CN109260480A - A kind of chitosan nano meter level acoustic contrast agent and the preparation method and application thereof carrying adriamycin - Google Patents
A kind of chitosan nano meter level acoustic contrast agent and the preparation method and application thereof carrying adriamycin Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The present invention relates to a kind of chitosan nano meter level acoustic contrast agents and the preparation method and application thereof for carrying adriamycin, the chitosan nano meter level acoustic contrast agent prepared by the present invention for carrying adriamycin, it is using chitosan as shell membrane, adriamycin (DOX) and perfluoropropane gas are wrapped in inside chitosan shell membrane, partial size is 300~900nm, average grain diameter is 641nm (polymer dispersity index PDI:0.256), in nano-scale range, tumor blood vessels wall gap can be passed through, there is targeting and high efficiency for the treatment of tumour.The nanoscale ultrasound contrast agents have stronger enhancing visualization capabilities, and it can maintain to develop in a longer period of time, entrapment efficiency and drugloading rate also with higher, biological safety is high, combining ultrasonic targeting destroys microvesicle (UTMD) and can significantly improve the recovery rate of DOX and reduce tumour cell survival rate, is conducive to the treatment of tumor disease.
Description
Technical field
The present invention relates to a kind of chitosan nano meter level acoustic contrast agents and the preparation method and application thereof for carrying adriamycin, belong to
In ultrasound molecular Imaging Technology field.
Background technique
With the fast development of ultrasound molecular imaging technique and bio-nanotechnology, nanoscale ultrasound contrast agents development is fast
Speed.Nanoscale ultrasound contrast agents are one of the research hotspot of molecular imaging field in recent years and targeting drug delivery system
One of the important research direction of (Targeted drug delivery system, TDDS), can effectively overcome traditional tumour
The disadvantages of drug therapy targeting is poor, whole body toxic side effect is big.It is different from traditional micron order acoustic contrast agent (blood pool imaging agent),
Nanoscale ultrasound contrast agents have very strong penetration power, can pass through tumor blood vessels wall gap, and development and application is also advantageous
In further development targeting, high efficiency, miniaturization and the new contrast-enhanced ultrasound agent with auxiliary therapeutic action.Nanoscale ultrasound
Contrast agent is the important directions and tumor-targeting of the development of ultrasound molecular iconography, visualization and the new direction precisely treated.
Shell membrane materials are the key elements for determining acoustic contrast agent performance.Currently, multiple material is used for acoustic contrast agent
Preparation, comprising: lipid, high molecular polymer, surfactant etc., but have some disadvantages.Lipid ultrasonic contrast agent
It has good stability, imaging effect is good, but price costly and to kidney and liver has potential toxicity, and there are safeties
Problem;High molecular polymer such as lactic acid/co-glycolic acid (PLGA) etc. can prepare relatively stable acoustic contrast agent, prolong
Long Diagnostic Time, but since contrast agent shell is harder, elasticity is poor, and imaging is poor, needs higher ultrasonic output frequency, may
It can normal tissue generation non-therapeutic loss;Surfactant acoustic contrast agent is imaged, but stability is poor, is not easy to be repaired
Decorations.Chitosan material is non-toxic, nonirritant, not only has good biocompatibility and biodegradability, also has only
Special physiology and pharmacological activity, it is most important that its free amino and hydroxyl isoreactivity group rich in has good
Modified is a kind of ideal acoustic contrast agent Shell membrane materials.Chinese patent literature CN106139174A provides a kind of package
The preparation method based on chitosan derivatives nanoscale ultrasound contrast agents of liquid fluorocarbon is by acylation reaction to carboxymethyl shell
Glycan is modified, and synthesis has amphiphilic positive hexanoyl carboxymethyl chitosan, and liquid fluorocarbon is added on this basis, using super
The nanoscale ultrasound contrast agents being made of liquid fluorocarbon kernel and chitosan derivatives shell are made in sound emulsification method.This method institute
High molecular material is safe and non-toxic, and contrast agent preparation process is easy, and operating condition is mild.But the nanometer of this method preparation
Grade acoustic contrast agent is nano-liquid droplet state, is allowed to that liquid-gas phase transition occurs it is still necessary to certain environmental stimuli in practical application, and
And not for further study to its targeting and drug-arrier feature and evaluation.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of chitosan nano meter level acoustic contrast agents for carrying adriamycin
And preparation method thereof, nanoscale ultrasound contrast agents are prepared by Shell membrane materials of chitosan, which has stronger enhancing aobvious
As ability, higher entrapment efficiency and drugloading rate, biological safety height.
The present invention also provides the above-mentioned chitosan nano meter level acoustic contrast agents for carrying adriamycin in treatment tumour and preparation
Application in anti-tumor drug.
Term explanation:
Adriamycin: Doxorubicin (DOX) is a kind of antitumor antibiotics, can inhibit the synthesis of RNA and DNA, to RNA
Inhibiting effect it is most strong, antitumor spectra is wider, has an effect to kinds of tumors, belongs to cell cycle nonspecific agent (CCNSA), all to various growths
The tumour cell of phase has killing effect.
Room temperature: 25 ± 2 DEG C.
The technical solution of the present invention is as follows:
A kind of chitosan nano meter level acoustic contrast agent carrying adriamycin, the nanoscale ultrasound contrast agents are using chitosan as shell
Film, is enclosed with adriamycin and gaseous fluorine carbon inside chitosan shell membrane, and the partial size of the nanoscale ultrasound contrast agents is 300~
900nm, wherein the mass ratio of chitosan and adriamycin is 1:1~4.
The preparation method of the above-mentioned chitosan nano meter level acoustic contrast agent for carrying adriamycin, steps are as follows:
(1) it disperses palmitinic acid, lecithin in phosphate buffer and suspension is made, chitosan is added into suspension
And adriamycin, 25 DEG C are incubated for 20~40mim altogether, obtain incubation product;
(2) it is filled with gaseous fluorine carbon to obtained be incubated in product of step (1), 1~5min of ultrasonic vibration, must emulsify at room temperature
Product;
(3) emulsification product made from step (2) is stood in 4 DEG C, time of repose >=2h takes subnatant after layering, dilute
Low-speed centrifugal afterwards takes lower liquid, as carries the chitosan nano meter level acoustic contrast agent of adriamycin.
Preferred according to the present invention, the mass ratio of step (1) palmitinic acid and lecithin is 1:2, in the suspension
The concentration of palmitinic acid is 0.4~0.5g/L;Wherein palmitinic acid purity >=99% is purchased from sigma, product number 200-312-9;Ovum
Phospholipid purity >=99% is purchased from sigma, product number 232-715-0.
It is preferred according to the present invention, the component of phosphate buffer described in step (1) are as follows: Na2HPO48mM,
KH2PO42mM, pH 7.2-7.4.
Preferred according to the present invention, the mass ratio of chitosan described in step (1) and adriamycin is 1:1~4, the suspension
The concentration of chitosan is 0.4~0.6g/L in liquid;Wherein the molecular weight of chitosan is 160kDa, is purchased from sigma, product number
MFCD00161512;Adriamycin purity >=98% is purchased from sigma, product number 246-818-3.
It is further preferred that the mass ratio of the chitosan and adriamycin is 1:2,
Preferred according to the present invention, gaseous fluorine carbon described in step (2) is perfluoropropane gas.
Preferred according to the present invention, diluted multiple described in step (3) is 5~10 times.
Preferred according to the present invention, low-speed centrifugal described in step (3) is that 500rpm is centrifuged 5min.
The above-mentioned chitosan nano meter level combining with ultrasound contrast agent ultrasound targeting for carrying adriamycin destroys microvesicle in treatment tumour
In application.
It is anti-swollen in preparation that the above-mentioned chitosan nano meter level combining with ultrasound contrast agent ultrasound targeting for carrying adriamycin destroys microvesicle
Application in tumor medicine.
Technical characterstic of the invention: palmitinic acid and lecithin facilitate the dissolution of chitosan, are conducive to after chitosan dissolution
The preparation for being incubated for product obtains with chitosan being outer by the incubation product of chitosan and adriamycin by the way of ultrasonic emulsification
Shell, using adriamycin and gaseous fluorine carbon as the emulsification product of kernel, after standing, the biggish acoustic contrast agent of partial size is distributed in
Layer, the relatively small nanoscale ultrasound contrast agents of partial size are distributed in lower layer, take subnatant through dilution and low-speed centrifugal, obtain partial size
Smaller and uniform nanoscale ultrasound contrast agents.
The utility model has the advantages that
1, the chitosan nano meter level acoustic contrast agent prepared by the present invention for carrying adriamycin, be using chitosan as shell membrane, Ah
Mycin (DOX) and perfluoropropane gas are wrapped in inside chitosan shell membrane, and partial size is 300~900nm, average grain diameter 641nm
(polymer dispersity index PDI:0.256) can pass through tumor blood vessels wall gap, for controlling for tumour in nano-scale range
Treating has targeting and high efficiency.
2, there is the chitosan nano meter level acoustic contrast agent prepared by the present invention for carrying adriamycin stronger enhancing to image energy
Power, and can maintain to develop in a longer period of time, entrapment efficiency and drugloading rate also with higher, biological safety
Height, irradiation intensity 0.5W/cm2, irradiation time is 30sec or 60sec, the equal > 80% of cells survival rate, and it is aobvious to be suitable for ultrasound
The adjuvant treatment of shadow and tumor disease.
3, on the one hand the chitosan nano meter level ultrasound for carrying adriamycin can be enhanced in the way of ultrasound wave irradiation in the present invention
Contrast agent development, the acoustic horn effect that on the other hand can be generated by ultrasound wave irradiation promote adriamycin more to enter tumour cell
It is interior, realize diagnosis and treatment integration.
4, the chitosan nano meter level combining with ultrasound contrast agent ultrasound targeting prepared by the present invention for carrying DOX destroys microvesicle
(UTMD) recovery rate of DOX can be significantly improved and reduce tumour cell survival rate, be conducive to the treatment of tumor disease.
Detailed description of the invention
Fig. 1 is the light microscopic figure for carrying the chitosan nano meter level acoustic contrast agent of DOX;
Fig. 2 is the grain-size graph for carrying the chitosan nano meter level acoustic contrast agent of DOX;
Fig. 3 is external self-control ultrasound enhancing display;
Fig. 4 is to carry the chitosan nano meter level acoustic contrast agent of DOX development figure in vitro;
Fig. 5 is to carry the chitosan nano meter level acoustic contrast agent of DOX to develop change over time tendency chart in vitro;
Fig. 6 is the Evaluation of Biocompatibility that the chitosan nano meter level acoustic contrast agent of DOX is carried under different ultrasound parameters;
Fig. 7 is the flow cytometry figure that DOX absorbs situation in the chitosan nano meter level acoustic contrast agent for carry DOX;
Fig. 8 is to carry the chitosan nano meter level acoustic contrast agent of DOX to MCF-7 Breast Cancer Cell survival rate influence diagram.
Specific embodiment
The present invention will be further explained with reference to the examples below, but protection scope of the present invention is not limited to that.
Palmitinic acid is purchased from sigma, product number 200-312-9;Lecithin is purchased from sigma, product number 232-715-0;
Chitosan is purchased from sigma, product number MFCD00161512;Adriamycin is purchased from sigma, product number 246-818-3.
The present embodiment is related to drug and reagent unless otherwise specified, is ordinary commercial products.
Embodiment 1: the preparation of the chitosan nano meter level acoustic contrast agent of DOX is carried
A kind of preparation method for the chitosan nano meter level acoustic contrast agent carrying adriamycin, steps are as follows:
(1) it weighs palmitinic acid and lecithin is scattered in phosphate buffer (Na2HPO48mM, KH2PO42mM, pH7.2)
In, it is spare that mixing is made suspension, and in the suspension, the concentration of palmitinic acid is 0.45g/L, and the concentration of lecithin is 0.9g/
L;Chitosan (molecular weight 160kDa) and adriamycin are added into above-mentioned suspension, chitosan and adriamycin are in suspension
Concentration is respectively 0.5g/L and 1g/L, and 25 DEG C are incubated for 30mim altogether, obtains incubation product;
(2) it is filled with perfluoropropane gas to obtained be incubated in product of step (1), ultrasonic vibration 2min, obtains newborn at room temperature
Change product;
(3) emulsification product made from step (2) is placed in 4 DEG C of standing 2h, subnatant is taken after layering, after 5 times of dilution
500rpm low-speed centrifugal 5min takes lower liquid, as carries the chitosan nano meter level acoustic contrast agent (DOX-NB) of adriamycin.
The chitosan nano meter level acoustic contrast agent for carrying adriamycin for taking above-mentioned preparation, is added drop-wise on glass slide after dilution,
By the apparent form of optical microphotograph sem observation contrast agent, as a result as shown in Figure 1, visible contrast agent under 1000 × optical microscopy
Spherical in shape, uniform particle diameter, be uniformly dispersed no aggregation;
The chitosan nano meter level acoustic contrast agent for carrying adriamycin for taking above-mentioned preparation, after dilution, uses nanometer laser grain
Degree and Zeta potential analyzer detection acoustic contrast agent partial size, as a result as shown in Fig. 2, the partial size of contrast agent be 300~
900nm, the average grain diameter of contrast agent are 641nm (polymer dispersity index PDI:0.256), surface potential is 67.12 ±
2.1mW。
Embodiment 2
A kind of preparation method for the chitosan nano meter level acoustic contrast agent carrying adriamycin, steps are as follows:
(1) it weighs palmitinic acid and lecithin is scattered in phosphate buffer (Na2HPO48mM, KH2PO42mM, pH7.3)
In, it is spare that mixing is made suspension, and in the suspension, the concentration of palmitinic acid is 0.4g/L, and the concentration of lecithin is 0.8g/L;
Chitosan (molecular weight 160kDa) is added into above-mentioned suspension and adriamycin, chitosan and adriamycin are dense in suspension
Degree is respectively 0.4g/L and 1.6g/L, and 25 DEG C are incubated for 25mim altogether, obtain incubation product;
(2) it is filled with perfluoropropane gas to obtained be incubated in product of step (1), ultrasonic vibration 3min, obtains newborn at room temperature
Change product;
(3) emulsification product made from step (2) is placed in 4 DEG C of standing 4h, subnatant is taken after layering, after 6 times of dilution
500rpm low-speed centrifugal 5min takes lower liquid, as carries the chitosan nano meter level acoustic contrast agent (DOX-NB) of adriamycin.
The chitosan nano meter level acoustic contrast agent manufactured in the present embodiment for carrying adriamycin is under 1000 × optical microscopy
Spherical in shape, uniform particle diameter, be uniformly dispersed no aggregation, and the partial size of contrast agent is 300~900nm.
Embodiment 3
A kind of preparation method for the chitosan nano meter level acoustic contrast agent carrying adriamycin, steps are as follows:
(1) it weighs palmitinic acid and lecithin is scattered in phosphate buffer (Na2HPO48mM, KH2PO42mM, pH7.4)
In, it is spare that mixing is made suspension, and in the suspension, the concentration of palmitinic acid is 0.5g/L, and the concentration of lecithin is 1.0g/L;
Chitosan (molecular weight 160kDa) is added into above-mentioned suspension and adriamycin, chitosan and adriamycin are dense in suspension
Degree is respectively 0.6g/L and 1.8g/L, and 25 DEG C are incubated for 35mim altogether, obtain incubation product;
(2) it is filled with perfluoropropane gas to obtained be incubated in product of step (1), ultrasonic vibration 4min, obtains newborn at room temperature
Change product;
(3) emulsification product made from step (2) is placed in 4 DEG C of standing 6h, subnatant is taken after layering, after 8 times of dilution
500rpm low-speed centrifugal 5min takes lower liquid, as carries the chitosan nano meter level acoustic contrast agent (DOX-NB) of adriamycin.
The chitosan nano meter level acoustic contrast agent manufactured in the present embodiment for carrying adriamycin is under 1000 × optical microscopy
Spherical in shape, uniform particle diameter, be uniformly dispersed no aggregation, and the partial size of contrast agent is 300~900nm.
The entrapment efficiency and drugloading rate of embodiment 4:DOX-NB detects
Example 1~3 stood when preparing DOX-NB after subnatant, using microplate reader detection subnatant in underlying liquid
Absorbance of the body at 480nm calculates the DOX mass dissociated in bottom liquid according to standard curve, further according to following formula meter
Calculate the entrapment efficiency (EE%) and drugloading rate (LD%) of DOX-NB:
EE%=(the initial DOX mass of DOX mass/addition being loaded with) × 100%.
LD%=(quality for the DOX mass/addition chitosan nano meter level acoustic contrast agent being loaded with) × 100%
Wherein,
The free DOX mass of the initial DOX mass-of DOX mass=addition being loaded with,
The quality of the chitosan nano meter level acoustic contrast agent of addition is the gross mass for the initial feed being added, including shell gathers
Sugar, adriamycin, palmitinic acid and lecithin.
Calculated result is as shown in table 1, the entrapment efficiency of DOX-NB prepared by the present invention 50% or more, drugloading rate >=
59.38mg DOX/g contrast agent.
1 difference of table carries the encapsulation rate and drugloading rate of the chitosan ultrasonic nano grade contrast agent of DOX
The external supersonic development capability of embodiment 5:DOX-NB detects
Make 4% agar gel model by oneself, the enhancing visualization capabilities of DOX-NB prepared by observation embodiment 1, operating device is such as
It is 1.0 × 10 by concentration shown in Fig. 36The DOX-NB of bubbles/mL is added in the circular hole of agar gel model, is examined using clinic
Disconnected Ultrasound Instrument (LOGIQ E9;GE, USA) detection ultrasonic development ability, major parameter are as follows: frequency 9.0MHz;Mechanical index:
0.12;Focal length: 3.0cm;Dynamic range: 60dB, two dimension are observed with ultrasonic contrast pattern synchronization, and parameter setting remains unchanged.
Image document is stored with Ultrasound Instrument inter workstation, image is as shown in figure 4, image and enhance imaging modality in grayscale
Under, it can show clearly image;The analysis of Image J software obtains the gray value of image, as a result as shown in figure 5, the above knot
Fruit all shows that the contrast agent has stronger ultrasonic development ability, and development can be maintained within the long period (15min).
Embodiment 6: the biological safety detection of chitosan nano meter level acoustic contrast agent
Chitosan nano meter level acoustic contrast agent is prepared according to preparation method described in embodiment 1, the difference is that step
(1) adriamycin is not added in.The chitosan nano meter level acoustic contrast agent for taking above-mentioned preparation, using CCK-8 reagent measure its
Biological safety in MCF-7 cell line, concrete operations are as follows: by MCF-7 cell inoculation in 96 orifice plates, containing 10%FBS's
DMEM is culture medium, and the density of cell is 1.0 × 104A/hole, 37 DEG C, 5%CO2It is cultivated in incubator for 24 hours, cell is adherent, will
Culture medium is changed to the fresh 10%FBS DMEM culture medium containing above-mentioned chitosan nano meter level acoustic contrast agent, DMEM culture medium
The volume fraction of middle chitosan nano meter level acoustic contrast agent is respectively 0,10%, 20%, 30%, and carries out ultrasound wave irradiation, ultrasound
Radiation treatment is as shown in the table:
The different ultrasound wave irradiation processing mode of table 2
After irradiation, 37 DEG C, 5%CO248h is cultivated in incubator, then washs cell with PBS, is added and is contained 10 μ L
The fresh 10%FBS DMEM culture medium of CCK-8 reagent continues to be incubated for 1-4h, detects its light at 480nm wavelength with microplate reader
Absorption value.As a result as shown in Figure 6: as volume fraction≤30% of chitosan nano meter level acoustic contrast agent, irradiation intensity 0.5W/
cm2When, the equal > 80% of group of cells survival rate;It improves when by irradiation intensity to 1.0W/cm2When, chitosan nano meter level ultrasonic contrast
Volume fraction≤20% of agent, cells survival rate show within the scope of certain ultrasound intensity, the radiography also 80% or more
Agent cytotoxicity is small, and biological safety is good.
Embodiment 7:DOX-NB joint targeting destroys the influence that microvesicle (UTMD) takes rate to DOX feeding
DOX-NB prepared by Example 1 detects MCF-7 cell to the uptake ratio of DOX in DOX-NB, and steps are as follows: will
MCF-7 cell is with every hole 2.5 × 105The density of a cell is seeded in 6 orifice plates adherent overnight, replaces fresh 10%FBS
DMEM culture medium, in 10%FBS DMEM culture medium respectively containing free DOX, DOX-NB, free DOX and UTMD, DOX-NB and
UTMD, wherein the volume fraction of DOX-NB in the medium is 20%, and the content of each group DOX is consistent.37 DEG C, 5%CO2
1h is cultivated in incubator, gives ultrasound wave irradiation or without ultrasound wave irradiation processing, processing mode is as follows:
Table 3. includes ultrasound wave irradiation processing mode different in the DMEM culture medium of heterogeneity
Ultrasound wave irradiation after treatment collects MCF-7 cell and carries out Flow cytometry, as a result as shown in fig. 7, result
Show: DOX-NB combined U TMD group DOX ingestion efficiency is significantly higher than control group and free DOX combined U TMD group, and irradiation time
Ingestion efficiency when 60sec is higher than ingestion efficiency when irradiation time 30sec.
Embodiment 8:DOX-NB joint targeting destroys the effect of microvesicle (UTMD) to MCF-7 cell
DOX-NB prepared by Example 1, measures effect of its combined U TMD to MCF-7 cell using CCK-8 reagent, in advance
Survey application value of the DOX-NB in terms of oncotherapy.Concrete operations are as follows: by MCF-7 cell inoculation in 96 orifice plates, to contain
The DMEM of 10%FBS is culture medium, and the density of cell is 1.0 × 104A/hole, 37 DEG C, 5%CO2It is incubated overnight in incubator, carefully
Born of the same parents are adherent, replace fresh 10%FBS DMEM culture medium, in culture medium respectively containing free DOX, DOX-NB, free DOX and
UTMD, DOX-NB and UTMD, wherein the volume fraction of DOX-NB in the medium is 20%, and the content of each group DOX keeps one
It causes, gives ultrasound wave irradiation or without ultrasound wave irradiation processing, processing mode is as shown in table 3;In addition, not contain the 10% of other compositions
The MCF-7 cell of FBS DMEM culture medium culture is blank control, no ultrasound wave irradiation processing.
After ultrasound wave irradiation, 37 DEG C, 5%CO2It is cultivated 24 hours in incubator, then washs cell with PBS, addition contains
There is the fresh 10%FBS DMEM culture medium of 10 μ LCCK-8 reagents, continues to be incubated for 1-4h, be examined at 480nm wavelength with microplate reader
Its absorbance value is surveyed, calculates cells survival rate, as a result as shown in figure 8, as the result is shown: DOX-NB combined U TMD is in ultrasound intensity
0.5W/cm2, irradiation time be 60sec when, the survival rate of MCF-7 cell is minimum, be 2.2 ± 0.9%, hence it is evident that be lower than DOX-NB
Group (21.0 ± 2.2%) and free DOX group (6.4 ± 0.7%), difference have statistical difference (p < 0.01), show DOX-NB
Combined U TMD can effectively reduce the survival rate of tumour cell under conditions of ultrasound wave irradiation, be applied to oncotherapy.
Claims (10)
1. a kind of chitosan nano meter level acoustic contrast agent for carrying adriamycin, which is characterized in that the nanoscale ultrasound contrast agents
Using chitosan as shell membrane, adriamycin and gaseous fluorine carbon, the grain of the nanoscale ultrasound contrast agents are enclosed with inside chitosan shell membrane
Diameter is 300~900nm, wherein the mass ratio of chitosan and adriamycin is 1:1~4.
2. a kind of preparation method of chitosan nano meter level acoustic contrast agent for carrying adriamycin described in claim 1, feature
It is, steps are as follows:
(1) disperse palmitinic acid, lecithin in phosphate buffer and suspension be made, into suspension be added chitosan and Ah
Mycin, 25 DEG C are incubated for 20~40mim altogether, obtain incubation product;
(2) it is filled with gaseous fluorine carbon to obtained be incubated in product of step (1), 1~5min of ultrasonic vibration, must emulsify production at room temperature
Object;
(3) emulsification product made from step (2) is stood in 4 DEG C, time of repose >=2h takes subnatant after layering, low after dilution
Speed centrifugation, takes lower liquid, as carries the chitosan nano meter level acoustic contrast agent of adriamycin.
3. preparation method as claimed in claim 2, which is characterized in that the mass ratio of step (1) palmitinic acid and lecithin
For 1:2, concentration of the palmitinic acid in suspension is 0.4~0.5g/L;Wherein palmitinic acid purity >=99%, purity of lecithin
>=99%.
4. preparation method as claimed in claim 2, which is characterized in that the component of phosphate buffer described in step (1) are as follows:
Na2HPO48mM, KH2PO42mM, pH 7.2-7.4.
5. preparation method as claimed in claim 2, which is characterized in that the quality of chitosan and adriamycin described in step (1)
Than for 1:1~4, concentration of the chitosan in suspension is 0.4~0.6g/L;Wherein, the molecular weight of chitosan is
160kDa, adriamycin purity >=98%.
6. preparation method as claimed in claim 2, which is characterized in that gaseous fluorine carbon described in step (2) is perfluoropropane gas
Body.
7. preparation method as claimed in claim 2, which is characterized in that diluted multiple described in step (3) is 5~10 times.
8. preparation method as claimed in claim 2, which is characterized in that low-speed centrifugal described in step (3) is 500rpm centrifugation
5min。
9. the chitosan nano meter level combining with ultrasound contrast agent ultrasound targeting described in claim 1 for carrying adriamycin destroys microvesicle and exists
Treat the application in tumour.
10. the chitosan nano meter level combining with ultrasound contrast agent ultrasound targeting described in claim 1 for carrying adriamycin destroys microvesicle
Application in preparation of anti-tumor drugs.
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