CN103638534A - Nanometer lipid ultrasonic contrast agent and preparation method thereof - Google Patents
Nanometer lipid ultrasonic contrast agent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a nanometer lipid ultrasonic contrast agent and a preparation method thereof. According to the method, the nanometer lipid ultrasonic contrast agent which is small in particle size and can develop stably is prepared by adopting lipid film dispersion and mechanical oscillation methods, is good in outer shell crushing resistance, is difficult to crack under the condition of low mechanical index, is extremely low in solubility and diffusion rate in a solution, can perform microcirculation pouring well, can last longer time in blood, has favorable acoustic property and stability, can obviously enhance the heart, kidney, liver and tumor development by in-vivo radiography, and long in radiography time which is more than 30 minutes.
Description
Technical field
The invention belongs to field of nano material preparation, be specifically related to a kind of preparation method of nano-lipid acoustic contrast agent.
Background technology
Ultrasound imaging techniques is an extensive use, the imaging of medical mode that Noninvasive and cost are low, however common ultra sonic imaging is not if by contrast agent, and resolution is lower.Compare with conventional Ultrasound inspection, ultrasonic contrast has dynamically, shows in real time, continuously the advantages such as internal organs essence and focus blood vessel framework and perfused tissue situation, the more important thing is, compare with additive method, easy, the easy repetition of ultrasonic contrast, cheapness, "dead", without nephrotoxicity, safe.Nowadays, ultrasonic contrast has been listed three kinds of conventional diagnostic imaging methods of liver in together with enhanced CT, M, and this technology also extended to the application of a plurality of internal organs, as kidney, pancreas, spleen, thyroid, mammary gland, blood vessel etc., greatly promoted the development of clinical ultrasound diagnosis.
The growth of tumor, invasion and attack, transfer depend on the formation of new vessels.Thereby tumor vessel is realized rapid growth by increasing oxygen and nutrition supply, invasion and transfer, yet, tumor tissues medium vessels is abundant, blood vessel wall gap is wider, poor structural integrity, the tumor vascular endothelial cell aperture ranges of a lot of malignant tumor is 380～780nm approximately, this depends on different tumor cells, simultaneously, tumor tissues lymphatic return is poor, cause macromole class material and lipid granule to there is selectivity high-permeability and anelasticity, this phenomenon is known as the enhancing infiltration retention effect (EPR of solid tumor tissue, enhanced permeability and retention effect).The EPR effect of tumor vascular system can allow the macromolecular drugs such as liposome, macromolecule micelle and gene delivery in the middle of tumor tissues, can be used for tumor tissues diagnostic imaging and treatment.Therefore, developer or anticancer therapeutic agent can be exactly to have a little particle diameter in order to the basic demand of targeted imaging and treatment by tumor vessel.
Yet, the acoustic contrast agent of use is mainly microbubble contrast clinically at present, the shell membrane microbubble structure forming for wrapping up certain gas, shell membrane can prevent mutual fusion between microbubble and the release of internal gas, thereby has strengthened the stability of contrast agent, microbubble diameter 1-8 μ m, can not see through blood vessel wall arrival and be positioned at the cell that exceeds blood capillary, as many cancerous cell, so they can only, as developer in blood pond, limit the Clinics and Practices to the outer disease of its blood vessel.Along with further going deep into of nanotechnology research, the acoustic contrast agent of novel submicron order just engenders, comprise and having and the liposome of organizing different acoustic responses, microemulsion, nano-emulsion and some nano-particle, these nanoparticles are as acoustic contrast agent, because its unique dimensional effect can have targeting and therapeutical effect [Rapoport N simultaneously, Gao Z & Kennedy A.Multifunctional Nanoparticles for Combining Ultrasonic Tumor Imaging and Targeted Chemotherapy[J] .J Natl Cancer Inst, 2007, 99 (14): 1095-1106].Experimental studies have found that, the acoustic contrast agent that diameter is less than 900um has good targeting [Hughes M S, Marsh J N & Hall C S, et al.Acoustic Characterization in Whole Blood and Plasma of Site-Targeted Nanoparticle Ultrasound Contrast Agent for Molecular Imaging[J] .J Acoust Soc Am, 2005,117 (2): 964-972].At present the acoustic contrast agent of 1-7um is considered to the good size of development effect, if but contrast agent small-sized can enter tumor tissues through the irregular blood vessel wall of cancer new vessels and develop to picture.
In recent years, develop many new methods and technology manufacture and take acoustic contrast agent [the Wheatley M A that nanoparticle is basic target tumor, Forsberg F & Dube N, et al.Surfactant-Stabilized Contrast Agent On the Nanoscale for Diagnostic Ultrasound Imaging[J] .Ultrasound Med Biol, 2006,32 (1): 83-93].In several research, various shell membranes (phospholipid or polymer) and core (gas have been prepared, liquid or solid) nanoscale ultrasound contrast agents, and demonstrate good contrast and strengthen [Marxer E E, Brussler J & Becker A, et al.Development and Characterization of New Nanoscaled Ultrasound Active Lipid Dispersions as Contrast Agents[J] .Eur J Pharm Biopharm, 2011,77 (3): 430-437].Study on nano-scale contrast agents is for tumor imaging, is mainly due to its high tissue rate of exosmosing, and in the increase of tumor region contrast agent, can reach gratifying imaging.Liposome has as advantages such as nontoxic, biodegradable, non-immunogenicities because of it, has extensively been used as the carrier that transports of medicine or gene.The matrix material of these types is also used to the gas that parcel has sound reflecting, there is now the contrast agent of several lipid films to be applied to central [the Goertz D E of clinical ultrasound diagnosis by routine, de Jong N & van der Steen A F.Attenuation and Size Distribution Measurements of Definity and Manipulated Definity Populations[J] .Ultrasound Med Biol, 2007,33 (9): 1376-1388].The basic demand of desirable bubble acoustic contrast agent comprises: can pass through blood capillary, have similar erythrocytic hemodynamics feature; Good stability, has good permeability and suitable surface tension, and can continue enough time after entering human body; Can produce abundant harmonic wave; The gas of shell membrane material and parcel is non-stimulated, damage or toxic and side effects.At present, existing multiple material is used as the coated fertilizer of microbubble, is mainly divided into following a few class: lipid, surfactant-based, protein-based and polymer class.Along with the appearance of various new materials and the raising of preparation method, can prepare the microbubble contrast of simultaneously loading gas, gene or medicine, realizing ultrasonic development strengthens when diagnosing, also can further reach the several functions such as transmission medicine, targeting location and disease treatment, become gradually the focus of ultrasound medicine research.Gas componant in bubble contrast agent mainly comprises nitrogen, sulfur hexafluoride, perfluoropropane, perfluorinated butane, perflenapent, perflexane etc.After the microbubble contrast intravenous injection of parcel air enters in human body, air can be dissolved in the middle of blood very soon, determined that its persistent period is short, easily break, cause angiographic diagnosis also not finish, microbubble disappears, thereby limited the time of observing and diagnosing in clinical practice, in addition, under ultrasonic energy effect, due to contraction and the expansion of bubble, can further accelerate the destruction of microbubble.Parcel high density noble gas (not soluble in water or blood) is the main thin and soft bubble of adventitia, and stabilization time is long, and vibration and echoing characteristics are good.
Along with the development of nano material technology of preparing, the size of bubble contrast agent and stability can be controlled, become the useful tool of clinical ultrasound radiography.When the size of microvesicle is reduced to nanoscale,, having there is the character that some are unique in the molecular characterization generation great change of contrast agent, comprises long half time, high surface reaction, and absorption affinity is strong, enzyme resistance degeneration etc.All these attributes are conducive to new study on nano-scale contrast agents in application medically.Studies show that Pluronic block copolymer can stabilized nanoscale particle, control their size, interact with lipid film, change the mobility of lipid or the elasticity of lipid cell-shell, prevent that particle from the effect such as being engulfed by RE system.[the Krupka T M such as Tianyi M, Solorio L & Wilson R E, et al.Formulation and Characterization of Echogenic Lipid-Pluronic Nanobubbles[J] .Mol Pharm, 2010, 7 (1): 49-59] 5 kinds of Pluronic block copolymer (L31 of molecular weight ranges 1100～4600 have been studied, L61, L81, L64 and P85) join hydration in lipidic shell lipid film, after be filled with pfc gas, result shows that the interaction of all the other lipid films can significantly reduce the size of bubble, the most important thing is, although result of study shows it is a kind of bubble of nanoscale, their stability and and echo be in vitro and in vivo without prejudice, consequent nano bubble is more suitable for sending for tumor Enhanced Imaging and successive treatment gene or medicine.
Contrast-enhanced ultrasound technique be by acoustic contrast agent after vein injects in human body, utilize contrast agent that rear scattered echoes is strengthened, thereby obviously improve resolving power, sensitivity and the specific technology of ultrasonic diagnosis.Along with the improvement of ultrasonic instrument performance and the appearance of novel acoustic contrast agent, ultrasonic contrast is widely used in fields such as the diseases such as cardiac muscle, liver, gallbladder, pancreas, spleen, kidney, pelvic cavity viscera and belly cavity tumor, breast tumor, thyroid and superficial lymph knots.Ultrasonic contrast is detecting aspect occupying lesion, etiologic diagnosis and judgement tumor promotion, the enhancing result that it obtains can compare favourably with enhanced CT or MRI, and it is little that it has institute's using dosage, to human body without obvious toxic-side effects and allergic phenomena, radiationless, easy and simple to handle, Real-time and Dynamic, advantage that can rechecking.Contrast-enhanced ultrasound technique, except conventional radiography harmonic imaging, also has low mechanical index imaging, batch (-type) ultra sonic imaging, energy contast harmonic imaging, contrast agent explosion imaging, is excited the methods such as acoustic emission imaging, back pulse harmonic imaging.Low mechanical index imaging refers to that its mechanical index MI, lower than 0.15 o'clock, is called low mechanical index when adopting the ultrasonic of transmitting.Adopt the radiography that the ultrasound wave of this energy while being broken up lower than acoustic contrast agent carries out to be called low mechanical index radiography.This method can realize the continuous harmonic imaging of blood flow, also can reduce the interference of tissue harmonic.This research has adopted real-time radiography to be matched to the inside and outside ultrasonic contrast that picture shadowgraph technique (CnTI) carries out nanometer acoustic contrast agent, the method that CnTI is used frequency domain to process, during transmitting, only launch " pure " fundamental signal, during reception, the main signal of processing second harmonic, when extracting contrast agent harmonic signal and eliminate the linear first-harmonic composition of tissue echo, can strengthen the resolving power of contrast agent, improve signal to noise ratio, improve the contrast of acoustic picture quality and lifting image, make the border of focus and angiography than more clear [the Frinking P J of conventional image, Bouakaz A & Kirkhorn J, et al.Ultrasound Contrast Imaging:Current and New Potential Methods[J] .Ultrasound Med Biol, 2000, 26 (6): 965-975].The echo-signal that acoustic contrast agent produces ultrasound wave is the physical basis of ultrasonic contrast diagnosing image, its principle is the compressibility of bubble, contrast agent shows as the asymmetric resonance motion of " expanding-compress-expand again-recompress " under ul-trasonic irradiation, gas more easily compresses than biological tissues such as soft tissues, therefore when microbubble is subject to pulsed ultrasonic wave irradiation, microbubble has experienced the pucker & bloat process replacing, there is first-harmonic, second harmonic, the echo response of the various complexity such as subharmonic, cause complicated microbubble contrast ultra sonic imaging mechanism, new contrast-enhanced ultrasound agent and shadowgraph technique provide powerful measure for ultrasonic contrast quantitative study.
Current clinical use acoustic contrast agent average diameter is several microns, and the granule that blood vessel endothelium gap in morbid state allows diameter to be less than 700nm at the most passes, therefore micron order acoustic contrast agent can not penetration rate of blood endothelial tube gap, has greatly weakened its ability being used for the treatment of.Therefore need to prepare the blood perfusion information that is no longer confined to only obtain tissue, but by improving the specificity of ultrasonoscopy the contrast agent developing to treatment field.But along with acoustic contrast agent is from single diagnostic function to the multi-functional realization such as treat in conjunction with gene or medicine, the research of microbubble and application are just becoming more and more extensive, the acoustic contrast agent of therefore, prepare that particle diameter is less, stable in properties, Echoenhance are effective is very important.Study on nano-scale contrast agents particle diameter is less, gives their extremely strong penetration powers, therefore becomes in recent years the focus of research.
Summary of the invention
Technical problem: the invention provides a kind of targeted developing outside tumor vessel of realizing, apply to the nano-lipid acoustic contrast agent in tumor tissues diagnostic imaging and treatment, the method for the above-mentioned nano-lipid acoustic contrast agent of preparation simple, that easily go is provided simultaneously.
Technical scheme: the preparation method of nano-lipid acoustic contrast agent of the present invention, comprises the steps:
(1) according to mol ratio 80:12:8～85:10:5, take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000, mix and be placed in container;
(2) mixed liquor that adds chloroform and methanol in the mixture making to step (1), the mass volume ratio of mixture and mixed liquor is 0.0005g/ml～0.001g/ml, in mixed liquor, the volume ratio of chloroform and methanol is 1:1～2:1, then carry out ultrasonic concussion, until the said mixture matter in container is fully dissolved;
(3) container that is placed with step (2) and makes compounding substances is satisfied with in rotary evaporator, under 50～55 ℃, rotating speed 80～100r/min, evacuation condition, move, organic solvent in container is fully volatilized, until container bottle wall forms one deck white, uniform lipid membrane;
(4) according to mass volume ratio 1.5～2 mg/ml, in the phosphate buffer of 0.01～0.1mol/L, add blocked polyethers F-68, then joined in the container that is attached with lipid membrane that step (2) obtains, after lipin dissolving thin film, ultrasonic dispersion is until bottle wall thin film comes off completely, and making it form phospholipid concentration is the lipid suspension of 3～5 mg/ml;
(5) the lipid suspension obtaining is joined in container, be filled with after sulfur hexafluoride gas, vibrate until lipid suspension is creamy white, sticky, opaque;
(6) after standing, discard upper foam, after low-speed centrifugal, make the layering of lipid suspension, discard the microbubble that upper strata particle diameter is larger, take off the milky white liquid rinsing 3 to 5 times of layer, the resuspended nano-lipid contrast agent that obtains.
Nano-lipid acoustic contrast agent of the present invention, prepares according to the method described above.
Nano-lipid acoustic contrast agent of the present invention, can be outside ultra sonic imaging, particularly tumor vessel application in targeted developing and treatment, particularly tumor tissues targeting and locating therapy.
Beneficial effect: the present invention compared with prior art, has the following advantages:
The present invention adopts film dispersion method to add mechanical oscillation legal system for the agent of nanoscale lipid ultrasonic contrast, and the microbubble of preparation has been carried out to structural characterization and study on the stability.By rat and mice with tumor are carried out to ultrasonic contrast, observe the video picture situation of the rat heart, liver, excess of the kidney matter Echoenhance situation and tumor, and compare with the development effect of common micron order lipid contrast agent.Adopt the standby nanoscale lipid contrast agent of mechanical oscillation legal system, be spheroidal outward appearance under Electronic Speculum, expoeridium one deck lipid film, is inside filled with bright SF6 gas, good dispersion degree, and particle size measurer shows that mean diameter is 413.8nm, zeta potential value is-23.39mV.EDS analysis result shows the gas componant fluorine that contains parcel in nanoscale lipid bubble and sulfur and the elements such as carbon, nitrogen, oxygen and phosphorus that form lipid film.Nanoscale lipid contrast agent is 1 grade to the cytotoxicity of L-929 cell, belongs to cell avirulence category, without haemolysis, occurs.External supersonic develops and shows that nanoscale lipid contrast agent shows the ultrasound contrast similar with the microbubble of micron order lipid contrast agent and sound Novi and strengthens ability.After body interimage intravenous injection nanoscale lipid contrast agent, and compare obvious, the lasting enhancing video picture as seen of rat heart, liver and kidney before radiography.The analysis of time-density curve of transplanted tumor ultrasonic contrast shows, nanoscale lipid contrast agent is compared with common lipid microbubble contrast agent, and peak time evening, peak strength is lower slightly, but that radiography strengthens the persistent period is longer.After body circulation 15min, common microvesicle is substantially eliminated in tumor vessel, and nanoscale lipid bubble still has part to be positioned at tumor tissues region.This nano-lipid acoustic contrast agent can be realized the outer targeted developing of tumor vessel, can apply in tumor tissues diagnostic imaging and treatment.
In the inventive method, adopt to adopt thin film to disperse to add the nanoscale ultrasound contrast agents that mechanical oscillation method has successfully been prepared surface band negative charge, the method simply, easily go.Nanoscale ultrasound contrast agents has good biocompatibility, and no cytotoxicity and haemolysis occur.External supersonic development evaluation experimental shows, under identical ultra sonic imaging condition, lipid Na Pao shows with micron order lipid contrast agent and the similar ultrasound contrast of commercial sound Novi microbubble and strengthens ability.Body interimage, after intravenous injection study on nano-scale contrast agents, and compares before radiography, obvious, the lasting enhancing video picture as seen of rat heart, liver and kidney.The analysis of time-density curve of transplanted tumor ultrasonic contrast shows, nanoscale lipid bubble is compared with common lipid microbubble contrast agent, and peak time is more late, and peak strength is lower slightly, and the enhancing persistent period is longer.After body circulation 15min, common microvesicle is substantially eliminated in tumor vessel, and nanoscale lipid bubble still has part to be positioned at tumor tissues region.This nanoscale ultrasound contrast agents has good image potentiation, because its particle diameter is little, can pass tumor neogenetic capillary endothelium gap, can realize the outer targeted developing of tumor vessel, can apply in tumor tissues diagnostic imaging and treatment.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of nano-lipid contrast agent.
Fig. 2 is the average GTG result of calculation of ultrasonoscopy ROI.
Fig. 3 is nanoscale ultrasound contrast agents and micron order acoustic contrast agent tumor ultrasonic contrast time-density curve.
The specific embodiment
Below by embodiment, the present invention program is done further and illustrated.
1 main agents: distearyl acyl group lecithin (DSPC); Diphenyl phosphoryl azide (DPPA); DSPE-PEG 2000 (DSPE-PEG2000); Sulfur hexafluoride gas (SF6); PluronicF-68; RPMI1640 culture medium, calf serum; Tetrazolium bromide (MTT, AMRESCO); Dimethyl sulfoxide (DMSO, Sigma company).
The preparation of 2 nano-lipid contrast agent and detection
The preparation of 2.1 nano-lipid contrast agent
Embodiment 1:
Adopt lipid membrane to disperse to add mechanical oscillation legal system for nano-lipid contrast agent, comprise the steps:
(1) according to mol ratio 80:12:8, take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000, mix and be placed in container;
(2) mixed liquor that adds chloroform and methanol in the mixture making to step (1), the mass volume ratio of mixture and mixed liquor is 0.0005g/ml, in mixed liquor, the volume ratio of chloroform and methanol is 2:1, then carries out ultrasonic concussion, until the said mixture matter in container is fully dissolved;
(3) container that is placed with step (2) and makes compounding substances is satisfied with in rotary evaporator, under 50 ℃, rotating speed 100r/min, evacuation condition, move, organic solvent in container is fully volatilized, until container bottle wall forms one deck white, uniform lipid membrane;
(4) according to mass volume ratio 2 mg/ml, in the phosphate buffer of 0.1mol/L, add blocked polyethers F-68, then joined in the container that is attached with lipid membrane that step (2) obtains, after lipin dissolving thin film, ultrasonic dispersion is until bottle wall thin film comes off completely, and making it form phospholipid concentration is the lipid suspension of 3 mg/ml;
(5) the lipid suspension obtaining is joined in container, be filled with after sulfur hexafluoride gas, vibrate until lipid suspension is creamy white, sticky, opaque;
(6) after standing, discard upper foam, after low-speed centrifugal, make the layering of lipid suspension, discard the microbubble that upper strata particle diameter is larger, take off the milky white liquid rinsing 3 to 5 times of layer, the resuspended nano-lipid contrast agent that obtains.
Embodiment 2, and basic procedure step is with embodiment 1, and difference is as follows:
In step (1), according to mol ratio 83:11:6, take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000 and prepare nano-lipid contrast agent;
The mixture making according to step (1) in step (2) and the mass volume ratio of chloroform methanol mixed liquor are that 0.0008g/ml prepares nano-lipid contrast agent, and the volume ratio of chloroform and methanol is 1.5:1.
In step (3), step (2) is made to mixture and be satisfied with in rotary evaporator, under 51 ℃, rotating speed 80r/min, evacuation condition, move.
In step (4), according to mass volume ratio 1.5 mg/ml, in the phosphate buffer of 0.01mol/L, add blocked polyethers F-68, then joined in the container that is attached with lipid membrane that step (2) obtains, after lipin dissolving thin film, it is the lipid suspension of 4 mg/ml that ultrasonic dispersion forms phospholipid concentration.
In the present embodiment, the ratio of all the other operations and material composition is all identical with embodiment 1.
In step (1), according to mol ratio 85:10:5, take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000 and prepare nano-lipid contrast agent;
The mixture making according to step (1) in step (2) and the mass volume ratio of chloroform methanol mixed liquor are that 0.001g/ml prepares nano-lipid contrast agent, and the volume ratio of chloroform and methanol is 1:1.
In step (3), step (2) is made to mixture and be satisfied with in rotary evaporator, under 55 ℃, rotating speed 90r/min, evacuation condition, move.
In step (4), according to mass volume ratio 1.8 mg/ml, in the phosphate buffer of 0.05mol/L, add blocked polyethers F-68, then joined in the container that is attached with lipid membrane that step (2) obtains, after lipin dissolving thin film, it is the lipid suspension of 5 mg/ml that ultrasonic dispersion forms phospholipid concentration.
In the present embodiment, the ratio of all the other operations and material composition is all identical with embodiment 1.
2.2 electron microscopic morphology detect
Take out the nano-lipid contrast agent of a small amount of preparation, drip and have film copper mesh, make electron microscopic sample, under H-600 type transmission electron microscope (transmission electron microscope, TEM), observe.Self-control nano-lipid contrast agent is milky suspension, and under transmission electron microscope, nano-lipid contrast agent is spheroidal outward appearance, and expoeridium one deck lipid film is inside filled with bright SF6 gas, good dispersion degree (Fig. 1).It is (1.19 ± 0.11) * 10 that blood counting instrument is surveyed nanometer microvesicle mean concentration
9/ ml.At-4 ℃ of refrigerators, protect one week, contrast agent particle diameter, distribute still more evenly, have no obvious adhesion agglomerating.
2.3 scanning electron microscope energy disperse spectroscopy (SEM-EDS) phenetic analysis
The nano-lipid contrast agent that takes a morsel and prepare, getting appropriate suspension drips on copper mesh, under the SEM visual field, select arbitrarily several visuals field, with energy disperse spectroscopy, its composition is analyzed, the visible gas fluorine of parcel and the elements such as carbon, nitrogen, oxygen and phosphorus of sulfur and composition lipid film of wherein containing, confirms that nano-lipid contrast agent is successfully prepared.
2.4 particle diameters and zeta potentiometric analysis
Get respectively the nano-lipid contrast agent that PBS buffer for 2mL (pH7.4) diluted and be placed in cuvette, with laser particle size analyzer (Brookhaven instrument company), carry out particle diameter, Zeta potential mensuration, application dynamic light scattering software carries out date processing, record mean diameter, polydispersity index and surface potential, each sample repeats 3 times.By laser particle size analyzer, nano-lipid contrast agent average diameter being detected is 413.8 ± 12.0nm, is unimodal shape, narrow diameter distribution; And the average diameter 1763.7 ± 185.4nm of micron lipid contrast agent.In pH7.4 neutral environment, the zeta potential value of nano-lipid contrast agent is-23.39 ± 0.96mV, and the zeta current potential of micron lipid contrast agent is-1.6 ± 3.09mV.
The stability of 2.5 nano-lipid contrast agent and concentration determination
4 ℃ of Refrigerator stores, observe its stability after one week; Application blood counting instrument is measured the concentration of nano-lipid contrast agent under light microscopic, 5 middle lattice of count plate are received and are steeped sum, line ball Na Pao only counts left side and top, calculates as follows: Na Pao number/ml=(5 middle lattice are received and steeped sum/5) * 25 * extension rate * 10
4/ ml.
Cell toxicity test-the mtt assay of 3 nano-lipid contrast agent
The cultivation of 3.1L-929 cell
L-929 cell is inoculated in containing in the RPMI1640 culture fluid of 10% calf serum, at 37 ℃, in the incubator of saturated humidity, 5%CO2, cultivates, and every 2-3 days goes down to posterity once, the trophophase cell of taking the logarithm during experiment.
The cytotoxicity experiment of 3.2 nano-lipid contrast agent (MTT)
The take the logarithm L-929 cell of trophophase, routine is digested to cell suspension and adjusts cell concentration to 5 * 10
4/ mL, with 100 μ L/ holes, inoculate 96 well culture plates, put 37 ℃, in the incubator of 5%CO2, cultivate, after 24 hours, discard stock solution, add various different phosphate lipid concentrations (1 μ g/ml, 5 μ g/ml, 10 μ g/mL and 15 μ g/mL) RPMI1640 culture fluid, negative control is RPMI-1640, positive control is 0.7% polyacrylamide monomer solution, establish 8, multiple hole for every group, continue to cultivate after 72 hours, with inverted microscope, observe the morphological change of L929 cell, follow every hole and add 20 μ LMTT, old terms is cultivated 4 hours, discard liquid in hole, add DMSO150 μ L/ hole, shake after ten minutes, in immune microplate reader, measure the absorbance at 492nm place.Be calculated as follows the relative rate of increase of cell, following (the relative growth rate of computing formula, RGR): RGR%=experimental group OD average/negative control group OD average * 100%, and by table 1 regulation, RGR value is converted to 6 order reactions, experimental result is that 0 or 1 order reaction is qualified, experimental result is the combination cellular morphology overall merit of 2 order reactions, and experimental result is that 3-5 order reaction is defective.
Table 1RGR and toxicity grading conversion table
| Reaction | The relative rate of increase (RGR%) |
| 0 grade | ≥100 |
| 1 grade | 75-99 |
| 2 grades | 50-74 |
| 3 grades | 25-49 |
| 4 grades | 1-24 |
| 5 |
0 |
Under inverted microscope, observe and find, add after various different phosphate lipid concentrations (1 μ g/ml, 5 μ g/ml, 10 μ g/mL and 15 μ g/mL) RPMI1640 culture fluid, L-929 Growth of Cells situation is compared and no significant difference with negative control group, positive controls situation is completely different, in dosing, after 24 hours, be visible cell become gradually circle, come off, after 72 hours substantially without survivaling cell.The culture fluid effect L-929 cell of different phosphate lipid concentration is after 72 hours, and its RGR lists in respectively table 2.Result shows that this nano-lipid contrast agent is 1 grade to L-929 cytotoxicity, and positive control is 4 grades, and combining form is learned to observe and can be judged that nano-lipid contrast agent should belong to cell avirulence category.
Table 2 nano-lipid contrast agent toxicity assessment MTT experimental result
| Group | OD value | RGR(%) | Toxicity grading |
| Negative control group | 1.43±0.12 | 100 | 0 |
[0067]?
| 1 μ g/mL phospholipid concentration | 1.33±0.01 | 93.49±0.08 | 1 |
| 5 μ g/mL phospholipid concentrations | 1.26±0.01 | 88.99±0.08 | 1 |
| 10 μ g/mL phospholipid concentrations | 1.16±0.05 | 81.56±0.07 | 1 |
| 15 μ g/mL phospholipid concentrations | 1.11±0.03 | 78.36±0.07 | 1 |
| Positive controls | 0.15±0.03 | 10.20±0.02 | 4 |
The hemolytic test of 4 nano-lipid contrast agent
From healthy new zealand rabbit auricular vein, extract blood 10mL, add 20g/L potassium oxalate 0.5mL, by every 8mL, add 10mL normal saline to obtain the fresh anticoagulant Sanguis Leporis seu oryctolagi of dilution, 0.2mL Sanguis Leporis seu oryctolagi diluent is added in 10mL distilled water.Nano-lipid contrast agent is pressed 1 μ g/ml with normal saline, 5 μ g/ml, 10 μ g/mL, and 15 μ g/mL configuration; Normal saline is as negative control, and distilled water is as positive control, every group of 3 test tubes.Detected materials will be housed, all test tubes of normal saline and each 10mL of distilled water are put into 37 ℃ of water-baths, after pre-temperature 30min, take out, respectively add dilution anticoagulant Sanguis Leporis seu oryctolagi 0.2mL, shake up gently, put into again 37 ℃ of water-bath relayings warm 60min that continues insurance, each pipe solution is placed in to the centrifugal (2500g/min of dry centrifuge tube, 5min), from every pipe, getting supernatant moves in cuvette, on spectrophotometer, measure respectively the OD value at 545nm place, positive controls OD value should be (0.8 ± 0.3), negative control group OD value should be not more than 0.03, hemolysis rate (%)=(testing sample OD average-negative control OD average)/(positive control OD average-negative control OD average) * 100, if hemolysis rate <5%, illustrative material is without haemolysis, meet the hemolytic test requirement of medical material.
Each experimental group is listed in table 3 at the absorbance at 545nm place.According to formula: hemolysis rate (%)=Dt-Dnc/Dpc-Dnc, the hemolysis rate that calculates variable concentrations nano-lipid contrast agent is respectively 0.782%, 0.615%, 0.407% and 0.328%, all much smaller than 5%, can think to test and use nano-lipid contrast agent without haemolysis, meet the hemolytic test requirement of medical material.
Table 3 variable concentrations nano-lipid contrast agent hemolytic test result
| Group | OD value | Hemolysis rate (%) |
| Negative control group | 0.015±0.002 | - |
| 15 μ g/mL phospholipid concentrations | 0.021±0.001 | 0.782 |
| 10 μ g/mL phospholipid concentrations | 0.020±0.001 | 0.615 |
| 5 μ g/mL phospholipid concentrations | 0.018±0.001 | 0.407 |
| 1 μ g/mL phospholipid concentration | 0.017±0.001 | 0.328 |
| Positive controls | 0.822±0.016 | - |
5 nano-lipid contrast agent external supersonic imagings
In experiment, adopt homemade model to carry out in vitro, nano-lipid contrast agent liquid used fully mixes in PBS, is injected in the silica gel tube of diameter 1.0cm, and is fixed on (distance from bottom is 6 centimetres) in water tank.Ultrasonic imaging device is TechnosMPX(Bai Sheng company, Italy), ultrasound machine has been equipped with a linear array transducer (LA532E), ultrasonic contrast pattern is the specific pattern of the nondestructive contrast (harmonic imaging of contrast, CnTI) and low acoustical power (mechanical index=0.089), when changing into Flash pattern, MI is 0.5.Use ImageJ software to carry out the meansigma methods of sample to carry out quantitative analysis.Select a border circular areas (ROI) as area-of-interest, automatically calculate the average gray value in each ROI.Bubble concentration is respectively 1.8-1.9 * 10
8individual bubble/milliliter (data that obtain from initial hematimeter).Contrast agent, before per injection, must suspend again, and probe and silica gel tube be filling ultrasonic coupling agent therebetween; Observe development effect.Compare with the commercial acoustic contrast agent sound Novi of lower micron of lipid contrast agent of similarity condition and clinical practice (Italian Bracco company) external supersonic imaging, instrument all conditions is set to same standard simultaneously.
External supersonic experiment shows, under CnTITM pattern, nanoscale lipid contrast agent ultrasonoscopy is fine and closely woven strong echo, and echo luminous point distribution uniform, the microbubble brightness of its brightness and micron order lipid contrast agent and sound Novi is similar, when video picture condition is from CnTI state (MI is 0.089), be that ultrasonic contrast pattern is while entering into the high mechanical index state of two-dimentional GTG (MI is 0.5), its ultrasonogram moment becomes approximate echoless, and perusal lipid Na Pao has become approximate transparent limpid liquid by milky suspension, corresponding oval ROI in figure is averaged to gray count and see that (each sample repeats 3 times Fig. 2, experimental result represents with meansigma methods and standard deviation) shown in.
Ultra sonic imaging in 6 nano-lipid contrast agent bodies
Adopt Italian Bai Sheng TechnosMPX colorful Doppler ultrasound diagnostic apparatus to carry out ultrasonic contrast experiment in nano-lipid contrast agent body, random built-in Contrast-enhanced Ultrasound coupling imaging technique, probe model is LA532E, and in all animals and angiographic procedure, various Photo conditions comprise that field depth, gain, TGC etc. all remain unchanged.Laboratory animal employing body weight is that the Yunnan elder brother of 250 scholar 50g is SD rat, before checking, rat fasting water is more than 8 hours, to reduce the impact of gastrointestinal peristalsis on image viewing, 10% chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia, dorsal position is fixed on rat extremity dull and stereotyped upper, removes the Mus hair of surveying position, the impact of the ultrasonic reflection of avoiding air on skin and Mus hair on ultra sonic imaging, fixed needle after tail venipuncture success, retains venous access with heparin.Nano-lipid contrast agent is diluted to finite concentration, first conventional two dimension, Doppler Color Flow Image Study rat liver situation, choose and show rat heart, liver and the good tangent plane of kidney, use iron stand static probe, to guarantee the variation along with the time, the ultrasonoscopy obtaining is the same anatomical position of rat.Ultrasonic contrast mode condition (mechanical index: 0.089, gain: 50, focus: 1.5cm) remain constant in all experiments were process.Through tail vein, press 0.lml/Kg dosage bolus contrast agent, use rapidly subsequently 1ml normal saline flushing, on diasonograph, observe immediately rat heart, liver and kidney ultrasonic development imaging situation, and the ultrasonoscopy of real time record gained.Per injection interval at least 30 minutes, and apply Flash technology and remove remaining contrast agent microbubble.
Ultrasonic development experiment shows the enhancing of developing successively of the visible Hepatic artery of liver after injection nano-lipid contrast agent normal saline solution, portal vein, hepatic vein in body, in blood vessel, start to occur strong catapoint, and there is the rapid filling of contrast agent, and spread to liver parenchyma, liver GTG strength-enhanced, be the high echo of tiny even point-like, radiography is the still enhancing as seen of liver parenchyma echo after 30 minutes, and the trend reducing gradually after first increase appears in ultrasonoscopy brightness.In heart and kidney body, obviously strengthening also all appears in ultrasonic development.
The tumor ultrasonic contrast of 7 nano-lipid acoustic contrast agents strengthens
The foundation of 7.1 hepatocellular carcinoma in nude mice models
In vitro culture SMMC7721 hepatoma carcinoma cell, the good cell of the trophophase form of taking the logarithm, discards a bottle interior culture fluid, PBS uses 0.25% trypsinization after rinsing, with the RPMll640 culture fluid containing 10% calf serum, stop digestion, after collecting cell centrifugal (1000rpm, 5min).In superclean bench, take out nude mice, 70% ethanol disinfection nude mice right fore axillary region skin, extracts about 0.2ml with lml syringe and (with normal saline, adjusts concentration with cell quantity 2 * 10
6individual) cell suspension injects that nude mice right fore is subcutaneous, and injection point, apart from entry point 1cm, all forms skin mound, and after inoculation, aseptic cotton carrier is gently compressed into pin mark and is prevented cell suspension seepage.Postvaccinal nude mice is put into mouse cage, through 2-3 week, in the subcutaneous tuberosity that occurs grain of rice size of visible nude mice right fore, treat that diameter of tumor reaches 1cm left and right and begins for ultrasonic contrast.
The tumor ultrasonic contrast of 7.2 nano-lipid acoustic contrast agents strengthens
Respectively nano-lipid acoustic contrast agent and common microbubble contrast agent are diluted to finite concentration, regulate color ultrasonic devices, two-dimensional ultrasound checks Subcutaneous tumor in advance, selects to proceed to CNTI radiography pattern after maximum tangent plane.Mechanical index (MI) 0.089, through tail venous access bolus (0.lml/Kg) acoustic contrast agent, after injection, use immediately normal saline flushing passage, dynamically observe acoustic contrast agent the development of Subcutaneous tumor echo is strengthened to situation, gather respectively the image of 1min, 1.5min after injection of contrast medium, 3min, 6min, 9min, 12min, 15min, the sequencing of two kinds of contrast-medium injection is random, and injection contrast agent at once plays whole process and records dynamic image, until contrast agent is cleaned up.At upper a kind of acoustic contrast agent acoustic image, disappear latter approximately 30 minutes, in same nude mice, with same method, inject another kind of acoustic contrast agent, observe as stated above the enhancing situation of contrast agent to tumor echo, nano-lipid acoustic contrast agent and common microbubble contrast agent are carried out to data quantitative analysis at the image of tumor, and adopt the method for self cross-reference to compare the two.
6 mice with tumor are after tail vein is taken up in order of priority the nano-lipid contrast agent and micron order acoustic contrast agent of bolus same dose, two kinds of contrast agent all can obviously strengthen subcutaneous transplantation tumor, in visible transplanted tumor blood vessel, there is strong catapoint, and full rapidly, contrast agent around gradually to central part filling, and is full of rapidly whole transplanted tumor from tumor, GTG intensity strengthens rapidly, rear beginning is slowly cleaned up, and transplanted tumor GTG intensity also weakens gradually, in experimentation without animal dead.Transplanted tumor ultrasonic contrast time-density curve result of study shows, compare with micron order acoustic contrast agent, study on nano-scale contrast agents transplanted tumor ultrasonic contrast peak strength low (P<0.001), peak time evening (P<0.001), but the enhancing persistent period is long, after body circulation 15min, the gray value of nanoscale lipid bubble is apparently higher than common microvesicle (P<0.001), illustrate that common microvesicle is substantially eliminated in tumor vessel, and nanoscale lipid bubble still have part be stranded in tumor tissues region (table 4, Fig. 3).
Table 4 nanoscale ultrasound contrast agents and micron order acoustic contrast agent are to the comparison of the ultrasonic enhancing video picture of tumor (x ± s)
| Contrast agent kind | Peak time (second) | Peak strength | Strengthen the persistent period (dividing) |
| Nanoscale ultrasound contrast agents | 91.17±2.79 | 27.28±0.72 | 31 |
| Micron order acoustic contrast agent | 56.67±1.63 | 32.78±1.17 | 16 |
Claims (2)
1. a preparation method for nano-lipid acoustic contrast agent, the method comprises the steps:
(1) according to mol ratio 80:12:8 ~ 85:10:5, take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000, mix and be placed in container;
(2) mixed liquor that adds chloroform and methanol in the mixture making to described step (1), the mass volume ratio of mixture and mixed liquor is 0.0005 g/ml ~ 0.001g/ml, in described mixed liquor, the volume ratio of chloroform and methanol is 1:1 ~ 2:1, then carry out ultrasonic concussion, until the said mixture matter in container is fully dissolved;
(3) container that is placed with described step (2) and makes compounding substances is satisfied with in rotary evaporator, under 50 ~ 55 ℃, rotating speed 80 ~ 100r/min, evacuation condition, move, organic solvent in container is fully volatilized, until container bottle wall forms one deck white, uniform lipid membrane;
(4) according to mass volume ratio 1.5 ~ 2 mg/ml, in the phosphate buffer of 0.01 ~ 0.1mol/L, add blocked polyethers F-68, then joined in the container that is attached with lipid membrane that described step (2) obtains, after lipin dissolving thin film, ultrasonic dispersion is until bottle wall thin film comes off completely, and making it form phospholipid concentration is the lipid suspension of 3 ~ 5 mg/ml;
(5) the lipid suspension obtaining is joined in container, be filled with after sulfur hexafluoride gas, vibrate until lipid suspension is creamy white, sticky, opaque;
(6) after standing, discard upper foam, after low-speed centrifugal, make the layering of lipid suspension, discard the microbubble that upper strata particle diameter is larger, take off the milky white liquid rinsing 3 to 5 times of layer, the resuspended nano-lipid contrast agent that obtains.
2. a nano-lipid acoustic contrast agent, is characterized in that, this contrast agent according to claim 1 method prepares.
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| CN104622848A (en) * | 2015-02-13 | 2015-05-20 | 西安交通大学 | Plasma activation encapsulated micro-bubbles |
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| WO2021258756A1 (en) * | 2020-06-24 | 2021-12-30 | 南京超维景生物科技有限公司 | Single-blood vessel ultrasonic blood perfusion imaging method and device |
| CN115646938A (en) * | 2022-11-19 | 2023-01-31 | 智程半导体设备科技(昆山)有限公司 | Method for strengthening megasonic cleaning of silicon wafer by utilizing nano bubbles with film coating |
| CN116549018A (en) * | 2023-05-22 | 2023-08-08 | 华润武钢总医院 | Three-dimensional ultrasonic super-resolution method based on nano liquid drops |
| CN116549018B (en) * | 2023-05-22 | 2024-02-02 | 华润武钢总医院 | Three-dimensional ultrasonic super-resolution method based on nano liquid drops |
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