CN109646676A - The preparation method of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response - Google Patents
The preparation method of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response Download PDFInfo
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- A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
- A61K41/0033—Sonodynamic cancer therapy with sonochemically active agents or sonosensitizers, having their cytotoxic effects enhanced through application of ultrasounds
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Abstract
The present patent application belongs to biomedicine technical field, specifically discloses a kind of preparation method of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response, comprising the following steps: (1) synthesis has the adipose membrane of IR780；(2) PFP is added in the adipose membrane that hydration step (1) obtains, and sound sensitiser IR780-NDs is made after acoustic shock, centrifugation under ice bath.Using sound sensitiser made from this preparation method can multi-level deep penetration tumour, and be uniformly distributed in tumour.
Description
Technical field
The present patent application belongs to biomedicine technical field, specifically discloses the more of the Mitochondrially targeted acoustic response of one kind
The preparation method of level tumour deep penetration sound sensitiser.
Background technique
Mitochondria plays important work in the conduction of Apoptosis, apoptosis and mitochondria activity oxygen signal
With.Therefore, mitochondria is as the target spot right and wrong of anticancer drug often with attractive.
Sound motivation therapy is a kind of alternative medicine for treating cancer based on ultrasonic (US), sound sensitiser, sound is quick
Agent is in the research of oncotherapy, the main predicament that sound sensitiser faces to be how sound sensitiser to be selectively enriched to tumor area
Domain, and how sound sensitiser is delivered to the undesirable tumour deep regional of perfusion from intravascular.It " is seeped since tumor vessel is distinctive
Structure enhances EPR effect for leakage ", allow sound sensitiser in tumour passively accumulation (accumulation it is more, be more conducive to controlling for tumour
Treat), but accumulation efficiency is lower, so only leaning on passive accumulation of the sound sensitiser in tumour, cannot reach control tumour completely
The ideal effect for the treatment of.Also, sound sensitiser is poor to the penetrating power of tumour deep regional (so that it is in tumor tissues, blood vessel week
Side and tumour deep regional are unevenly distributed), sound sensitiser is greatly reduced to the therapeutic effect of tumour.
Summary of the invention
The purpose of the present invention is to provide a kind of multi-level tumour deep penetration sound sensitisers of Mitochondrially targeted acoustic response
Preparation method, it is low to solve accumulation efficiency of the existing sound sensitiser in tumour, and to the penetrating power of tumour deep regional
Poor, the problem of being unevenly distributed in tumour.
In order to achieve the above object, base case of the invention are as follows:
The preparation method of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response, comprising the following steps:
(1) synthesis has the adipose membrane of IR780；
(2) PFP is added in the adipose membrane that hydration step (1) obtains, and sound sensitiser IR780-NDs is made.
The principle and beneficial effect of this base case are:
IR-780 used in this preparation method is iodide (No. cas is 207399-07-3), and PFP is perflenapent
(No. cas is 375-61-1).Internal packet is just made by emulsion process simple, easy to operate and strong experimental repeatability for this preparation method
PFP is carried, shell modifies the sound sensitiser of IR780.
Firstly, being irradiated after this sound sensitiser is squeezed into blood vessel using US, the dispersal direction of this sound sensitiser are as follows: intravascular → blood
The tumor tissues on pipe periphery → tumour deep can be gathered in tumor tissues inner height, and can multi-level deep penetration tumour,
It is uniformly distributed in tumour, concrete principle is as follows:
1. US irradiation can inherently make the displacement of substance generation particle, to influence week since US is a kind of mechanical wave
Confining pressure power (i.e. generation cavitation effect), so that sound sensitiser is pushed away wave source, promote sound sensitiser along blood vessel periphery tumor tissues →
The diffusion of tumour deep direction.
2. PFP generates ADV effect under US irradiation, one side PFP becomes gas from liquid, forms microvesicle, formation it is micro-
Bubble further enhances cavitation effect, promotes process 1.；On the other hand, ADV effect can be destroyed between tumor vascular endothelium
Gap makes vascular endothelial gap become larger, and enters the sound sensitiser more in blood circulation in the tumor tissues on blood vessel periphery,
And tumour deep is entered by the tumor tissues on blood vessel periphery.
Other than ADV effect can promote this sound sensitiser to be spread by the tumor tissues → tumour deep on blood vessel periphery,
IR780 can also promote this sound sensitiser and be spread by the tumor tissues → tumour deep on blood vessel periphery.
Secondly, this sound sensitiser can also inducing apoptosis of tumour cell, concrete reason is as follows: 1. US irradiates IR780, IR780
It generates active oxygen (ROS), ROS can result in cytotoxicity, and (IR780 can reach without US irradiation to be moved to tumour deep
Effect, but active oxygen will not be generated).2. US can direct inducing apoptosis of tumour cell.
Secondly, this sound sensitiser, which does not need additional chemical coupling, can be targeted to mitochondria.
Secondly, this sound sensitiser can also multi-modality imaging (ultrasonic US, living body fluorescent FL and optoacoustic PA imaging), PFP is in US
US imaging can be used for from unique phase transformation ability that liquid becomes gas (forming microvesicle) under irradiation；IR780 is strong near infrared region
Absorption can be used in FL imaging and PA imaging.Multi-modality imaging can monitor the distribution situation of sound sensitiser each internal organs in vivo, refer to
Lead sound dynamic therapy.
To sum up, this sound sensitiser can reach multiplication enhancing tumour deep SDT, can direct inducing apoptosis of tumour cell, energy
It is enough that Mitochondrially targeted SDT is carried out to tumour, can multi-modality imaging, effectively ensure this sound sensitiser to the therapeutic effect of tumour.
In addition, being nanoscale when this sound sensitiser is in liquid, sufficiently stable, under US irradiation, this sound sensitiser is by nanoscale liquid
Drop is changed into microbubble, this sound sensitiser has been effectively ensured to the therapeutic effect of tumour in microbubble.
Further, after PFP is added in step (2), sound sensitiser IR780-NDs is made after acoustic shock, centrifugation under ice bath.In ice bath
Lower acoustic shock, the operating procedure of centrifugation are relatively simple, convenient for the popularization and application of this preparation method.
Further, the step (1) synthesizes adipose membrane using DPPC, DSPE-PEG2000, cholesterol and IR780, and DPPC is
Dipalmitoylphosphatidylcholine, DSPE-PEG2000 are distearoylphosphatidylethanolamine-polyethylene glycol 2000.DPPC,
DSPE-PEG2000 and three kinds of raw materials of cholesterol are relatively conventional, convenient for the popularization and application of this preparation method.
Further, in mass ratio, DPPC:DSPE-PEG2000: cholesterol: IR780=8-16:2-6:2-6:1-3.Encapsulating
Efficiency is higher, this sound sensitiser is better in the targeting of mitochondria and tumour, better to the therapeutic effect of tumour, in the above mass ratio
Under, encapsulation rate of the IR780 in sound sensitiser IR780-NDs can reach 83%.
Further, in mass ratio, DPPC:DSPE-PEG2000: cholesterol: IR780=12:4:4:1.In the above mass ratio
Under, the performance of sound sensitiser IR780-NDs obtained is preferable.
Further, the intensity of the acoustic shock of the step (2) is 100-150W.Under the conditions of the above acoustic shock, this sound sensitiser pair
PFP to contain efficiency higher.
Detailed description of the invention
Fig. 1 is the light microscopic figure of sound sensitiser IR780-NDs made from the embodiment of the present invention 1；
Fig. 2 is the optical microscope image of present invention experiment one；
Fig. 3 is the fluorescence intensity of the SOSG probe of present invention experiment two；
Fig. 4 is the living body fluorescent image of present invention experiment six.
Specific embodiment
It is further described below by specific embodiment:
Embodiment 1
A kind of preparation method of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response, specifically include with
Lower step:
(1) synthesis has the adipose membrane of IR780: DPPC, DSPE-PEG2000, cholesterol and IR780 are mixed (in mass ratio
It for 12:4:4:1), after being dissolved in 5ml chloroform, is transferred in round-bottomed flask and carries out rotary evaporation, form adipose membrane；
(2) the PBS aquation adipose membrane for using 4ml, is added the PFP of 400 μ L, under ice bath, in the acoustic shock condition that intensity is 125W
Lower acoustic shock 5min (the mode acoustic shock 5min to shake 5s, suspend 5s), is finally centrifuged (6000rpm, 5min) and sound sensitiser is made
IR780-NDs。
Embodiment 2-9 and comparative example 1-2
The difference of embodiment 2-5 and embodiment 1 is only that the mass ratio of DPPC, DSPE-PEG2000, cholesterol and IR780
Difference, embodiment 6-9 the difference from embodiment 1 is that acoustic shock intensity, referring specifically to the following table 1.
Comparative example 1 is the blank nano-liquid droplet (hereinafter referred to as NDs) of the unmodified IR780 of shell, the system of comparative example 1 (NDs)
The difference of Preparation Method and this sound sensitiser IR780-NDs, which are only that in synthesis adipose membrane step, is not added with IR780.
Comparative example 2 is that shell modifies IR780, but inside contains blank liposome (the hereinafter referred to as IR780- of PFOB
PFOB)。
Table 1
Conclusion:
(1) DPPC:DSPE-PEG2000: cholesterol: IR780 mass ratio in comparative example 3=7:1:1:1 < 8-16:
2-6:2-6:1-3；DPPC:DSPE-PEG2000 in comparative example 7: cholesterol: the mass ratio of IR780=17:7:7:1 > 8-16:
2-6:2-6:1-3, in the case where PFP is quantitative, DPPC:DSPE-PEG2000: cholesterol: the mass ratio > 8-16:2- of IR780
When 6:2-6:1-3, the PFP contained in sound sensitiser is very few；DPPC:DSPE-PEG2000: cholesterol: the mass ratio < 8- of IR780
When 16:2-6:2-6:1-3, the balling ratio of sound sensitiser is lower.
The intensity of acoustic shock in comparative example 5=90W < 100W (intensity of acoustic shock is too small), acoustic shock in comparative example 6 it is strong
Degree=160W > 150W (intensity of acoustic shock is excessive), sound sensitiser size prepared by comparative example 5 is inhomogenous, and sound sensitiser balling ratio
It is lower, in 6 preparation process of comparative example, observes that PFP becomes gas from liquid, form microvesicle, be unfavorable for adipose membrane to the packet of PFP
It carries.
The size of sound sensitiser IR780-NDs made from embodiment 1-5 is uniform, and the PFP amount contained in adipose membrane is larger, and rouge
Film can reach 83% to the encapsulation rate of PFP.
(2) embodiment 1 (IR780-NDs) and comparative example 1 (NDs) are dissolved in water, IR780-NDs aqueous solution is in
Blue-green, NDs aqueous solution are creamy white.
(3) sound sensitiser IR780-NDs obtained by embodiment 1 is characterized, embodiments parameter is shown in Fig. 1:
1. from optical microscope image (Fig. 1) as can be seen that the size of this sound sensitiser IR780-NDs is uniform, rounded.
2. the partial size of this sound sensitiser can enter tumour through tumor vascular endothelium gap by EPR effect in 300nm or so
Tissue, tumour deep.
3. acquiring the ultraviolet spectrogram of this sound sensitiser IR780-NDs, this sound sensitiser IR780-NDs has maximum at 780nm
Absorption peak.
Experiment one
Sound sensitiser IR780-NDs made from embodiment 1 is dissolved in deionized water, it is quick that the sound that concentration is 2mg/mL is made
Agent IR780-NDs aqueous solution.
Ultrasound wave irradiation instrument is used to irradiate concentration for the sound sensitiser IR780-NDs aqueous solution of 2mg/mL.Different strong
Degree is (in 0.8,1.6,2.4,3.2 and 4.0W/cm2Irradiated 3 minutes under intensity) irradiation.
Conclusion:
(1) as shown in Fig. 2, optical microscope image is acquired, in 0.8W/cm2Ultrasound intensity under do not detect it is obvious
Microvesicle；When intensity reaches 1.6W/cm2When, microvesicle increasingly generates, and the quantity of microvesicle and degree of transformation are with US irradiation intensity
Increase and increases；In 2.4W/cm2Under intensity, sound sensitiser IR780-NDs almost all is converted into microvesicle, and part microvesicle is opened
Begin to rupture；In 3.2W/cm2Under intensity, microvesicle gradually ruptures, in 4.0W/cm2Under intensity, sound sensitiser IR780-NDs microvesicle is almost
All ruptures.
(2) the ultrasound image discovery for acquiring this sound sensitiser IR780-NDs, in 0.8W/cm2Ultrasound intensity under do not detect
Enhance signal to ultrasound, in 1.6-2.4W/cm2Ultrasound intensity under, ultrasonic contrast observes apparent ultrasound enhancing, and ultrasound is strong
The variation tendency of degree is identical as the forming process of microvesicle in conclusion (1).
(3) using the echo strength of ultrasonic analysis software measurement sound sensitiser IR780-NDs (for measuring the microvesicle number generated
Amount), echo strength is stronger, and the bubble that this sound sensitiser IR780-NDs is generated is more, finds after measurement: the quantitative knot of echo strength
Fruit is consistent with optical microscope image, the result of ultrasound image.
Under US irradiation, PFP generates ADV effect, and ADV effect can cause ultrasonic enhancing, this experiment one is found: microvesicle
The trend generate, ruptured is consistent with ultrasonically enhanced variation tendency, so being inferred to: containing in sound sensitiser IR780-NDs core
PFP to ADV effect, US imaging performance have notable contribution.
Experiment two
Since SOSG probe can be reacted with ROS, fluorescence can be generated under 488nm wavelength, it is possible to use SOSG
The ROS generated as detection probe detection.Respectively in US intensity (2.4W/cm2), irradiation time (0,30,60,90 and 120s)
Under the conditions of irradiate sound sensitiser IR780-NDs made from embodiment 1, record the fluorescence intensity (such as Fig. 3) of SOSG probe, wherein SOSG
The maximum fluorescence intensity of probe appears in 530nm, such as the following table 2.
Table 2
| Irradiation time/s | 0 | 30 | 60 | 90 | 120 |
| The fluorescence intensity of SOSG probe | 84.27801 | 135.8032 | 225.027 | 246.2628 | 280.3845 |
Conclusion:
Can be obtained by Fig. 3 and table 2, US irradiation time it is longer, IR780 generate ROS it is more, ROS generate it is more more can
This sound sensitiser IR780-NDs is enough improved to the cytotoxicity of tumour cell.
Experiment three
Under US irradiation, perfluorooctyl bromide (PFOB) cannot generate microvesicle, thus ADV effect cannot occur, so this hair
It is bright that penetration depth of the sound sensitiser IR780-NDs in tumour is assisted as a comparison to verify ADV effect using PFOB.
US irradiation embodiment 1 (IR780-NDs), comparative example 1 (NDs) and comparative example 2 (IR780-PFOB) are chosen, is adopted respectively
Embodiment 1 (IR780-NDs) and comparative example 2 (IR780-PFOB) are irradiated with US, and to embodiment 1 (IR780-NDs), comparative example
1 (NDs) and comparative example 2 (IR780-PFOB) carry out fluorescence analysis.
Conclusion:
Comparative example 1 (NDs), comparative example 2 (IR780-PFOB) and embodiment 1 (IR780-NDs) with a distance from blood vessel successively
Increase, wherein not being distributed only near vessels using the comparative example 1 (NDs) of US irradiation.The embodiment 1 irradiated using US
(IR780-NDs) it is distributed in the place far from blood vessel, this proves that ADV effect and ultrasound wave irradiation can induce sound sensitiser deep at many levels
Tumour is penetrated, is uniformly distributed in tumour.
Experiment four
The intake feelings of sound sensitiser IR780-NDs in the cell are studied by confocal laser scanning microscopy and flow cytometry
Condition.In different incubation time (1h, 2h, 3h and 4h) observations mouse mastopathy cell (hereinafter referred to as 4T1 cell) to embodiment 1
(IR780-NDs) and the intake situation of comparative example 1 (NDs).
Conclusion:
Confocal Images show that the IR780-NDs of 4T1 cell peripheral will be significantly more than the NDs of 4T1 cell peripheral, this proof
IR780-NDs can be effectively by 4T1 cellular uptake, but NDs is poor by the intake situation of 4T1 cell.
In addition, for embodiment 1 (IR780-NDs), when the fluorescence intensity of flow cytometer measurement is with total incubation
Between increase and significantly increase, focusing results are consistent together, and also demonstrating IR780-NDs can be effectively by 4T1 cellular uptake.
Experiment five
The three-dimensional nodule modeling tumour that tumor perfusion is uneven, cell density is high, interstitial fluid pressure increases is constructed, for grinding
This sound sensitiser IR780-NDs is studied carefully to the penetration capacity of tumour.
Conclusion:
The sound sensitiser IR780-NDs of a large amount of DiI labels issues red fluorescence, penetrates tumour core sphere, and be distributed in whole
A sphere.But comparative example 1 (NDs) is only adhered to tumour spherome surface.
Experiment six
To 4T1 tumor-bearing mice be injected intravenously embodiment 1 (IR780-NDs), and in different time points (0.5h, 1h, 2h, 4h,
6h and for 24 hours) carry out living body fluorescent imaging.And after 24 hours, external fluorescence imaging is carried out to the major organs of mouse.
Conclusion:
As shown in figure 4, there is apparent fluorescence signal in mouse tumor region, with pushing away for time after injection 0.5-24 hours
Move tumor region signal enhancing.
After 24 hours, carry out external fluorescence imaging discovery to the major organs of mouse: the fluorescence of the tumor tissues of mouse is most
By force, the fluorescence signal of accumulation will be significantly stronger than other organs (heart, liver, spleen, lung, kidney).Main organs and tumour are taken to carry out pathology
It checks, a large amount of sound sensitiser IR780-NDs is gathered in tumor tissues, and NDs is mainly gathered in liver and spleen.
Experiment seven
Using Mito Tracker (there is green fluorescence) labeled mitochondria, DiI (there is red fluorescence) label embodiment 1
(IR780-NDs) and comparative example 1 (NDs), using Laser Scanning Confocal Microscope to embodiment 1 (IR780-NDs) and comparative example 1 (NDs)
Subcellular localization be monitored and compare.
Conclusion:
(1) in the presence of IR780-NDs, in mitochondria region detection to significant red fluorescence.When image weight
When folded, red fluorescence and green fluorescence good knitting illustrate that IR780-NDs has Mitochondrially targeted ability；
(2) in the presence of NDs, there is no apparent red fluorescence in mitochondria region.
Result above prove effective in tumour of sound sensitiser IR780-NDs accumulate, multi-level deep penetration and as fluorescence
Imaging contrast agent and the ideal characterisitics that mitochondria can be targeted to.
What has been described above is only an embodiment of the present invention, and the common sense such as well known specific structure and characteristic are not made herein in scheme
Excessive description.It should be pointed out that for those skilled in the art, under the premise of not departing from the present invention, can also make
Several modifications and improvements, these also should be considered as protection scope of the present invention, these all will not influence the effect that the present invention is implemented
And patent practicability.
Claims (6)
1. the preparation method of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response, which is characterized in that including
Following steps:
(1) synthesis has the adipose membrane of IR780；
(2) PFP is added in the adipose membrane that hydration step (1) obtains, and sound sensitiser IR780-NDs is made.
2. the preparation side of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response according to claim 1
Method, which is characterized in that after PFP is added in step (2), sound sensitiser IR780-NDs is made after acoustic shock, centrifugation under ice bath.
3. the preparation side of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response according to claim 1
Method, which is characterized in that the step (1) synthesizes adipose membrane using DPPC, DSPE-PEG2000, cholesterol and IR780.
4. the preparation side of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response according to claim 3
Method, which is characterized in that in mass ratio, DPPC:DSPE-PEG2000: cholesterol: IR780=8-16:2-6:2-6:1-3.
5. the preparation side of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response according to claim 4
Method, which is characterized in that in mass ratio, DPPC:DSPE-PEG2000: cholesterol: IR780=12:4:4:1.
6. the preparation side of the multi-level tumour deep penetration sound sensitiser of Mitochondrially targeted acoustic response according to claim 2
Method, which is characterized in that the intensity of the acoustic shock of the step (2) is 100-150W.
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