CN103638534B - A kind of nano-lipid acoustic contrast agent and preparation method - Google Patents
A kind of nano-lipid acoustic contrast agent and preparation method Download PDFInfo
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Abstract
The invention discloses a kind of nano-lipid acoustic contrast agent and preparation method, it is little for particle diameter that the method adopts lipid membrane dispersion to add mechanical oscillation legal system, the nano-lipid contrast agent of Absorbable organic halogens video picture, its shell crushing resistance is good, not easily break under low mechanical index condition, and dissolubility in the solution and diffusibility are extremely low, well can carry out microcirculatory perfusion, long period can be continued in blood, there is good acoustic properties and stability, show that nano-lipid acoustic contrast agent obviously can strengthen heart by body interimage, kidney, liver and tumor imaging, and contrast time is long, sustainable more than 30min.
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, Noninvasive and the low medical imaging modalities of cost, but ordinary ultrasonic imaging is not if by contrast agent, and resolution is lower.Compared with conventional Ultrasound inspection, ultrasonic contrast has dynamically, show internal organs essence and the advantage such as focus blood vessel framework and perfused tissue situation in real time, continuously, the more important thing is, compared with additive method, easy, the easy repetition of ultrasonic contrast, cheapness, "dead", high without nephrotoxicity, safety.Nowadays, ultrasonic contrast has listed three kinds of conventional image diagnostic methods of liver in together with enhanced CT, M, and this technology has also extended to the application of multiple internal organs, as kidney, pancreas, spleen, thyroid, mammary gland, blood vessel etc., greatly promote the development of clinical ultrasound diagnosis.
The growth of tumor, invasion and attack, transfer depend on the formation of new vessels.Tumor vessel is by increasing oxygen and nutrition supply thus realizing increasing fast, invasion and transfer, but, tumor tissues medium vessels enriches, blood vessel wall gap is wider, poor structural integrity, the tumor vascular endothelial cell aperture ranges about 380 ~ 780nm of a lot of malignant tumor, this depends on different tumor cells, simultaneously, tumor tissues lymphatic return is poor, macromole class material and lipid granule is caused to have selectivity high-permeability and anelasticity, this phenomenon is referred to as the enhancing infiltration retention effect (EPR of solid tumor mass, enhancedpermeabilityandretentioneffect).The EPR effect of tumor vascular system can allow, in the middle of the macromolecular drug such as liposome, macromolecule micelle and gene delivery to tumor tissues, to 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.
But, current acoustic contrast agent mainly microbubble contrast clinically, for wrapping up the shell membrane microbubble structure that certain gas is formed, shell membrane can prevent the release of mutual fusion between microbubble and internal gas, thus enhances the stability of contrast agent, microbubble diameter 1-8 μm, can not arrive through blood vessel wall and be positioned at the cell exceeding blood capillary, as many cancerous cell, so they as developer in blood pond, can only limit the Clinics and Practices to the outer disease of its blood vessel.Along with going deep into further of nanotechnology research, the acoustic contrast agent of novel submicron order just engenders, comprise the liposome had from organizing different acoustic response, microemulsion, nano-emulsion and some nano-particle, these nanoparticles are as acoustic contrast agent, because the dimensional effect of its uniqueness can have targeting and therapeutical effect [RapoportN simultaneously, GaoZ & KennedyA.MultifunctionalNanoparticlesforCombiningUltraso nicTumorImagingandTargetedChemotherapy [J] .JNatlCancerInst, 2007, 99 (14): 1095-1106].Experimental studies have found that, the acoustic contrast agent that diameter is less than 900um has good targeting [HughesMS, MarshJN & HallCS, etal.AcousticCharacterizationinWholeBloodandPlasmaofSite-TargetedNanoparticleUltrasoundContrastAgentforMolecularI maging [J] .JAcoustSocAm, 2005,117 (2): 964-972].The acoustic contrast agent of current 1-7um is considered to the good size of development effect, if but contrast agent small-sized, then can enter tumor tissues through the irregular blood vessel wall of cancer new vessels and develop to picture.
In recent years, develop the acoustic contrast agent [WheatleyMA of the many new target tumor of Method and Technology manufacture based on nanoparticle, ForsbergF & DubeN, etal.Surfactant-StabilizedContrastAgentOntheNanoscalefor DiagnosticUltrasoundImaging [J] .UltrasoundMedBiol, 2006,32 (1): 83-93].In several research, various shell membrane (phospholipid or polymer) and core (gas are prepared, liquid or solid) nanoscale ultrasound contrast agents, and demonstrate good contrast strengthen [MarxerEE, BrusslerJ & BeckerA, etal.DevelopmentandCharacterizationofNewNanoscaledUltras oundActiveLipidDispersionsasContrastAgents [J] .EurJPharmBiopharm, 2011,77 (3): 430-437].Study on nano-scale contrast agents is used for tumor imaging, mainly because its high tissue exosmoses rate, can reach gratifying imaging in the increase of tumor region contrast agent.Liposome has as advantages such as nontoxic, biodegradable, non-immunogenicities because of it, is extensively used as the transport carrier of medicine or gene.The matrix material of these types is also used to wrap up the gas with sound reflecting, the contrast agent of several lipid film is had now to be applied to [GoertzDE in the middle of clinical ultrasound diagnosis by routine, deJongN & vanderSteenAF.AttenuationandSizeDistributionMeasurements ofDefinityandManipulatedDefinityPopulations [J] .UltrasoundMedBiol, 2007,33 (9): 1376-1388].The basic demand of desirable bubble acoustic contrast agent comprises: by blood capillary, has similar erythrocytic hemodynamics feature; Good stability, has good permeability and suitable surface tension, and can duration sufficient after entering human body; Abundant harmonic wave can be produced; The gas of Shell membrane materials 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 material and the raising of preparation method, the microbubble contrast of simultaneously loading gas, gene or medicine can be prepared, realize ultrasonic development strengthen carry out diagnosing while, also can reach the several functions such as transmission medicine, targeting location and disease treatment further, become the focus of ultrasound medicine research gradually.Gas componant in bubble contrast agent mainly comprises nitrogen, sulfur hexafluoride, perfluoropropane, perfluorinated butane, perflenapent, perflexane etc.The microbubble contrast intravenous injection of parcel air enters after in human body, air can be dissolved in the middle of blood very soon, determine its persistent period short, easily break, cause angiographic diagnosis also not terminate, microbubble disappears, thus limit in clinical practice the time observed and diagnose, in addition, due to the contraction of bubble and expansion under ultrasonic energy effect, the destruction of microbubble can be accelerated further.Parcel high density noble gas (not soluble in water or blood) is the thin and bubble of softness of main adventitia, and stabilization time is long, vibrate and echoing characteristics good.
Along with the development of nano material preparation technology, the size of bubble contrast agent and stability can be controlled, and become the useful tool of clinical ultrasound radiography.When the size of microvesicle is reduced to nanoscale, there is the character of some uniquenesses, comprised long half time, high surface reaction in the molecular characterization generation great change of contrast agent, and absorption affinity is strong, enzyme resistance degeneration etc.All these attributes are conducive to the application medically of new study on nano-scale contrast agents.Research display Pluronic block copolymer can stabilized nanoscale particle, controls their size, interacts with lipid film, the change mobility of lipid or the elasticity of lipid cell-shell, prevents particle from the effect such as to be engulfed by RE system.[the KrupkaTM such as TianyiM, SolorioL & WilsonRE, etal.FormulationandCharacterizationofEchogenicLipid-Plur onicNanobubbles [J] .MolPharm, 2010, 7 (1): 49-59] have studied 5 kinds of Pluronic block copolymer (L31 of molecular weight ranges 1100 ~ 4600, 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 significantly can 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 is injected after in human body through vein by acoustic contrast agent, utilizes contrast agent that rear scattered echoes is strengthened, thus significantly improve the resolving power of ultrasonic diagnosis, the technology of Sensitivity and Specificity.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 in 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, can the advantage of rechecking.Contrast-enhanced ultrasound technique, except the radiography harmonic imaging of routine, also has low mechanical index imaging, batch (-type) ultra sonic imaging, energy contast harmonic imaging, contrast agent explosion imaging, is excited the method such as acoustic emission imaging, back pulse harmonic imaging.Low mechanical index imaging refer to when adopt launch ultrasonic, its mechanical index MI lower than 0.15 time, be called low mechanical index.The radiography adopting the ultrasound wave of this energy when being broken up lower than acoustic contrast agent to carry out is 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 have employed real-time radiography and is matched to the inside and outside ultrasonic contrast that picture shadowgraph technique (CnTI) carries out nanometer acoustic contrast agent, CnTI uses the method for frequency domain process, during transmitting, only launch " pure " fundamental signal, during reception, the signal of main process second harmonic, the linear fundamental composition of tissue echo is eliminated while extracting contrast agent harmonic signal, the resolving power of contrast agent can be strengthened, improve signal to noise ratio, improve the contrast of acoustic picture quality and lifting image, make the border of focus and angiography [FrinkingPJ more clear than conventional image, BouakazA & KirkhornJ, etal.UltrasoundContrastImaging:CurrentandNewPotentialMet hods [J] .UltrasoundMedBiol, 2000, 26 (6): 965-975].Acoustic contrast agent is the physical basis of ultrasonic contrast diagnosing image to the echo-signal that ultrasound wave produces, its principle is the compressibility of bubble, the asymmetric resonance motion that contrast agent shows as under ul-trasonic irradiation " expansion-compression-reflation-recompression ", gas more easily compresses than biological tissues such as soft tissues, therefore when microbubble is subject to pulsed ultrasonic wave irradiation, microbubble experienced by pucker & bloat process alternately, 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 are that ultrasonic contrast quantitative study provides powerful measure.
Current Clinical practice acoustic contrast agent average diameter is several microns, and the granule that the 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, greatly reduces the ability that it is used for the treatment of.Therefore need to prepare the blood perfusion information being no longer confined to only obtain tissue, but by improve ultrasonoscopy specificity and to treatment field development contrast agent.But along with acoustic contrast agent carries out the multi-functional realization such as treating from single diagnostic function in conjunction with gene or medicine, the investigation and application of microbubble is just becoming more and more extensive, therefore, the acoustic contrast agent that particle diameter is less, stable in properties, Echoenhance are effective is prepared very important.Study on nano-scale contrast agents particle diameter is less, gives their extremely strong penetration powers, therefore becomes the focus of research in recent years.
Summary of the invention
Technical problem: the invention provides one and can realize the outer targeted developing of tumor vessel, apply to the nano-lipid acoustic contrast agent in tumor tissues diagnostic imaging and treatment, provide the method for the above-mentioned nano-lipid acoustic contrast agent of simple, easy preparation simultaneously.
Technical scheme: the preparation method of nano-lipid acoustic contrast agent of the present invention, comprises the steps:
(1) take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000 according to mol ratio 80:12:8 ~ 85:10:5, mixing is placed in container;
(2) in the obtained mixture of step (1), add the mixed liquor of chloroform and methanol, 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 ultrasonic vibration is carried out, until the said mixture matter in container is fully dissolved;
(3) container being placed with the obtained compounding substances of step (2) is satisfied with in rotary evaporator, 50 ~ 55 DEG C, rotating speed 80 ~ 100r/min, to run under vacuumized conditions, 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, blocked polyethers F-68 is added in the phosphate buffer of 0.01 ~ 0.1mol/L, what then joined that step (2) obtains is attached with in the container of lipid membrane, after lipin dissolving thin film, ultrasonic disperse until bottle wall thin film comes off completely, make it be formed liquid suspension that phospholipid concentration is 3 ~ 5 mg/ml;
(5) join in container by the liquid suspension obtained, after being filled with sulfur hexafluoride gas, vibration is until liquid suspension is creamy white, sticky, opaque;
(6) discard upper foam after leaving standstill, make liquid suspension layering after low-speed centrifugal, discard the microbubble that upper strata particle diameter is larger, take off the milky white liquid rinsing 3 to 5 times of layer, resuspendedly namely obtain nano-lipid contrast agent.
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 application in the outer targeted developing of ultra sonic imaging, particularly tumor vessel 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 has carried out structural characterization and study on the stability to the microbubble of preparation.By carrying out ultrasonic contrast to rat and mice with tumor, observing the video picture situation of the rat heart, liver, excess of the kidney matter Echoenhance situation and tumor, and comparing with the development effect of common micro level lipid contrast agent.Adopt the nanoscale lipid contrast agent that mechanical oscillation legal system is standby, in 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 be 413.8nm, zeta potential value is-23.39mV.EDS analysis result shows the elements such as carbon, nitrogen, oxygen and the phosphorus containing gas componant fluorine and sulfur and the composition lipid film wrapped up in nanoscale lipid bubble.Nanoscale lipid contrast agent is 1 grade to the cytotoxicity of L-929 cell, belongs to cytotoxic category, occurs without haemolysis.External supersonic development display 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, compared with before radiography, the enhancing video picture that rat heart, liver and kidney are visible obviously, lasting.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 is late, and peak strength is lower slightly, but the radiography 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 tissue sections.This nano-lipid acoustic contrast agent can realize the outer targeted developing of tumor vessel, can apply in tumor tissues diagnostic imaging and treatment.
In the inventive method, the dispersion of employing thin film adds the nanoscale ultrasound contrast agents that mechanical oscillation method has successfully prepared surface band negative charge, and the method is simple, easy.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 and strengthens ability with micron order lipid contrast agent and the similar ultrasound contrast of commercial sound Novi microbubble.Body interimage, after intravenous injection study on nano-scale contrast agents, compared with before radiography, the enhancing video picture that rat heart, liver and kidney are visible obviously, lasting.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 tissue sections.This nanoscale ultrasound contrast agents has good Imaging enhanced effect, 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.
Detailed description of the invention
Below by embodiment the present invention program done and illustrate further.
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 dispersion to add mechanical oscillation legal system for nano-lipid contrast agent, comprise the steps:
(1) take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000 according to mol ratio 80:12:8, mixing is placed in container;
(2) in the obtained mixture of step (1), add the mixed liquor of chloroform and methanol, 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 vibration, until the said mixture matter in container is fully dissolved;
(3) container being placed with the obtained compounding substances of step (2) is satisfied with in rotary evaporator, 50 DEG C, rotating speed 100r/min, to run under vacuumized conditions, 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, blocked polyethers F-68 is added in the phosphate buffer of 0.1mol/L, what then joined that step (2) obtains is attached with in the container of lipid membrane, after lipin dissolving thin film, ultrasonic disperse until bottle wall thin film comes off completely, make it be formed liquid suspension that phospholipid concentration is 3 mg/ml;
(5) join in container by the liquid suspension obtained, after being filled with sulfur hexafluoride gas, vibration is until liquid suspension is creamy white, sticky, opaque;
(6) discard upper foam after leaving standstill, make liquid suspension layering after low-speed centrifugal, discard the microbubble that upper strata particle diameter is larger, take off the milky white liquid rinsing 3 to 5 times of layer, resuspendedly namely obtain nano-lipid contrast agent.
Embodiment 2, basic procedure step is with embodiment 1, and difference is as follows:
Take distearyl acyl group lecithin according to mol ratio 83:11:6 in step (1), diphenyl phosphoryl azide, DSPE-PEG 2000 prepare nano-lipid contrast agent;
Be that 0.0008g/ml prepares nano-lipid contrast agent according to the mass volume ratio of the obtained mixture of step (1) and chloroform methanol mixed liquor in step (2), the volume ratio of chloroform and methanol is 1.5:1.
In step (3), obtained for step (2) mixture is satisfied with in rotary evaporator, 51 DEG C, rotating speed 80r/min, to run under vacuumized conditions.
In step (4), according to mass volume ratio 1.5 mg/ml, blocked polyethers F-68 is added in the phosphate buffer of 0.01mol/L, what then joined that step (2) obtains is attached with in the container of lipid membrane, after lipin dissolving thin film, ultrasonic disperse forms the liquid suspension that phospholipid concentration is 4 mg/ml.
In the present embodiment, the ratio of all the other operations and material composition is all identical with embodiment 1.
Embodiment 3, basic procedure step is with embodiment 1, and difference is as follows:
Take distearyl acyl group lecithin according to mol ratio 85:10:5 in step (1), diphenyl phosphoryl azide, DSPE-PEG 2000 prepare nano-lipid contrast agent;
Be that 0.001g/ml prepares nano-lipid contrast agent according to the mass volume ratio of the obtained mixture of step (1) and chloroform methanol mixed liquor in step (2), the volume ratio of chloroform and methanol is 1:1.
In step (3), obtained for step (2) mixture is satisfied with in rotary evaporator, 55 DEG C, rotating speed 90r/min, to run under vacuumized conditions.
In step (4), according to mass volume ratio 1.8 mg/ml, blocked polyethers F-68 is added in the phosphate buffer of 0.05mol/L, what then joined that step (2) obtains is attached with in the container of lipid membrane, after lipin dissolving thin film, ultrasonic disperse forms the liquid suspension that phospholipid concentration is 5 mg/ml.
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, obtained electron microscopic sample, observes under H-600 type transmission electron microscope (transmissionelectronmicroscope, TEM).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 surveys nanometer microvesicle mean concentration
9/ ml.Protect one week at-4 DEG C of refrigerators, contrast agent particle diameter, distribution are still comparatively even, have no obviously adhesion agglomerating.
2.3 scanning electron microscope energy disperse spectroscopy (SEM-EDS) phenetic analysis
Take a morsel the nano-lipid contrast agent prepared, get appropriate suspension to drip on copper mesh, several visual field is selected arbitrarily under the SEM visual field, with energy disperse spectroscopy, its composition is analyzed, the elements such as visible gas fluorine wherein containing parcel and the carbon of sulfur and composition lipid film, nitrogen, oxygen and phosphorus, confirm that nano-lipid contrast agent is successfully prepared.
2.4 particle diameters and zeta potentiometric analysis
The nano-lipid contrast agent getting 2mL PBS buffer (pH7.4) respectively diluted is placed in cuvette, particle diameter, Zeta potential mensuration is carried out with laser particle size analyzer (Brookhaven instrument company), application dynamic light scattering software carries out date processing, record mean diameter, polydispersity index and surface potential, each sample repeats 3 times.Detect that nano-lipid contrast agent average diameter is 413.8 ± 12.0nm by laser particle size analyzer, in 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 DEG C of Refrigerator stores, observed its stability after one week; Application blood counting instrument measures the concentration of nano-lipid contrast agent under light microscopic, count plate 5 middle lattice are received and are steeped sum, on the left of line ball Na Pao only counts and top, calculate 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 the RPMI1640 culture fluid containing 10% calf serum, and at 37 DEG C, cultivate in the incubator of saturated humidity, 5%CO2, every 2-3 days goes down to posterity once, trophophase cell of taking the logarithm during experiment.
The cytotoxicity experiment (MTT) of 3.2 nano-lipid contrast agent
Take the logarithm the L-929 cell of trophophase, conventional digestion becomes cell suspension and adjusts cell concentration to 5 × 10
4/ mL, 96 well culture plates are inoculated with 100 μ L/ holes, put 37 DEG C, cultivate in the incubator of 5%CO2, stock solution is discarded after 24 hours, add various different phosphate lipid concentration (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 the polyacrylamide monomer solution of 0.7%, often group establishes 8, multiple hole, continue cultivation after 72 hours, the morphological change of L929 cell is observed with inverted microscope, then every hole adds 20 μ LMTT, old terms cultivates 4 hours, discard liquid in hole, add DMSO150 μ L/ hole, shake after ten minutes, immune microplate reader measures the absorbance at 492nm place.Be calculated as follows the relative rate of increase of cell, following (the relativegrowthrate 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 0 or 1 order reaction is qualified, experimental result be 2 order reactions in conjunction with cellular morphology overall merit, experimental result is 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 grades | 0 |
Observe under inverted microscope and find, after adding various different phosphate lipid concentration (1 μ g/ml, 5 μ g/ml, 10 μ g/mL and 15 μ g/mL) RPMI1640 culture fluid, L-929 cell growth status is compared and no significant difference with negative control group, positive controls situation is then completely different, dosing after 24 hours namely visible cell become circle gradually, come off, then substantially without survivaling cell after 72 hours.The culture fluid effect L-929 cell of different phosphate lipid concentration is after 72 hours, and its RGR lists in table 2 respectively.It is 1 grade that result shows this nano-lipid contrast agent to L-929 cytotoxicity, and positive control is 4 grades, and combining form observation can judge that nano-lipid contrast agent should belong to cytotoxic category.
Table 2 nano-lipid contrast agent toxicity assessment MTT experiment result (
n=8)
| Group | OD value | RGR(%) | Toxicity is graded |
| Negative control group | 1.43±0.12 | 100 | 0 |
| 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
Extract blood 10mL from healthy new zealand rabbit auricular vein, add 20g/L potassium oxalate 0.5mL, add by every 8mL the fresh anticoagulant Sanguis Leporis seu oryctolagi that 10mL normal saline obtains dilution, 0.2mL Sanguis Leporis seu oryctolagi diluent is added in 10mL distilled water.Nano-lipid contrast agent normal saline presses 1 μ g/ml, 5 μ g/ml, 10 μ g/mL, and 15 μ g/mL configure; Normal saline is as negative control, and distilled water, as positive control, often organizes 3 test tubes.Detected materials will be housed, 37 DEG C of water-baths put into by all test tubes of normal saline and each 10mL of distilled water, take out after pre-temperature 30min, respectively add dilution anticoagulant Sanguis Leporis seu oryctolagi 0.2mL, shake up gently, put into 37 DEG C of water-bath relayings again to continue insurance warm 60min, each pipe solution is placed in the dry centrifugal (2500g/min of centrifuge tube, 5min), getting supernatant from every pipe moves in cuvette, spectrophotometer measures the OD value at 545nm place respectively, 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 lists in table 3 at the absorbance at 545nm place.According to formula: hemolysis rate (%)=Dt-Dnc/Dpc-Dnc, the hemolysis rate calculating variable concentrations nano-lipid contrast agent is respectively 0.782%, 0.615%, 0.407% and 0.328%, all much smaller than 5%, can think that experiment nano-lipid contrast agent is without haemolysis, meets 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 vitro in experiment, adopt homemade model to carry out, 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 is equipped with a linear array transducer (LA532E), ultrasonic contrast pattern is the specific pattern of the nondestructive contrast (harmonic imaging of contrast, and low acoustical power (mechanical index=0.089) CnTI), when changing into Flash pattern, MI is 0.5.ImageJ software is used to carry out carrying out quantitative analysis to the meansigma methods of sample.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 from initial hematimeter obtains).Contrast agent is before per injection, and necessary settling flux, pops one's head in and silica gel tube filling ultrasonic coupling agent therebetween; Observe development effect.Compare with commercial acoustic contrast agent sound Novi (Italian Bracco company) the external supersonic imaging of micron lipid contrast agent under similarity condition and clinical practice, 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), namely when ultrasonic contrast pattern enters into two-dimentional GTG height mechanical index state (MI is 0.5), its ultrasonogram becomes approximate echoless instantaneously, and perusal lipid Na Pao is become the clear liquid of similar transparent by milky suspension, oval ROI corresponding in the figure gray count that is averaged is shown in that (each sample repeats 3 times to Fig. 2, experimental result meansigma methods and standard deviation represent) shown in.
Ultra sonic imaging in 6 nano-lipid contrast agent bodies
Italian Bai Sheng TechnosMPX colorful Doppler ultrasound diagnostic apparatus is adopted 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 condition comprises field depth, gain, TGC etc. and all remains unchanged.Laboratory animal employing body weight is the Yunnan elder brother system SD rat of 250 scholar 50g, Rat Fast water more than 8 hours before checking, to reduce the impact of gastrointestinal peristalsis on image viewing, 10% chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia, rat extremity are fixed on flat board by dorsal position, remove the Mus hair at detection position, avoid the ultrasonic reflection of air on skin and Mus hair on the impact of 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 the good tangent plane of display rat heart, liver and kidney, use iron stand static probe, to ensure the change along with the time, the ultrasonoscopy obtained is the same anatomical position of rat.Ultrasonic contrast mode condition (mechanical index: 0.089, gain: 50, focus: 1.5cm) remains constant in all experiments process.Press 0.lml/Kg dosage bolus contrast agent through tail vein, use rapidly 1ml normal saline flushing subsequently, on diasonograph, observe rat heart, liver and kidney ultrasonic development imaging contexts immediately, 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.
The visible Hepatic artery of liver after ultrasonic development experiment display injection nano-lipid contrast agent normal saline solution in body, portal vein, hepatic vein develop enhancing successively, Ink vessel transfusing starts to occur strong catapoint, and there is the rapid filling of contrast agent, and spread to liver parenchyma, liver GTG intensity enhancing, in fine uniform point-like height echo, radiography after 30 minutes liver parenchyma echo still strengthen as seen, the trend that reduces gradually after first increasing 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 cell that trophophase form of taking the logarithm is good, discards culture in glassware liquid, PBS uses 0.25% trypsinization after rinsing, digestion is stopped, after collecting cell centrifugal (1000rpm, 5min) with the RPMll640 culture fluid containing 10% calf serum.Take out nude mice in superclean bench, 70% ethanol disinfection nude mice right fore axillary region skin, extracts about 0.2ml with lml syringe and (adjusts concentration with cell quantity 2 × 10 with normal saline
6individual) to inject nude mice right fore subcutaneous for cell suspension, 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 prevented cell suspension seepage.Postvaccinal nude mice is put into mouse cage, through 2-3 week in the subcutaneous tuberosity occurring grain of rice size of visible nude mice right fore, treats that diameter of tumor reaches about 1cm 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, leading B-ultrasound examination Subcutaneous tumor, proceeds to CNTI imaging mode after selecting maximum tangent plane.Mechanical index (MI) 0.089, through tail venous access bolus (0.lml/Kg) acoustic contrast agent, normal saline flushing passage is used immediately after injection, dynamic observation acoustic contrast agent strengthens situation to the development of Subcutaneous tumor echo, gather the image of 1min, 1.5min, 3min, 6min, 9min, 12min, 15min after injection of contrast medium respectively, the sequencing of two kinds of contrast-medium injection is random, inject contrast agent and at once play omnidistance record dynamic image, till contrast agent is cleaned up.After upper a kind of acoustic contrast agent acoustic image disappears about 30 minutes, another kind of acoustic contrast agent is injected in same nude mice same method, observe contrast agent as stated above to the enhancing situation of tumor echo, at the image of tumor, data quantitative analysis is carried out to nano-lipid acoustic contrast agent and common microbubble contrast agent, and adopts the method for self cross-reference to compare the two.
6 mice with tumor are taken up in order of priority after the nano-lipid contrast agent of bolus same dose and micron order acoustic contrast agent through tail vein, two kinds of contrast agent all can obviously strengthen subcutaneous transplantation tumor, there is strong catapoint in visible transplanted tumor Ink vessel transfusing, and fill rapidly, contrast agent from around tumor gradually to central part filling, and be full of rapidly whole transplanted tumor, GTG intensity strengthens rapidly, rear beginning is slowly cleaned up, and transplanted tumor GTG intensity also weakens, gradually without animal dead in experimentation.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 has some retention in tumor tissue sections (table 4, Fig. 3).
Table 4 nanoscale ultrasound contrast agents and micron order acoustic contrast agent comparing (x ± s) the ultrasonic enhancing video picture of tumor
| 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) take distearyl acyl group lecithin, diphenyl phosphoryl azide, DSPE-PEG 2000 according to mol ratio 80:12:8 ~ 85:10:5, mixing is placed in container;
(2) in the obtained mixture of described step (1), add the mixed liquor of chloroform and methanol, the mass volume ratio of mixture and mixed liquor is 0.0005g/ml ~ 0.001g/ml, in described mixed liquor, the volume ratio of chloroform and methanol is 1:1 ~ 2:1, then ultrasonic vibration is carried out, until the said mixture matter in container is fully dissolved;
(3) container being placed with the obtained compounding substances of described step (2) is placed in rotary evaporator, 50 ~ 55 DEG C, rotating speed 80 ~ 100r/min, to run under vacuumized conditions, 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, blocked polyethers F-68 is added in the phosphate buffer of 0.01 ~ 0.1mol/L, what then joined that described step (3) obtains is attached with in the container of lipid membrane, after lipin dissolving thin film, ultrasonic disperse until bottle wall thin film comes off completely, make it be formed liquid suspension that phospholipid concentration is 3 ~ 5 mg/ml;
(5) join in container by the liquid suspension obtained, after being filled with sulfur hexafluoride gas, vibration is until liquid suspension is creamy white, sticky, opaque;
(6) discard upper foam after leaving standstill, make liquid suspension layering, discard the microbubble that upper strata particle diameter is larger after low-speed centrifugal, the milky white liquid rinsing of taking off layer removes lipid bubble 3 to 5 times, resuspendedly namely obtains nano-lipid contrast agent.
2. a nano-lipid acoustic contrast agent, is characterized in that, this contrast agent according to claim 1 method prepares.
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