image detection and drug-loading treatment effects is characterized by comprising the following steps: the method comprises the following steps:
s1: DPPC, DSPE-PEG2000-NH 2 Dispersing shell membrane raw materials consisting of DSPG and CH in chloroform to obtain a mixture I; DPPC, DSPE-PEG2000-NH 2 The mass ratio of DSPG to CH is 20:7:3:4;
s2: adding a mixed solution containing gold nanorod solution and triazamidine or a solution containing gold nanorod into the mixture I to obtain a mixture II, and performing primary sound vibration treatment on the mixture II;
s3: performing rotary evaporation on the mixture II subjected to the first sound vibration treatment to obtain a film; the power of the first sound vibration treatment is 60w, the time is 1min, and a discontinuous sound vibration method is adopted;
s4: dissolving the film by using a buffer solution to obtain a mixture III;
s5: adding perfluoropentane into the mixture III under ice bath condition, and performing secondary sound vibration treatment to obtain suspension; the power of the second sound vibration treatment is 60w, the time is 3min, and a discontinuous sound vibration method is adopted;
S6: centrifuging the suspension to obtain a solid phase; resuspending the solid phase with a solution of 4-morpholinoethanesulfonic acid to obtain a resuspension;
s7: activating CNA35 using EDC and NHS to obtain a CNA35 solution;
s8: adding CNA35 solution into the heavy suspension, and incubating under ice bath and shaking conditions to obtain a connection product;
s9: and cleaning the connection product, centrifuging to obtain nanoparticles, and re-suspending the nanoparticles by using a buffer solution to obtain a nanoparticle solution.
5. The method for preparing the collagen-targeted contrast agent with multi-modal image detection and drug-loading treatment effects according to claim 4, wherein the method comprises the following steps: in S1, the dosage ratio of the shell membrane raw material to the chloroform is 34mg:15ml.
6. The method for preparing the collagen-targeted contrast agent with multi-modal image detection and drug-loading treatment effects according to claim 4, wherein the method comprises the following steps: in S2, the volume ratio of the mixed solution containing gold nanorods and triazamidine to chloroform is 0.5:15; the concentration of the gold nanorods in the mixed solution is 1.0mg/ml, and the concentration of the triazamidine is 12mg/ml.
7. The method for preparing the collagen-targeted contrast agent with multi-modal image detection and drug-loading treatment effects according to claim 4, wherein the method comprises the following steps: in S5, the volume ratio of perfluoropentane to chloroform is 0.2:15; in S6, the concentration of the 4-morpholinoethanesulfonic acid solution is 0.1M, and the pH value is 8.0; in S7, the mass ratio of EDC to NHS is 3:1, and the pH value of the CNA35 solution is 5.2; in S8, the pH of the CNA35 solution was adjusted from 5.2 to 8.0, and the CNA35 solution was added to the resuspension.
8. Use of a collagen-targeted contrast agent with multimodal imaging detection and drug-loaded therapeutic effect according to any one of claims 2-3 for the preparation of a therapeutic drug for myocardial fibrosis.
9. Use of a collagen-targeted contrast agent with multimodal imaging detection and drug-loaded therapy according to any one of claims 1-3 for the preparation of a contrast agent for myocardial fibrosis.
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