CN113730607A - Preparation method of nano drug delivery system carrying oxygen-carrying perfluoropropane and indocyanine green - Google Patents
Preparation method of nano drug delivery system carrying oxygen-carrying perfluoropropane and indocyanine green Download PDFInfo
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- CN113730607A CN113730607A CN202111062639.9A CN202111062639A CN113730607A CN 113730607 A CN113730607 A CN 113730607A CN 202111062639 A CN202111062639 A CN 202111062639A CN 113730607 A CN113730607 A CN 113730607A
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a nano drug delivery system carrying oxygen perfluoropropane and indocyanine green, which comprises the following steps: s1: preparing a PLGA dichloromethane solution, an MTX solution, an ICG ultrapure water solution and a polyvinyl alcohol ultrapure water solution; s2: performing sound vibration to obtain a first-stage emulsion and a second-stage emulsion; s3: stirring the secondary emulsion obtained in the step S2, centrifuging at a high speed, repeatedly washing the precipitate with ultrapure water and centrifuging at a high speed twice, washing until the supernatant is clear, then suspending in the ultrapure water, and storing at 4 ℃ for later use; the nanoparticle prepared by the method has definite loaded active ingredients and stable pharmaceutical characteristics, has good rheumatoid arthritis affected joint targeted distribution effect, and has positive effects on abnormal synovium hyperplasia and local hypoxia.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a nano drug delivery system carrying oxygen perfluoropropane and indocyanine green.
Background
Rheumatoid Arthritis (RA) is a common chronic and systemic inflammatory disease induced by an autoimmune mechanism, and is mainly manifested as symmetrical, progressive and invasive polyarticular synovial inflammation, if not treated timely and correctly, the involved polyarticular synovial inflammation can be persistently and repeatedly attacked, so that articular cartilage and subchondral bone are damaged, joint deformity and function loss are finally caused, the rheumatoid arthritis has extremely high disability rate, the individual health is greatly influenced, and the economic and social burdens are obviously increased.
Currently, the treatment of RA still takes the main goals of early receiving disease-improving antirheumatic drugs (DMARDs) therapy, reducing or preventing joint damage and maintaining the integrity and function of joints, wherein Methotrexate (MTX) is the first-line treatment drug, is superior to other DMARDs in the aspects of alleviating symptoms and signs, reducing disability, delaying the destruction of imaging structures and the like, plays a very important role in inhibiting synovial hyperplasia, inflammatory response and continuously controlling diseases in early RA, and is the current classic first-line drug for treating RA. Although MTX is used alone or in combination with antirheumatic drugs for treating most RA patients, the systemic drug administration lacks the targeting property of pathological synovial tissues, and the drug is distributed in other tissues to cause serious systemic adverse reactions; in addition, approximately 30% of RA patients lack an effective response to MTX treatment, resulting in chronic, persistent arthritis. Therefore, the MTX-carrying novel multifunctional drug delivery system for targeting pathological joint synovium is explored and constructed, so that the pathological synovium is accurately targeted and 'cut', the treatment effect is improved, the adverse reaction of the MTX system is reduced, and meanwhile, the dynamic monitoring and the real-time curative effect evaluation of the pathological synovium are very necessary.
In recent years, indocyanine green (ICG) enhanced Fluorescence Optical Imaging (FOI) has achieved certain success in imaging therapy, for example, CN202010306885.3 discloses a multimode targeted nanobubble carrying AMD070 and ICG, which discloses a housing, AMD070 is connected to the outside of the housing, a nanobubble structure of indocyanine green and bio-inert gas is wrapped in the housing, perfluoropropane is further disclosed as the bio-inert gas, the multimode targeted nanobubble can pass through the wall of tumor vessel and enter the gap of tumor tissue, so that precise imaging and therapy can be achieved in tumor tissue, the reason for ultrasound contrast enhanced imaging is that microbubble gas is contained in the housing, and is easier to compress than surrounding biological media, and has stronger echo reflection performance in an ultrasound field, so that ultrasound signals can be significantly enhanced, and the more gas contained in the housing, the greater signal enhancing capacity is, which also means that indocyanine green has very practical application in imaging therapy, however, the specific lesion of the tumor is aimed at, and the conditions required by clinical application are very harsh, for example, because the gap of the new tumor vascular wall is between 380 and 780nm, only the particles with the particle size of less than 700nm are allowed to pass through the tumor vascular wall to enter the gap of the tumor tissue, and the targeted nanobubbles need to enter the gap of the tumor tissue to play a role, so that the particle size of the targeted nanobubbles is required to be 400 and 600nm, and thus, although indocyanine green is clinically and practically applied to imaging treatment, the aspects of targeting, penetrability, effectiveness and the like need to be considered, comprehensive consideration is needed, the conditions are also harsh, and the application in the aspect of targeted imaging treatment is greatly limited.
RA joint inflammation is one of diseases which plague human beings all the time, the strong advantages of indocyanine green are utilized, the application of indocyanine green can be inferred to have profound influence on the imaging targeted therapy of rheumatoid arthritis, the application evaluation is verified on an animal model at present, and a preliminary clinical result also indicates that the imaging method can be used for evaluation and clinical evaluation of the RA joint inflammation and has good consistency with the results of nuclear magnetic resonance and ultrasonic imaging. Therefore, FOI based on indocyanine green is expected to become an effective means for dynamically monitoring the treatment effect of RA arthritis, and has great application prospect in real-time adjustment of treatment schemes and realization of individualized accurate treatment targets, but is only verified on animal models at present, so that a drug-loading system capable of improving harsh conditions in the application process, improving the activity of load components and stabilizing the pharmacological characteristics is researched and developed, and the FOI based on indocyanine green has important significance in promoting the application of indocyanine green in targeted imaging treatment of RA arthropathy.
Disclosure of Invention
The invention provides a preparation method of a nano drug delivery system carrying oxygen perfluoropropane and indocyanine green, aiming at the defects of the prior art.
The method is realized by the following technical scheme:
the preparation method of the nano drug delivery system carrying oxygen-carrying perfluoropropane and indocyanine green is characterized by comprising the following steps:
s1: dissolving PLGA in dichloromethane to obtain a PLGA dichloromethane solution, dissolving MTX in a PBS buffer solution with the pH value of 7-9 to obtain an MTX solution, and respectively dissolving ICG and polyvinyl alcohol with the concentration of 3-8% in ultrapure water to respectively obtain ultrapure water solutions;
s2: oxygenating medical oxygen into perfluoropropane for 1-10min to obtain oxygen-carrying perfluoropropane, treating the oxygen-carrying perfluoropropane and the ICG ultrapure water solution and the MTX solution obtained in the step S1 by using an ultrasonic vibroseis to obtain emulsion, adding the emulsion into a PLGA dichloromethane solution, oxygenating for 1-10min, and then performing vibroseis to obtain primary emulsion; adding a polyvinyl alcohol aqueous solution into the primary emulsion, and performing sound vibration to obtain a secondary emulsion;
s3: stirring the second-stage emulsion of S2 for 2-8h, centrifuging at high speed, repeatedly washing the precipitate with ultrapure water and centrifuging at high speed twice, washing until the supernatant is clear, suspending in ultrapure water, storing at 4 deg.C for use, stirring the second-stage emulsion, and centrifuging to volatilize dichloromethane to the maximum
Further, the weight and volume ratio of the PLGA to the dichloromethane is as follows: the mL is 80-120: 1-8.
Further, the weight and volume ratio of the ICG to the ultrapure water is as follows: 1-5 in mL: 0.5-1.5.
Further, the weight and volume ratio of the MTX to the PBS is as follows: the mL is 10-20: 1-5.
Further, the ultrasonic vibro-acoustic treatment in the step S2 is to mix the oxygen-carrying perfluoropropane, the ICG ultrapure aqueous solution and the MTX solution in proportion and then to perform intermittent vibration for 80 to 100 seconds by using the ultrasonic vibro-acoustic treatment, wherein the intermittent vibration is repeated by vibrating for 3 to 8 seconds and stopping for 3 to 8 seconds.
Further, the step S2 includes adding the emulsion into the PLGA dichloromethane solution, oxygenating, and performing intermittent vibration for 2-8min by using an ultrasonic vibroseis, wherein the intermittent vibration is performed for 3-10S and is stopped for 3-10S, so as to obtain the primary emulsion by repeated vibroseis.
Further, the sound vibration in the step S2 is to obtain a secondary emulsion by adding the polyvinyl alcohol aqueous solution to the primary emulsion and performing intermittent vibration for 1 to 5 minutes by using an ultrasonic sound vibration apparatus, wherein the intermittent vibration is performed for 2 to 10 seconds and stopped for 2 to 10 seconds, and thus the secondary emulsion is obtained by repeating the sound vibration.
Further, the high speed centrifugation in S3 is performed at 10000-.
Further, the whole process from S1 to S3 was carried out in an ice bath and protected from light.
Further, a nano drug delivery system carrying oxygen perfluoropropane and indocyanine green is applied to diagnosis and treatment integrated clinical transformation of rheumatoid arthritis joint lesion under multi-modal fluorescence imaging and ultrasonic imaging lesion detection.
Has the advantages that:
1) the novel multifunctional nanoparticles carrying oxygen perfluoropropane, indocyanine green and methotrexate are prepared by an improved double-emulsification method, and the nanoparticles prepared by the method are clear in loaded active ingredients, stable in pharmaceutic characteristics and good in rheumatoid arthritis affected joint targeted distribution effect.
2) The stability of the ICG encapsulated by the nanoparticles is obviously improved compared with that of free ICG, and the fluorescence optical imaging and photodynamic action of the diseased joint are improved.
3) The nanoparticles of the oxygen-carrying perfluoropropane, indocyanine green and methotrexate prepared by the invention show good multi-mode fluorescence imaging and ultrasonic imaging of rheumatoid arthritis pathological joints, show the effect of improving illness state and rheumatism resistance through pharmacological action and photodynamic action, and are expected to realize diagnosis and treatment integrated clinical transformation application of RA joint pathological changes under the navigation of fluorescence imaging and ultrasonic imaging.
4) The ICG and the oxygen-carrying fluorocarbon (PFP) are jointly encapsulated in the nano drug delivery system, so that on one hand, the stability of the ICG solution is increased, the blood circulation time of the ICG solution is prolonged, on the other hand, oxygen is provided for the ICG photoacoustic power effect, the double effects of the ICG fluorescence imaging and the light-sound kinetic treatment are simultaneously exerted, and meanwhile, the encapsulated oxygen-carrying fluorocarbon has the effect of an ultrasonic contrast agent.
5) Synovial hyperplasia and local hypoxia of affected joints play an important role in the pathogenesis of RA, and in addition, hyperproliferation of RA fibroblast-like synovial cells (FLSs) leads to increased oxygen consumption and local hypoxia of the microenvironment, resulting in synovial inflammation, pro-inflammatory cell infiltration, angiogenesis and cartilage degradation. The processes are mutually promoted to form a positive feedback circulation to jointly promote the progress of RA. Therefore, the treatment for effectively improving the local hypoxia microenvironment RA in the treatment process has important significance and clinical value.
6) The oxygen-carrying PFP causes the conversion from liquid drops to bubbles under the stimulation of near-infrared laser, NPs are converted into micro bubbles, and the photodynamic action is exerted. When exposed to low intensity focused acoustic waves, the microbubbles collapse and cause cavitation, releasing oxygen and ICG and producing an sonodynamic response.
Drawings
FIG. 1 is a schematic diagram of a preparation method of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles;
FIG. 2 is a schematic structural diagram and representation of multifunctional nanoparticles of oxygen-carrying perfluoropropane, indocyanine green and methotrexate;
FIG. 3 is detection of encapsulation efficiency and drug loading rate of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles;
FIG. 4 is an optical property test of the oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticle;
FIG. 5 is an in vitro ultrasonic imaging diagram of multifunctional nanoparticles of oxygen-carrying perfluoropropane, indocyanine green and methotrexate;
FIG. 6 is an in vitro photoacoustic imaging of oxygen-carrying perfluoropropane and indocyanine green and methotrexate multifunctional nanoparticles;
FIG. 7 is an in vivo targeted distribution fluorescence imaging experiment of oxygen-carrying perfluoropropane and indocyanine green and methotrexate multifunctional nanoparticles carried by normal rats and CIA rats;
FIG. 8 is an in vivo ultrasonic imaging experiment of oxygen-carrying perfluoropropane and indocyanine green and methotrexate multifunctional nanoparticles carried by normal rats and CIA rats;
FIG. 9 is an in vivo photoacoustic imaging experiment of oxygen-carrying perfluoropropane and indocyanine green and methotrexate multifunctional nanoparticles carried by normal rats and CIA rats;
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
The general steps of the drug-loaded nanoparticles (OIM @ NPs) carrying oxygen-loaded perfluoropropane and indocyanine green in the application are shown in a schematic preparation flow diagram shown in FIG. 1
Example 1
S1: dissolving 80mgPLGA in 1mL of dichloromethane, 1mgICG in 0.5mL of ultrapure water, 10mgMTX in 100 μ L of PBS buffer solution with pH 7, and adding polyvinyl alcohol to the ultrapure water to a final concentration of 3% for later use;
s2: slowly oxygenating a closed device filled with 1mL of perfluoropropane by medical oxygen for 1min to prepare PFP-O2, taking out 200 mu L of PFP-O2, and fully mixing an ICG solution and an MTX solution prepared by S1 for 80S (vibrating for 3S, stopping for 3S) by using an ultrasonic vibroseis; adding the emulsion after sound vibration into PLGA dichloromethane solution, oxygenating for 1min again, and performing sound vibration for 2min (vibrating for 3s, stopping for 3s) to obtain first-stage emulsion; adding 5% PVA water solution into the first-stage emulsion, and performing sound vibration for 1min (vibration for 2s, stopping for 2s) to obtain a second-stage emulsion;
s3: stirring the product obtained in S2 with a magnetic stirrer for 2h to fully volatilize dichloromethane, centrifuging at high speed for 2min (4 ℃, 10000rpm), repeatedly washing and centrifuging the precipitate with ultrapure water twice, washing until the supernatant is clear, then suspending in ultrapure water, and storing at 4 ℃ for later use. The preparation was carried out in ice bath and protected from light.
Example 2
S1: dissolving 120mgPLGA in 8mL of dichloromethane, 5mgICG in 1.5mL of ultrapure water, 50mgMTX in 500 μ L of PBS buffer solution with pH 9, adding polyvinyl alcohol to the ultrapure water to make the final concentration 8% for later use;
s2: slowly oxygenating a closed device filled with 1mL of perfluoropropane with medical oxygen for 10min to prepare PFP-O2, taking out 200 mu L of PFP-O2, and fully mixing an ICG solution and an MTX solution prepared by S1 for 100S (vibrating for 8S and stopping for 8S) by using an ultrasonic vibroseis; adding the emulsion after sound vibration into PLGA dichloromethane solution, oxygenating for 10min again, and performing sound vibration for 8min (vibration for 10s, stopping for 10s) to obtain first-stage emulsion; adding 5% PVA water solution into the first-stage emulsion, and vibrating for 5min (vibration for 10s, stop for 10s) to obtain second-stage emulsion;
s3: stirring the product obtained in S2 for 8h by using a magnetic stirrer to fully volatilize dichloromethane, centrifuging at high speed for 10min (4 ℃, 15000rpm), repeatedly washing and centrifuging the precipitate for two times by using ultrapure water, washing until the supernatant is clear, then suspending in the ultrapure water, and storing at 4 ℃ for later use. The preparation was carried out in ice bath and protected from light.
Example 3
S1: dissolving 50mgPLGA in 1.5mL of dichloromethane, 2mgICG in 1mL of ultrapure water, 3mgMTX in 400 μ L of PBS buffer solution with pH 7.4, adding polyvinyl alcohol to ultrapure water to make the final concentration 5% for later use;
s2: slowly oxygenating a closed device filled with 1mL of perfluoropropane by medical oxygen for 5min to prepare PFP-O2, taking out 200 mu L of PFP-O2, and fully mixing an ICG solution and an MTX solution prepared by S1 for 90S (vibrating for 5S, stopping for 5S) by using an ultrasonic vibroseis; adding the emulsion after the sound vibration into a PLGA dichloromethane solution, oxygenating for 5min again, and performing sound vibration for 3min (vibrating for 5s and stopping for 5s) to obtain a first-stage emulsion; adding 5% PVA water solution into the first-stage emulsion, and performing sound vibration for 3min (vibration for 5s, stopping for 5s) to obtain second-stage emulsion;
s3: the product from S2 was stirred for 4h using a magnetic stirrer to evaporate the dichloromethane sufficiently. Centrifuging at high speed for 5min (4 deg.C, 11000rpm), washing precipitate with ultrapure water repeatedly and centrifuging twice, washing until supernatant is clear, suspending in ultrapure water, and storing at 4 deg.C. The preparation was carried out in ice bath and protected from light.
Taking example 3 as an example, the drug-loaded nanoparticles (OIM @ NPs) carrying oxygen-carrying perfluoropropane and indocyanine green prepared in this example were subjected to detection analysis, and the results are as follows:
firstly, the pharmacological characteristics of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs) are characterized
The specific operation is as follows: 1) diluting OIM @ NPs by a certain multiple with ultrapure water, and observing the shape and size of the OIM @ NPs by using an inverted fluorescence microscope; 2) diluting OIM @ NPs by a certain multiple of ultrapure water, and observing the fine morphology of the OIM @ NPs by using a transmission electron microscope; 3) the particle size, distribution and potential of the particles are detected by a Malvern particle size analyzer.
The results are shown in FIG. 2: 1) OIM @ NPs are green milky liquids at normal temperature, as shown in FIG. 2. a; 2) under an inverted fluorescence microscope, the OIM @ NPs are spherical, have good dispersibility, are uniform and consistent, and show red fluorescence, which indicates that the ICG is successfully encapsulated in the nanoparticles, as shown in FIGS. 2.d and 2. e; 3) OIM @ NPs are observed under a transmission electron microscope to have smooth and spherical surfaces, are single dispersed nanoparticles and have obvious shell-core structures, as shown in FIGS. 2.b and 2. c; 4) OIM @ NPs have a particle size of 231.70. + -. 16.54nm and a potential of-6.00. + -. 1.84mV, as shown in FIGS. 2.f and 2. g.
② the encapsulation efficiency and drug-loading rate detection of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs)
The specific operation is as follows: 1) weighing ICG, dissolving in ultrapure water, accurately diluting to different concentrations, detecting the absorbance value of each concentration by using a UV-Vis spectrophotometer, and drawing a standard curve of the ICG aqueous solution; 2) weighing MTX, dissolving the MTX in PBS (phosphate buffer solution) with pH 7.4, accurately diluting to different concentrations, detecting peak areas under curves of various concentrations by using a high performance liquid chromatograph, and drawing a standard curve of the MTX solution; 3) in the process of preparing the nanoparticles, collecting supernatant liquid of all centrifugal processes, measuring the content of ICG and MTX by adopting an indirect measurement method, namely detecting the absorbance value in the supernatant liquid and the peak area under the curve by respectively using a UV-Vis spectrophotometer and a high performance liquid chromatograph, and then calculating the encapsulation efficiency and the drug loading capacity of the supernatant liquid by using the prepared standard curve.
The encapsulation efficiency and drug loading were calculated as follows:
encapsulation ratio (%) - (total amount of ICG used-amount of ICG in supernatant)/total amount of all ICGs × 100%; the drug loading was (total ICG used-amount of ICG in supernatant)/total amount of NPs × 100%.
The encapsulation efficiency and drug loading of MTX are the same.
The results are shown in FIG. 3 and show that: the encapsulation efficiency and the drug loading capacity of the ICG are 65.291 +/-1.442% and 2.374 +/-0.053% respectively; the encapsulation efficiency and the drug loading capacity of MTX are 42.179 +/-5.275% and 2.301 +/-0.288%, respectively.
Thirdly, optical characteristic detection of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs)
The specific operation is as follows: 1) diluting the prepared OIM @ NPs and the free ICG to a certain concentration by using ultrapure water, and respectively detecting absorption spectrograms of the OIM @ NPs and the free ICG within the range of 600-900 nm; 2) diluting the prepared OIM @ NPs and free ICG to certain concentration with ultrapure water, storing at 4 ℃, measuring the absorbance of the prepared OIM @ NPs and free ICG at 780nm every 3d, and continuously measuring for 15 days.
The results are shown in FIG. 4 and show that: the degradation rate of free ICG was faster than that of OIM @ NPs. The OIM @ NPs absorption intensity decreased by about 18% and the free ICG decreased by about 60% over a 15-day observation. The OIM @ NPs are shown to effectively improve the stability of the ICG.
In-vitro ultrasonic imaging of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs)
Preparing an in-vitro imaging gel model by using the ICG and MTX loaded nanoparticles, and specifically operating as follows: adding 8g of agarose gel powder into 400mL of ultrapure water, heating and stirring to completely dissolve the agarose gel powder and completely discharge bubbles, slowly pouring the agarose gel powder into a 200-mu L gun head box while the agarose gel powder is hot, inserting a gun head into the gun head box, and obtaining a gel model after the gel is cooled and solidified; the experiment was divided into three groups: PBS group, free ICG group, OIM @ NPs group, the experimental samples were added to the gel wells, respectively, and B-Mode and CEUS imaging was performed using an ultrasonic diagnostic imager. Then, after 1 minute of laser irradiation at 808nm of 1.5W/cm2, imaging was performed again. An imaging picture is acquired and analyzed for mean sound intensity using DYF software.
The results are shown in FIG. 5 and show that: after near-infrared laser irradiation, the OIM @ NPs group is obviously enhanced. The result shows that the ICG in the OIM @ NPs is excited by near infrared irradiation, so that the nuclear phase change of the PFP generates gas, and the ultrasonic imaging is enhanced.
In-vitro photoacoustic imaging picture of loaded oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs)
Preparing an in-vitro imaging gel model by using the ICG and MTX loaded nanoparticles, and specifically operating as follows: adding 8g of agarose gel powder into 400mL of ultrapure water, heating and stirring to completely dissolve the agarose gel powder and completely discharge bubbles, slowly pouring the agarose gel powder into a 200-mu L gun head box while the agarose gel powder is hot, inserting a gun head into the gun head box, and obtaining a gel model after the gel is cooled and solidified; the experiment was divided into three groups: and the PBS group, the free ICG group and the OIM @ NPs group respectively add experimental samples into gel holes, and collect photoacoustic imaging images by using a VEVO LAZR photoacoustic imaging system. Then, after 1 minute of laser irradiation at 808nm of 1.5W/cm2, imaging was performed again, and the results were analyzed.
The results are shown in fig. 6 and show that: the photoacoustic imaging of the PBS and OIM @ NPs groups did not differ significantly, they did not have an absorption peak in the NI range, and thus were not capable of photoacoustic imaging. The photoacoustic intensity of the OIM @ NPs group increased significantly after irradiation. Meanwhile, the photoacoustic intensity of OIM @ NPs is higher than that of free ICG. This suggests that OIM @ NPs, as a mediator of photoacoustic imaging, exhibit a more stable imaging effect in photoacoustic imaging than free ICG.
Sixthly, fluorescence imaging experiment of joint targeted distribution of oxygen-carrying perfluoropropane, indocyanine green and methotrexate (OIM @ NPs) in normal rats and CIA model rats
The specific operation is as follows: taking three normal rats and three CIA model rats respectively, injecting OIM @ NPs (3 mg/kg by MTX (maximum drug delivery) in tail vein, observing the distribution condition of the joints of the nanoparticles in the mouse body in a living body fluorescence imaging system of the small animal at 2h, 6h and 24h after injection, collecting images, and finally carrying out fluorescence intensity analysis.
The results are shown in FIG. 7: in the result of in-vivo fluorescence imaging of the small animals, the OIM @ NPs have the advantages that the fluorescence signals of the joint parts of the CIA rats are all stronger than those of the normal rats at 2h, 6h and 24h after injection, the highest value is achieved at 2h, and the fluorescence intensity quantitative analysis result further proves that the fluorescence intensity of the joint parts of the CIA rats is obviously higher than that of the normal rats after the OIM @ NPs are injected, so that the OIM @ NPs have obvious targeted distribution on the joint parts of rheumatoid arthritis lesions.
Seventhly, carrying oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs) in vivo ultrasonic imaging experiments of normal rats and CIA model rats
The method comprises the following specific operations: normal SD rats were randomly divided into 3 groups: saline group (i.e., NS group), ICG group, OIM @ NPs group; CIA rats were randomly divided into 3 groups: saline group (i.e., NS group), ICG group, OIM @ NPs group. Each group of rats received laser (1.5W/cm 25 min) -low intensity focused ultrasound (3W/cm 21 min) excitation 2h after OIM @ NPs injection. Respectively observing the joint and acquiring images by an ultrasonic diagnostic imager in a B-Mode and a CEUS Mode before excitation (namely 0h) and 2h, 6h and 24h after excitation, and finally processing the acquired images by DFY ultrasonic quantitative analysis software.
The results are shown in FIG. 8: FIGS. 8.a, 8.c and 8.e correspond to Normal Saline (NS), free ICG and OIM @ NPs groups, respectively, in normal rats, and FIGS. 8.b, 8.d and 8.f correspond to NS, free ICG and OIM @ NPs groups, respectively, in CIA rats; the ultrasound signals of the articular area in the laser-low intensity focused ultrasound excitation (i.e. 0h), B-Mode and CEUS modes after injection of the corresponding drugs (NS, ICG, OIM @ NPs) did not differ significantly between the 6 groups. After laser-low-intensity focused ultrasound excitation, the echo signals of joint parts of OIM @ NPs group are obviously enhanced in a CEUS mode, particularly in CIA rats, but the echo signals are not enhanced in the NS group and the ICG group. The OIM-NPs composition image enhancement can be obviously observed under the B-Mode, and the CIA rat imaging intensity is larger than that of a normal rat. The specific results of ultrasonic imaging intensity of the OIM-NPs group are as follows: EI in CIA rat was 11.22. + -. 1.39(0h), 60.05. + -. 2.73(2h), 32.14. + -. 3.68(6h), 3.03. + -. 2.93(24h) in CEUS mode, and EI in normal rat was 6.37. + -. 2.73(0h), 20.63. + -. 1.05(2h), 9.10. + -. 3.97(6h), 2.12. + -. 0.53(24h) in CEUS mode. In B-Mode, EI in CIA rat is 74.30 + -0.53 (0h), 93.10 + -2.63 (2h), 80.67 + -1.89 (6h), 54.89 + -1.89 (24h), and EI in normal rat is 57.02 + -1.39 (0h), 72.78 + -1.82 (2h), 62.48 + -0.53 (6h), 51.56 + -0.53 (24 h).
Photoacoustic imaging experiment of oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles (OIM @ NPs) in normal rats and CIA model rats
The method comprises the following specific operations: normal SD rats were randomly divided into 3 groups: saline group (i.e., NS group), ICG group, OIM @ NPs group; CIA rats were randomly divided into 3 groups: saline group (i.e., NS group), ICG group, OIM @ NPs group. Each group of rats received laser (1.5W/cm 25 min) -low intensity focused ultrasound (3W/cm 21 min) excitation 2h after OIM @ NPs injection. And respectively collecting photoacoustic imaging images by a VEVO LAZR photoacoustic imaging system 2h, 6h and 24h before excitation (namely 0h) and after excitation, and carrying out data analysis by self-contained software of a photoacoustic instrument.
The results are shown in FIG. 9: FIGS. 9.a, 9.c and 9.e correspond to Normal Saline (NS), free ICG and OIM @ NPs groups, respectively, for normal rats, and FIGS. 9.b, 9.d and 9.f correspond to NS, free ICG and OIM @ NPs groups, respectively, for CIA rats; in photoacoustic imaging, the NS group and the ICG group have no obvious imaging effect before and after laser-low intensity focused ultrasound excitation, while the OIM @ NPs group has good imaging effect before and after laser-low intensity focused ultrasound excitation, the PA intensity in CIA rats is 0.13 +/-0.00 (0h), 0.31 +/-0.03 (2h), 0.23 +/-0.02 (6h) and 0.11 +/-0.01 (24h), and the PA intensity in normal rats is 0.14 +/-0.01 (0h), 0.24 +/-0.02 (2h), 0.19 +/-0.02 (6h) and 0.10 +/-0.02 (24 h).
The imaging experiment results show that OIM @ NPs have longer circulation time in vivo and can better aggregate and distribute in joint parts. On the other hand, after OIM-NPs are injected, better PA imaging effect can be achieved 2h after laser-low intensity focused ultrasound excitation, and the navigation effect on later-stage in-vivo treatment is reflected.
In conclusion, the oxygen-carrying perfluoropropane, indocyanine green and methotrexate multifunctional nanoparticles with definite active ingredients, high drug loading capacity and good pharmaceutical characteristics are prepared, the products show more stable and excellent imaging effects than free ICG in-vitro ultrasonic imaging and photoacoustic imaging, and the products are expected to realize accurate treatment of rheumatoid arthropathy under the navigation of fluorescence imaging and ultrasonic imaging.
Claims (10)
1. The preparation method of the nano drug delivery system carrying oxygen-carrying perfluoropropane and indocyanine green is characterized by comprising the following steps:
s1: dissolving PLGA in dichloromethane to obtain a PLGA dichloromethane solution, dissolving MTX in a PBS buffer solution with the pH value of 7-9 to obtain an MTX solution, and respectively dissolving ICG and polyvinyl alcohol with the concentration of 3-8% in ultrapure water to respectively obtain ultrapure water solutions;
s2: oxygenating medical oxygen into perfluoropropane for 1-10min to obtain oxygen-carrying perfluoropropane, treating the oxygen-carrying perfluoropropane and the ICG ultrapure water solution and the MTX solution obtained in the step S1 by using an ultrasonic vibroseis to obtain emulsion, adding the emulsion into a PLGA dichloromethane solution, oxygenating for 1-10min, and then performing vibroseis to obtain primary emulsion; adding a polyvinyl alcohol aqueous solution into the primary emulsion, and performing sound vibration to obtain a secondary emulsion;
s3: and (3) stirring the secondary emulsion of S2 for 2-8h, centrifuging at a high speed, repeatedly washing the precipitate with ultrapure water and centrifuging at a high speed twice, washing until the supernatant is clear, then suspending in the ultrapure water, and storing at 4 ℃ for later use.
2. The method of claim 1, wherein the weight to volume ratio of PLGA to dichloromethane is as follows mg: the mL is 80-120: 1-8.
3. The method of claim 1, wherein the weight and volume ratio of ICG to ultrapure water is as follows in mg: 1-5 in mL: 0.5-1.5.
4. The method of claim 1, wherein the weight to volume ratio of MTX to PBS is in mg: the mL is 10-20: 1-5.
5. The method according to claim 1, wherein the step S2 is performed by mixing perfluoropropane with oxygen carrier, ICG ultra-pure aqueous solution and MTX solution in a ratio and intermittently vibrating for 80-100S with an ultrasonic vibrator, wherein the intermittent vibration is repeated by vibrating for 3-8S and stopping for 3-8S.
6. The method according to claim 1, wherein the step S2 is performed by performing the step of performing the step S2.
7. The method according to claim 1, wherein the step S2 is performed by repeating the step of performing the step S2.
8. The method according to claim 1, wherein the high speed centrifugation in S3 is performed at 10000-15000rpm for 2-10 min.
9. The method of claim 1, wherein the steps from S1 to S3 are performed in an ice bath and protected from light.
10. A nano drug delivery system carrying oxygen perfluoropropane and indocyanine green is characterized by being applied to diagnosis and treatment integrated clinical transformation for guiding rheumatoid arthritis joint lesions under multi-modal fluorescence imaging and ultrasonic imaging lesion detection.
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