CN108114290A - Preparation method that is a kind of while loading chemicals and the excretion body of nano material - Google Patents
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- CN108114290A CN108114290A CN201810004207.4A CN201810004207A CN108114290A CN 108114290 A CN108114290 A CN 108114290A CN 201810004207 A CN201810004207 A CN 201810004207A CN 108114290 A CN108114290 A CN 108114290A
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Abstract
The invention discloses a kind of while load the preparation method of chemicals and the excretion body of nano material, include the following steps：1) " classical differential supercentrifugation " is utilized to extract the excretion body of cell secretion from cell culture supernatant；2) curcumin in aqueous solution and nano ferriferrous oxide are loaded into excretion body simultaneously using the method for electroporation；3) OD260nm (RNA characteristic absorption peaks) verifications are measured to perforate successfully；4) perforate after by mixed liquor be immediately placed in cell incubator be incubated 1 it is small when, to promote excretion body film reparation；5) 100000g ultracentrifugations remove free curcumin and nano ferriferrous oxide, are carried the compound excretion body of curcumin and nano ferriferrous oxide simultaneously.This method is simple and practicable, success rate is high, and the capacity value of curcumin and nano ferriferrous oxide can meet inside and outside treatment in compound excretion body obtained and imaging requirements provide new instrument for the targeting diagnose and treat of a variety of chronic diseases including tumour.
Description
Technical field
The present invention relates to a kind of compound excretion bodies for having both magnetic resonance imaging and magnetic thermotherapeutic function and drug therapy effect.
Background technology
Nano ferriferrous oxide is one kind of nano material, have be readily synthesized, superparamagnetism, high saturation and magnetic intensity,
Good biocompatibility, low toxin, it is considered to be the production of a kind of good magnetic resonance imaging (MRI) contrast agent and magnetic thermotherapy
Hot material.Curcumin is a kind of vegetalitas polyphenolic substance, has stronger antitumor action.Experiment in vitro discovery, curcumin
There is apparent inhibitory action to the kinds of tumor cells growth including glioma, main mechanism is to inhibit tumour cell
Multiplication, invasion and attack, promote apoptosis of tumor cells, Antineoplastic angiogenesis etc., therefore curcumin is in the drug treatment of glioma
With broader prospect.However the two has the shortcomings that some are apparent, such as：Dissolubility, stabilization in blood circulation system
Property is poor, is easily swallowed by mononuclear macrophage, is often more gathered in liver and spleen and lymphoid tissue, the concentration of tumor locus compared with
It is low, influence imaging and antitumor effect.Material and/or drug are wrapped up with suitable carrier, improved in blood circulation
Dissolubility, stability, avoid/reduce immune clearance, and it is the pass for overcoming disadvantages mentioned above that can more specifically reach tumour cell
Key and the hot spot studied at present.Excretion body is the biological microcapsule bubble of cell derived, has stronger load capacity and relatively low
Immunogenicity is a kind of noticeable drug/material delivery vector.Research report excretion body can carry out different mesh in vitro
The load of object is marked, (is received including nucleic acid molecules (siRNA, miRNA etc.), chemicals (adriamycin, taxol etc.) and nano material
Meter Jin, Nanoscale Iron etc.).For for the tumour in tumour, especially deep, it is necessary to by means of imageological examination find lesion, it is auxiliary
Diagnosis, evaluation change of illness state are helped, so, it is just particularly important with treating the dynamic imaging to accompany, in order to reach to tumour
The concertedness and uniformity of diagnose and treat, embody the integration of diagnosis and treatment, the reality based on ripe theoretical foundation and Optimal improvements
Condition is tested, we have invented this imagings that can be used for tumour simultaneously and the preparation method of the compound excretion body for the treatment of.
The content of the invention
Technical problem：The object of the present invention is to provide a kind of while load the system of chemicals and the excretion body of nano material
Preparation Method.Compound excretion body obtained can play the effect of MRI imagings, magnetic thermotherapy and drug therapy simultaneously, for clinic later
Application study provides reliable experimental study data and theoretical foundation.
Technical solution：The excretion preparation that is a kind of while loading chemicals and nano material of the present invention is a kind of
Have both the compound excretion preparation of imaging and treatment function.
Be loaded with inside the excretion body available for neoplasm tracing, imaging and magnetic thermotherapy nano ferriferrous oxide and can
For the curcumin of oncotherapy；Wherein nano ferriferrous oxide is aqueous solution, and curcumin is dimethyl sulfoxide (DMSO) (DMSO) solution,
Its specific preparation process is as follows：
1) the excretion body of cell secretion is extracted from mouse macrophage Raw264.7 culture solutions, using classical differential
Supercentrifugation, obtained excretion weight are suspended from the PBS solution of the trehalose containing 50mM, and -80 DEG C save backup；
It 2) will be described in the addition of the nano ferriferrous oxide aqueous solution that prepared, curcumin DMSO solution and excretion body suspension
Trehalose PBS solution in, shake mixing, obtain sample；
3) the above-mentioned sample of respective volume is added in by electroporation apparatus requirement and electroporation ware specification, is put into electroporation apparatus,
The optimal perforation condition setup parameter groped according to preliminary experiment, into eleven punch 11；
4) sampled immediately after perforating, light absorption value of the sample at 260nm detected on ultraviolet-visible spectrophotometer,
And compared with puncherless sample, judge whether perforation succeeds；
5) sample is immediately placed in 1 hour of incubation in cell incubator after remaining perforation, to promote excretion body film reparation；
6) 1 it is small when after, by aforesaid liquid with 100,000-14 ten thousand g (RCF) centrifugal force ultracentrifugations twice, each 70-90min,
Most supernatant is abandoned, sediment is the excretion body for loading nano ferriferrous oxide and curcumin simultaneously, is referred to as compound excretion body；
7) above-mentioned compound excretion body is added in the burnt culture dish of copolymerization, and fluorescence microscopy Microscopic observation curcumin is from fluoresced green
Proof contains curcumin success, and above-mentioned excretion body prepares copper mesh specimen, in iron particle in electric Microscopic observation excretion body, it was demonstrated that nanometer
Ferroso-ferric oxide loads successfully；
8) compound excretion body is resuspended in PBS, with ferrosin Spectrophotometric Determination of Iron content, is surveyed with visible spectrophotometer
Determine turmeric cellulose content, excretion body content is surveyed with BCA methods (with protein refractometer)；
9) compound excretion body suspension is taken to carry out nano particle trace analysis detection NTA or dynamic optical reflective analysis DLS to survey
Its fixed particle size and distribution, and its size and shape characteristic, western blot detections are observed under transmission electron microscope TEM
The expression of excretion body film surface characteristic protein (compares) with excretion body before perforation；
9) above-mentioned compound excretion body suspension packing is put in -80 DEG C of refrigerators, sample week about once measure it is above-mentioned
Concentration and excretion body characteristics, the stability of evaluating combined excretion body；
The mouse macrophage Raw264.7 culture solutions are containing no excretion body serum or without serum.
In the step 2), 100 μ g- are separately added into the phosphate buffered saline solution PBS solution of 1ml trehaloses containing 50mM
300ug excretions body, 5-20 μ l nano ferriferrous oxides aqueous solutions and 10-30 μ l curcumins DMSO solution composition hybrid reaction body
System.
The excretion body is in terms of its protein content, and the concentration of curcumin DMSO solution is 10-30mg/ml, and nanometer four aoxidizes
The concentration of three water solutions is 1-10mg/ml.
In the step 3), electroporation conditions are：Voltage 100-700v, capacitance 125-500 μ F, discharge time 1-5ms are put
Electric number：1 time.
It is described 8) in, institute's iron content and the concentration of curcumin use UV-visible spectrophotometry in compound excretion body
It measures.
Advantageous effect：Compared with existing simple carrying medicament or the excretion body of simple load nano material, this research is used
Excretion body nano ferriferrous oxide and curcumin simultaneously, compound excretion body obtained on the one hand add loaded article dissolubility,
Stability, when reducing vivo applications by the phagocytosis of immune system and remove may；On the other hand, due to the compound excretion body
Maintain the grain size of its Nano grade and good mobility, during vivo applications, tumour can be carried out at the same time targeted imaging with
Targeted therapy ensures timely, the dynamic evaluation of curative effect, realizes concertedness and uniformity to tumor diagnosis and therapy, embodies
Diagnosis and treatment integrated Advanced Ideas.Meanwhile the invention has specific theoretical foundation,
Ripe experimental method and easy-to-use operating process.
Description of the drawings
Fig. 1 is the electron microscope of the nano ferriferrous oxide prepared with high-temperature cracking method；
Fig. 2 is nano particle trace analysis (NTA) measurement excretion body particle size and distribution map before electroporation；
Fig. 3 is nano particle trace analysis (NTA) measurement excretion body particle size and distribution map after electroporation；
Fig. 4 is transmission electron microscope (TEM) observation excretion body size and shape characteristic before electroporation；
Fig. 5 is transmission electron microscope (TEM) observation excretion body size and shape characteristic after electroporation；
Fig. 6 is excretion body characteristics memebrane protein (CD63) expression (Western blot) before and after electroporation.
Specific embodiment
Carrier and loaded article (excretion body, nano ferriferrous oxide and curcumin) are obtained first：
1) extraction of excretion body, purifying：Raw264.7 cell supernatant 300ml are collected, carry out differential hypervelocity as follows
Centrifugation：300g×10min→2000g×10min→10000g×30min→100000g×70min→100000g×
70min；Whole sediment is resuspended with 500 μ lPBS liquid, and 50 μ l sample BCA methods is taken to survey protein content.Used in the method for the present invention
Excretion body, also other admissible extractions obtain, and excretion body as drug and the common carrier of material, carries in the methods of the invention
Method and flow is taken to be not intended to limit the scope of the present invention..
2) preparation of nano ferriferrous oxide：
It is prepared using high temperature pyrolytic cracking (HTP)：1. weigh 0.7g ferric acetyl acetonades, 20mL benzyl oxides, 2mL oleic acid and the transfer of 6mL oleyl amines
Into 100mL three-necked round bottom flask, the glass dropper for being connected with argon gas is stretched into below liquid level, adjustment throughput plays stirring and makees
With；2. start heating schedule：220 DEG C are warming up to using 60min from room temperature and keep 60min, be warming up to system using 30min
290 DEG C and keep 30min；3. continuing to be passed through argon gas after heating and being allowed to cooled to room temperature, 30mL is added in into system
Absolute ethyl alcohol and ultrasonic disperse 10min；It 4. system is transferred to small beaker, is placed in strong magnets and stands 10min, utilize magnetic force
Effect obtains nanoscale ferroso-ferric oxide precipitation, after abandoning supernatant, after adding in 30mL ethyl alcohol and ultrasonic disperse 10min again, continues
It is put in magnet and stands 10min, after which repeats 3-4 times, abandon supernatant, 10mL n-hexanes are added in into precipitation, ultrasonic disperse obtains
To homodisperse nano ferriferrous oxide hexane solution；5. by the dilute hydrochloric acid solution mixing of the said goods and 20mL pH=4
After mechanical agitation 2h, into system add in acetone 10mL ultrasonic disperses it is uniform after, 360000g centrifuge washings for several times after, obtain water
The nano ferriferrous oxide solution of dissolubility (Electronic Speculum confirms grain size for 5nm or so).Four oxygen of nanometer employed in the method for the present invention
Change three-iron, also prepared by admissible other to obtain, and nano ferriferrous oxide possesses the radiography of magnetic resonance imaging function in the present invention
Agent and the heat production agent of magnetic thermotherapy, therefore its preparation method and flow are not intended to limit the scope of the present invention..
3) curcumin solution is prepared：24mg curcumin powder is weighed, is dissolved in 1mLDMSO, obtains 24mg/ml solution：
Then Nanoscale Iron and curcumin are loaded by excretion body by electroporation again：
4) excretion body, nano ferriferrous oxide aqueous solution, curcumin are added in by a certain percentage in trehalose PBS solution
DMSO solution；It after mixing, adds in electroporation ware, is put into electroporation apparatus, electroporation is carried out according to certain reaction condition.
5) perforate after excretion body through 100,000-14 ten thousand g (RCF) centrifugal force ultracentrifugations twice, each 70-90min, abandon to the greatest extent on
Clearly, sediment is the excretion body for loading nano ferriferrous oxide and curcumin simultaneously, is referred to as compound excretion body；
With reference to embodiment, the present invention is furture elucidated, it should be appreciated that following specific embodiments are only used for
It is bright the present invention rather than limit the scope of the invention.
Embodiment 1
100 μ g of excretion body, 10 μ of 4mg/mL nano ferriferrous oxides aqueous solution are added in 1mL50mM trehalose PBS solutions
L, 5 μ l of 24mg/mL curcumins DMSO solution；After mixing, add in 4mm (750 μ l of volume) electroporation ware, be put into electroporation apparatus,
According to following reaction condition：Voltage 100V, capacitance 125 μ F, discharge time 1ms, discharge time：1 time.It can will be suspended after perforation
Liquid is put into 1 hour of incubation in cell incubator, and followed by twice, each 80min carefully abandons most supernatant to 100000g ultracentrifugations,
The compound excretion body that precipitation is load nano ferriferrous oxide and curcumin is resuspended with PBS liquid, -80 DEG C of storages are subsequently examined
It surveys and tests；
Embodiment 2
100 μ g of excretion body, 10 μ of 4mg/mL nano ferriferrous oxides aqueous solution are added in 1mL50mM trehalose PBS solutions
L, 5 μ l of 24mg/mL curcumins DMSO solution；After mixing, add in 4mm (750 μ l of volume) electroporation ware, be put into electroporation apparatus,
According to following reaction condition：Voltage 400V, capacitance 250 μ F, discharge time 1ms, discharge time：1 time.It can will be suspended after perforation
Liquid is put into 1 hour of incubation in cell incubator, and followed by twice, each 80min carefully abandons most supernatant to 100000g ultracentrifugations,
The compound excretion body that precipitation is load nano ferriferrous oxide and curcumin is resuspended with PBS liquid, -80 DEG C of storages are subsequently examined
It surveys and tests；
Embodiment 3
100 μ g of excretion body, 10 μ of 4mg/mL nano ferriferrous oxides aqueous solution are added in 1mL50mM trehalose PBS solutions
L, 5 μ l of 24mg/mL curcumins DMSO solution；After mixing, add in 4mm (750 μ l of volume) electroporation ware, be put into electroporation apparatus,
According to following reaction condition：Voltage 700V, capacitance 500 μ F, discharge time 1ms, discharge time：1 time.It can will be suspended after perforation
Liquid is put into 1 hour of incubation in cell incubator, and followed by twice, each 80min carefully abandons most supernatant to 100000g ultracentrifugations,
The compound excretion body that precipitation is load nano ferriferrous oxide and curcumin is resuspended with PBS liquid, -80 DEG C of storages are subsequently examined
It surveys and tests；
Embodiment 4
100 μ g of excretion body, 30 μ of 4mg/mL nano ferriferrous oxides aqueous solution are added in 1mL50mM trehalose PBS solutions
L, 20 μ l of 24mg/mL curcumins DMSO solution；After mixing, add in 4mm (750 μ l of volume) electroporation ware, be put into electroporation apparatus,
According to following reaction condition：Voltage 100V, capacitance 125 μ F, discharge time 1ms, discharge time：1 time.It can will be suspended after perforation
Liquid is put into 1 hour of incubation in cell incubator, and followed by twice, each 80min carefully abandons most supernatant to 100000g ultracentrifugations,
The compound excretion body that precipitation is load nano ferriferrous oxide and curcumin is resuspended with PBS liquid, -80 DEG C of storages are subsequently examined
It surveys and tests；
Embodiment 5
100 μ g of excretion body, 30 μ of 4mg/mL nano ferriferrous oxides aqueous solution are added in 1mL50mM trehalose PBS solutions
L, 20 μ l of 24mg/mL curcumins DMSO solution；After mixing, add in 4mm (750 μ l of volume) electroporation ware, be put into electroporation apparatus,
According to following reaction condition：Voltage 400V, capacitance 250 μ F, discharge time 1ms, discharge time：1 time.It can will be suspended after perforation
Liquid is put into 1 hour of incubation in cell incubator, and followed by twice, each 80min carefully abandons most supernatant to 100000g ultracentrifugations,
The compound excretion body that precipitation is load nano ferriferrous oxide and curcumin is resuspended with PBS liquid, -80 DEG C of storages are subsequently examined
It surveys and tests；
Embodiment 6
100 μ g of excretion body, 30 μ of 4mg/mL nano ferriferrous oxides aqueous solution are added in 1mL50mM trehalose PBS solutions
L, 20 μ l of 24mg/mL curcumins DMSO solution；After mixing, add in 4mm (750 μ l of volume) electroporation ware, be put into electroporation apparatus,
According to following reaction condition：Voltage 700V, capacitance 500 μ F, discharge time 1ms, discharge time：1 time.It can will be suspended after perforation
Liquid is put into 1 hour of incubation in cell incubator, and followed by twice, each 80min carefully abandons most supernatant to 100000g ultracentrifugations,
The compound excretion body that precipitation is load nano ferriferrous oxide and curcumin is resuspended with PBS liquid, -80 DEG C of storages are subsequently examined
It surveys and tests；
It is noted that above-mentioned embodiment is only intended to clearly illustrate example, and not to the limit of embodiment
Fixed, there is no necessity and possibility to exhaust all the enbodiments.Each component being not known in the present embodiment is available
The prior art is realized.Without departing from the principle of the present invention, for those skilled in the art,
Several improvements and modifications can also be made, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (6)
- It is 1. a kind of while load chemicals and the excretion preparation of nano material, it is characterised in that：The excretion body Inside is loaded with the nano ferriferrous oxide available for neoplasm tracing and imaging and the curcumin available for oncotherapy；Wherein receive Rice ferroso-ferric oxide is aqueous solution, and curcumin is dimethyl sulfoxide (DMSO) DMSO solution, and specific preparation process is as follows：1) the excretion body of cell secretion is extracted from mouse macrophage Raw264.7 culture solutions, is exceeded the speed limit using classical differential Centrifugal process, obtained excretion weight are suspended from the PBS solution of the trehalose containing 50mM, and -80 DEG C save backup；2) the nano ferriferrous oxide aqueous solution prepared, curcumin DMSO solution and excretion body suspension are added in the sea In algae sugar PBS solution, mixing is shaken, obtains sample；3) the above-mentioned sample of respective volume is added in by electroporation apparatus requirement and electroporation ware specification, is put into electroporation apparatus, according to The optimal perforation condition setup parameter that preliminary experiment is groped, into eleven punch 11；4) sampled immediately after perforating, detect light absorption value of the sample at 260nm on ultra-violet and visible spectrophotometer, and with not Punch sample compares, and judges whether perforation succeeds；5) sample is immediately placed in 1 hour of incubation in cell incubator after remaining perforation, to promote excretion body film reparation；6) 1 it is small when after, by aforesaid liquid with 100,000-14 ten thousand g RCF centrifugal force ultracentrifugation 70-100min, abandon most supernatant, precipitate Object is the excretion body for loading nano ferriferrous oxide and curcumin simultaneously, is referred to as compound excretion body；7) above-mentioned compound excretion body is added in the burnt culture dish of copolymerization, and fluorescence microscopy Microscopic observation curcumin is proved from fluoresced green Curcumin success is contained, above-mentioned excretion body prepares copper mesh specimen, in iron particle in electric Microscopic observation excretion body, it was demonstrated that four oxygen of nanometer Change three-iron to load successfully；8) compound excretion body is resuspended in PBS, and with ferrosin Spectrophotometric Determination of Iron content, ginger is measured with visible spectrophotometer With protein refractometer, excretion body content is surveyed with BCA methods for flavine content；9) compound excretion body suspension is taken to carry out nano particle trace analysis detection NTA or dynamic optical reflective analysis DLS and measures it Particle size and distribution, and its size and shape characteristic, western blot detection excretions are observed under transmission electron microscope TEM Body film surface characteristic protein is expressed；10) above-mentioned compound excretion body suspension packing is put in -80 DEG C of refrigerators, samples once measure above-mentioned concentration week about And excretion body characteristics, the stability of evaluating combined excretion body.
- 2. preparation method that is according to claim 1 while loading chemicals and the excretion body of nano material, feature It is:The mouse macrophage Raw264.7 culture solutions are containing no excretion body serum or without serum.
- 3. preparation method that is according to claim 1 while loading chemicals and the excretion body of nano material, feature It is:In the step 2), 100 μ g-300ug are separately added into the phosphate buffered saline solution PBS solution of 1ml trehaloses containing 50mM Excretion body, 5-20 μ l nano ferriferrous oxides aqueous solutions and 10-30 μ l curcumins DMSO solution composition hybrid reaction system.
- 4. preparation method that is according to claim 2 while loading chemicals and the excretion body of nano material, feature It is:The excretion body is in terms of its protein content, and the concentration of curcumin DMSO solution is 10-30mg/ml, nano ferriferrous oxide The concentration of aqueous solution is 1-10mg/ml.
- 5. preparation method that is according to claim 1 while loading chemicals and the excretion body of nano material, feature It is:In the step 3), electroporation conditions are：Voltage 100-700v, capacitance 125-500 μ F, discharge time 1-5ms, electric discharge Number：1 time.
- 6. preparation method that is according to claim 1 while loading chemicals and the excretion body of nano material, feature It is:It is described 8) in, measure concentration of iron and curcumin concentration and use UV-visible spectrophotometry.
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| CN108956563A (en) * | 2018-06-21 | 2018-12-07 | 东南大学 | A method of multi-function metal nano-probe is synthesized with tumour cell biology in situ |
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