CN109364272A - Application of the nano enzyme corpusculum in catalysis photoacoustic imaging - Google Patents
Application of the nano enzyme corpusculum in catalysis photoacoustic imaging Download PDFInfo
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- corpusculum
- bionical
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- gqdzyme
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 74
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 74
- 238000003384 imaging method Methods 0.000 title description 29
- 238000006555 catalytic reaction Methods 0.000 title description 3
- 239000002872 contrast media Substances 0.000 claims abstract description 73
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- 238000002360 preparation method Methods 0.000 claims abstract description 16
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 10
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- 238000011068 loading method Methods 0.000 claims abstract description 4
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 claims description 56
- 238000000034 method Methods 0.000 claims description 23
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
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- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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Classifications
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- 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
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- 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
- A61K49/225—Microparticles, microcapsules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Acoustics & Sound (AREA)
- Physics & Mathematics (AREA)
- Radiology & Medical Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to a kind of bionical corpusculum of nano enzyme and its application for loading nano enzyme and azine di-ammonium salts contrast agent, the preparation method of the bionical corpusculum of the nano enzyme includes the following steps: that (1) prepares nano enzyme and azine di-ammonium salts contrast agent；(2) bionical corpusculum is prepared；(3) nano enzyme and azine di-ammonium salts complex contrast agent are packed into bionical corpusculum and prepare the bionical corpusculum of nano enzyme.The bionical corpusculum of the nano enzyme can prepare tumour light image-forming contrast medium or cancer target drug-delivery preparation.
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, in particular to a kind of loading carbon dots nano enzyme and azine diammonium
The bionical corpusculum of the nano enzyme of salt contrast agent and its application.
Background technique
Photoacoustic imaging combines the high contrast of pure optical imagery and the high-penetration depth advantage of pure ultrasonic imaging, Neng Gouti
It is a kind of current very promising imaging pattern for the imaging of tissue of high-resolution and high contrast.Photoacoustic imaging is made
Shadow agent is the key that determine photoacoustic imaging performance, it improves imaging contrast by the optics and acoustic characteristic of change lesion tissue
Degree and resolution ratio, become current biological iconography area research hot spot.Relatively common photoacoustic imaging contrast agent includes gold nano
Material, carbon nanotube, dyestuff nano material.With the fast development of nano material, nano enzyme is as the nanometer with enzymatic activity
The discovery of material promotes the further development of nano material family.It is this to have both nano material fundamental property and enzymatic energy
The characteristic of power makes it before the fields of biomedicine such as immune detection, diagnosing tumor and bio-sensing all show and are widely applied
Scape.Nano particle is only to be applied to laboratory research mostly at present, and many factors limit nanometer formulation from basic research to clinic
Conversion, such as transfer efficiency, biocompatibility and be used for a long time safety.Nearly 2 years, the international top periodical such as Nature connected
Continue the research and clinical conversion status of depth profiling Nano medication, and thinks that the biological safety of Nano medication is clinical turn
Change the major reason of failure.Therefore, develop a kind of nano enzyme photoacoustic imaging contrast agent that there can be higher biological safety, be
Realize the necessary condition of nano enzyme tumour photoacoustic imaging.
Ideal photoacoustic imaging contrast agent should have the following characteristics that first-class Photothermal Signals and acoustical signal transfer capability；
Delicately reflect the performance of the physiological structure of organism；Internal distribution and metabolic characteristics and excellent biocompatibility well.
Nano enzyme is with the potentiality that these features are all integrated into a kind of nano material.
The catalysis characteristics of nano enzyme peroxidase: it is being rich in H2O2Tumor region, can be with efficient catalytic azine di-ammonium salts
(ABTS) it is converted to the ABTS with near-infrared absorption ability+, absorbing the thermal energy discharged after luminous energy causes to inhale tumor tissues
Local temperature increases, and temperature causes to thermally expand and generate pressure wave after increasing, and then forms photoacoustic signal, passes through detection optoacoustic letter
The light absorption distributed image in tissue number can be reconstructed, realizes the photoacoustic imaging diagnosis of tumour.
Furthermore studies have shown that nano enzyme can further enhance photoacoustic signal in the stronger optical absorption near infrared region.
However due to the extremely complicated property of living organism, by performance of the well-designed nano-drug preparation in final clinical test
Often there are larger differences with preliminary in vitro experiments result, so being difficult to carry out clinical conversion.
Summary of the invention
In order to solve the problems, such as above-mentioned nano enzyme in practical applications, inventor herein is prepared using natural erythrocyte membrane
Bionical corpusculum carries out bionic coating to nano enzyme photoacoustic contrast agent, to promote stability of the contrast agent in hematological system,
And the biocompatibility of body.
Present invention firstly relates to a kind of bionical corpusculums of nano enzyme for preparing loading nano enzyme and azine di-ammonium salts contrast agent
Method, described method includes following steps:
(1) nano enzyme and azine di-ammonium salts contrast agent are prepared；
(2) bionical corpusculum is prepared；
(3) nano enzyme and ABTS complex contrast agent are packed into bionical corpusculum and prepare the bionical corpusculum of nano enzyme.
It is preferably carbon dots nano enzyme (GQDzyme) that nano enzyme is prepared described in step (1), and the contrast agent is preferably carbon
Point nano enzyme and ABTS complex, the carbon dots nano enzyme and ABTS complex the preparation method comprises the following steps:
1) polyacrylonitrile carbon fiber is added in the mixed solution of the concentrated sulfuric acid and nitric acid；Ultrasonic mixing dissolution, is warming up to
It 100 DEG C and stirs abundant；
2) it molecular cut off 3KD ultrafiltration solution and is lyophilized, GQDzyme is made；
3) by GQDzyme and ABTS mixed dissolution in aqueous solution, it is stirred overnight rear ultrafiltration, freeze-drying can obtain GQDzyme/
ABTS；
The method of bionical corpusculum is prepared described in step (2) are as follows:
1) red blood cell is added in the hypotonic buffer liquid of pre-cooling, mixing is placed on 1~2h in 4 DEG C, is allowed to complete hemolysis；
2) the centrifugation removal hemoglobin supernatant at 4 DEG C, precipitating are erythrocyte membrane；
3) repeated centrifugation is washed 3~5 times, and the biggish bionical corpusculum of partial size is made；
4) bionical corpusculum is ultrasonically treated, the bionical corpusculum that obtained partial size is about 400nm.
The method that GQDzyme/ABTS contrast agent is packed into bionical corpusculum described in step (3) are as follows:
Using physical impact mode by GQDzyme/ABTS contrast agent be loaded into bionical corpusculum obtain nano enzyme it is bionical
Corpusculum.
The invention further relates to the GQDzyme/ABTS contrast agent for using the method to prepare or comprising described
The bionical corpusculum of the nano enzyme of GQDzyme/ABTS contrast agent.
The particle size of the GQDzyme/ABTS contrast agent is about 10nm, and the particle size of the bionical corpusculum is about
Particle size for 100nm, the bionical corpusculum of the nano enzyme is about 50nm.
The invention further relates to the GQDzyme/ABTS contrast agent or receiving comprising the GQDzyme/ABTS contrast agent
The rice bionical corpusculum of enzyme is preparing the application in cancer target preparation, the cancer target preparation are as follows: fill oncotherapy preparation
It is loaded in the bionical corpusculum of nano enzyme, the oncotherapy preparation is antibody, polypeptide, aptamer or functional form
Albumen.Preferably, the functional form albumen is folic acid.
The invention further relates to the GQDzyme/ABTS contrast agent or receiving comprising the GQDzyme/ABTS contrast agent
Application of the rice bionical corpusculum of enzyme in preparation tumour light image-forming contrast medium, the tumour are solid tumor.
The tumour light image-forming contrast medium is the lesions position that solid tumor is marked by way of intravenously administrable
Contrast agent.
Detailed description of the invention
The preparation and characterization of the carbon dots nano enzyme photoacoustic contrast agent of the bionical corpusculum package of Fig. 1, excretion body:
Fig. 1 a carbon dots nano enzyme and ABTS photoacoustic contrast agent complex (GQDzyme/ABTS) transmission electron microscope image；
Fig. 1 b erythrocyte membrane is prepared into the lesser bionical corpusculum of excretion body of partial size by Hypotonic treatment；
The mode of Fig. 1 c physical impact loads the bionical corpusculum of excretion body of GQDzyme/ABTS complex；
Fig. 1 d is the ABTS of carbon dots nanometer enzymatic various concentration ABTS conversion+Optical absorption map at near-infrared；
Fig. 1 e GQDzyme/ABTS complex at 808nm light absorption with to H2O2Dependence；
Photoacoustce signal intensity and being positively correlated property of concentration in Fig. 1 f solution.
The nano enzyme photoacoustic imaging contrast agent study on the stability of Fig. 2, different dosage forms:
Fig. 2 a each group contrast agent photoacoustic imaging photo；
Fig. 2 b photoacoustce signal intensity changes over time figure.
The nano enzyme photoacoustic imaging contrast agent of the bionical corpusculum modification of excretion body of Fig. 3, special marking is to nasopharyngeal carcinoma cell
The endocytosis situation of recognition capability and nasopharyngeal carcinoma cell:
Fig. 3 a laser confocal imaging analyzes CNE-2 cell membrane and the expression of NIH3T3 cell membrane folacin receptor；
Fig. 3 b CNE-2 nasopharyngeal carcinoma cell is to nanometer enzyme granulate RM:GQDzyme/ABTS and FA-RM:GQDzyme/ABTS
(RM, erythrocyte membrane；FA-RM, the erythrocyte membrane of modified with folic acid) endocytosis laser confocal imaging；
The transmission electron microscope for the photoacoustic imaging contrast agent endocytosis that Fig. 3 c CNE-2 nasopharyngeal carcinoma cell modifies targeted molecular is imaged
Figure；
The optoacoustic that Fig. 3 d lysosome common location laser confocal imaging characterization CNE-2 nasopharyngeal carcinoma cell modifies targeted molecular
Image-forming contrast medium endocytosis enters intracellular situation.
Fig. 4 H2O2The nano enzyme photoacoustic imaging contrast agent of the bionical corpusculum modification of the excretion body of response is in nasopharyngeal carcinoma tumor-bearing mice
Intracorporal photoacoustic imaging:
2h before in Fig. 4 a contrast agent tail vein injection to tumor-bearing mice body or after injection, when 4h, 8h tumor locus optoacoustic at
As analysis；
The photoacoustic signal quantitative analysis of Fig. 4 b different time points tumor locus.
Fig. 5, distribution and corresponding Tissue distribution in different nano enzyme photoacoustic imaging contrast agent bodies are investigated by fluorescence imaging:
In Fig. 5 a 8h, different pharmaceutical real-time distribution result in Mice Body:
The fluorescent value of Fig. 5 b quantitative fluorescence analysis tumor tissue sections；
Fig. 5 c tumor-bearing mice in vitro tissue fluorescence imaging result；
The contrast agent fluorescence values distribution that Fig. 5 d is respectively organized.
Specific embodiment
The preparation and characterization of the nano enzyme photoacoustic contrast agent of the bionical corpusculum package of embodiment 1, excretion body
1, chemical oxidation stripping method prepares GQDzyme
In order to prepare the nano enzyme with catalase activity, we are using polyacrylonitrile carbon fiber as raw material, passing through
It learns oxidation stripping method and prepares GQDzyme, detailed process is as follows:
(1) 0.2g polyacrylonitrile carbon fiber is added in the mixed solution of the concentrated sulfuric acid (40mL) and nitric acid (12mL)；It is super
Sound mixed dissolution 2h is warming up to 100 DEG C of stirrings for 24 hours；
(2) GQDzyme is made after ultrafiltration (molecule interception 3000Da) freeze-drying.
As a result as shown in Figure 1a, the GQDzyme after freeze-drying can be uniformly dispersed in water again, is formed transparent light
Yellow solution shows that GQDzyme has good water solubility.GQDzyme size obtained by transmission electron microscope analysis is about 10nm, and
And size uniformity.
2, the absorption to ABTS is completed using electrostatic interaction and pi-pi accumulation active force to load, prepare GQDzyme/
ABTS developer
(1) 10mg GQDzyme is mixed into ultrasonic mixing dissolution 2h with the ABTS of 2mg in aqueous solution, is stirred overnight；
(2) ultrafiltration (molecule interception 3000Da), freeze-drying can obtain GQDzyme/ABTS.
ABTS is in H appropriate2O2The ABTS of light absorption is had near infrared light region by GQDzyme under effect+.In order to
Investigate the process and H2O2Relationship, we are in different H2O2In concentration ABTS and GQDzyme mixed aqueous solution, ABTS is detected+
Light absorption and H2O2The relationship of concentration.Further pass through no H2O2With the H of catalase (CAT) pre-treatment2O2Group verifies nanometer
Enzyme photoacoustic contrast agent is to H2O2Dependence.And utilize the H of various concentration2O2This dependence is investigated to photoacoustic signal power
Influence.As a result as shown in Fig. 1 d-f, in H2O2In the presence of, ABTS could be catalytically converted into ABTS by GQDzyme+,
And ABTS+Light absorption in the near infrared region is with H2O2Concentration increase, absorbance value gradually increases, photoacoustic signal also with
H2O2Being positively correlated property of concentration.
3, bionical corpusculum is prepared
(1) eyeball blood extracting method is plucked using mouse take BALB/c mouse whole blood 2mL；
(2) it is placed in 5mL to contain in the centrifuge tube of 0.5mL anti-coagulants (3.8wt.% sodium citrate solution), after being sufficiently mixed；
(3) 3000rpm is centrifuged 20min at 4 DEG C, absorbs and abandons upper layer blood tapetum；
(4) red blood cell of bottom is placed in the isotonic phosphate buffer liquid (pH 7.4) of 3 times of volumes of 4 DEG C of pre-coolings,
After resuspension centrifuge washing 3 times (15min × 5000rpm) at 4 DEG C；
(5) red blood cell after cleaning centrifugation delays according to the hypotonic Tris-HCl of 10mmol/L that pre-cooling is added in the ratio of 1:40
Fliud flushing (pH 7.4), needs to be slowly stirred during adding, and is subsequently placed at 1~2h in 4 DEG C of refrigerators, is allowed to complete hemolysis；
(6) 9000rpm is centrifuged 15min at 4 DEG C, removes hemoglobin supernatant, and precipitating is erythrocyte membrane；
(7) repeated centrifugation is washed 3~5 times, and the biggish bionical corpusculum of excretion body of partial size is made；
(8) by big partial size red blood cell corpusculum after rated power is 60W water bath sonicator 20min, obtained partial size is about
The bionical corpusculum of excretion body of 400nm.
Hemoglobin is removed by Hypotonic treatment and forms hollow imitated vesicle structure, forms the red blood cell vesica that partial size is about 3 μm,
It is discovered by experiment that as a result as shown in Figure 1 b, the red blood cell vesica of micron-scale can be changed to grain after water bath sonicator 20min
The bionical corpusculum of diameter~400nm excretion body.
4, GQDzyme/ABTS developer is loaded into bionical corpusculum
While bionical corpusculum more complete reservation bioactivity, it can be effectively reduced or be shielded its immunogenicity, improve life
Object compatibility avoids endothelium network from identifying and remove, promotes the water solubility and stability of nano particle, in significant extension body
Circulation time.Next, GQDzyme/ABTS developer is loaded using the bionical corpusculum being prepared, by the outer of obtained 100nm
Secreting the bionical corpusculum of body and GQDzyme/ABTS by concentration ratio is 1:1 blending, the continuous mistake of MINI squeezer for being 50nm by aperture
Film squeezes 7~10 times, forms the GQDzyme/ABTS (RM:GQDzyme/ABTS) for being coated with erythrocyte membrane.Utilize same side
Method, RM:GQDzyme/ABTS and FA by concentration ratio 1:10 be blended after by MINI squeezer squeeze 10~15 times, it is obtained label have
The RM:GQDzyme/ABTS of FA.
Transmission electron microscope statistics indicate that, nano grain surface significantly covers one layer of complete membrane structure after extruding, squeeze
Process does not also impact (such as Fig. 1 c) to the structure of nano enzyme.
The nano enzyme photoacoustic imaging contrast agent study on the stability of embodiment 2, different dosage forms
Nano enzyme photoacoustic imaging contrast agent is suspended in the PBS of pH 7.2 and cell culture fluid (DMEM) containing 10%FBS
In, it is fitted into hollow agar gel stick, 0h, 12h is acquired by photoacoustic imager, for 24 hours, photoacoustic signal when 48h, and draw
Photoacoustce signal intensity changes over time figure.As a result as shown in Fig. 2, with the time extension (within 48h), the light of three kinds of contrast agent
Without apparent downward trend, this illustrates our the bionical corpusculum packages of excretion body obtained for acoustic imaging image and photoacoustce signal intensity
Nano enzyme photoacoustic contrast agent there is good photoacoustic signal stability, photoacoustic signal will not extension at any time and quickly reveal
Bionical small external to excretion body, this can largely determine the feasibility of subsequent applications, lay a good foundation for clinical application.
The nano enzyme photoacoustic contrast agent targeting of embodiment 3, cell in vitro assessment of levels excretion body bionical corpusculum package and interior
Gulp down ability
Although the modification of the bionical corpusculum of excretion body can extend the circulation time of nano enzyme photoacoustic contrast agent in blood,
Realize the targeting conveying of nano particle, nano enzyme photoacoustic contrast agent should also have the ability for improving tumour cell intake.Folic acid
The major target class of molecule is folacin receptor, and the expression quantity of CNE-2 cell surface folacin receptor will directly affect the target of contrast agent
To ability and therapeutic effect.Therefore, we have detected CNE-2 cell surface integrin expression feelings using Laser Scanning Confocal
Condition, and use NIH 3T3 as non-tumor cell model (negative control group).As a result as best shown in figures 3 a and 3b, tumour cell CNE-2
Really the expression quantity than NIH 3T3 cell surface folacin receptor is high, this will be helpful to improve FA-RM:GQDzyme/ABTS and swell
The affinity of oncocyte CNE-2, to achieve the purpose that promote intake and improve drug effect.Nano particle is through endocytosis
Metabolism transport pathway into after intracellular plays key effect to the performance of its drug effect.According to datagram 3c and 3d analysis it is found that outer
Secrete the coated nano enzyme overwhelming majority of the bionical corpusculum of body and lysosome not common location.This is because the building bionical corpusculum of excretion body is red
There is cell membrane mobility to be easy to merge with tumor cell membrane, in addition because erythrocyte membrane flowing will increase nano enzyme contrast agent
With cell contact area, promote more folic acid in conjunction with tumor surface receptor, final nano enzyme contrast agent can escape lysosome
It is directly entered in tumour cell matter and discharges drug.
Embodiment 4, internal photoacoustic imaging investigate the diagnosing tumor ability of nano enzyme contrast agent
For the effect of research FA-RM:GQDzyme/ABTS tumour photoacoustic imaging in vivo, we successfully construct nasopharyngeal carcinoma lotus
After tumor mouse model, respectively with RM:ABTS, RM:GQDzyme, RM:GQDzyme/ABTS and FA-RM:GQDzyme/ABTS to small
Mouse carries out tail vein administration, by mouse with 2.5% isoflurane anesthesia, before the injection with 2h, 4h after injection, 8h, with optoacoustic at
As instrument acquires mouse images.As a result it as shown in figure 4, FA-RM:GQDzyme/ABTS is most in the distribution of tumor tissues, shows
Excellent tumor-targeting.The complex for further demonstrating GQDzyme and ABTS composition can be used as nano enzyme optoacoustic radiography
Agent generates photoacoustic signal, and the coating of the bionical corpusculum of excretion body of erythrocyte membrane building extends the blood of medicament-carried nano enzyme contrast agent
Liquid circulation time, effective endothelium network of escaping are removed, and have more time windows to enter tumor group using EPR effect
It knits.It finds simultaneously, the photo-thermal absorbent properties of GQDzyme in the near infrared region can also generate photoacoustic signal, can further increase
The photoacoustic signal of strong nano enzyme photoacoustic contrast agent.
The imaging effect of embodiment 5, intracorporal near-infrared fluorescence imaging verifying nano enzyme photoacoustic contrast agent
CNE-2 tumor-bearing mice passes through tail vein injection contrast agent (10 μ g/100 μ L), in different times point, anesthetized mice
And it is scanned using small animal living body imaging system.Last time internal body imaging data puts to death mouse after acquiring, and wins each small
Mouse tumour and tissue simultaneously do corresponding fluorescence imaging.As a result as shown in figure 5, the bionical corpusculum chain of excretion body assigns nano enzyme contrast agent
Good stealth ability, RM:GQDzyme/ABTS and FA-RM:GQDzyme/ABTS do not occur largely accumulating on Mouse Liver and
Situation in spleen.Due to the introducing of folate-targeted molecule, the ability of nano enzyme contrast agent target tumor is improved, therefore is increased
Its cumulant in tumor locus.
Finally, it should be noted that above embodiments are used only as helping skilled in the art to understand essence of the invention,
Limiting the scope of the present invention that it goes without doing.
Claims (10)
1. a kind of prepare the method for loading the bionical corpusculum of nano enzyme of nano enzyme and azine di-ammonium salts contrast agent, which is characterized in that
Described method includes following steps:
(1) nano enzyme and azine di-ammonium salts contrast agent are prepared；
(2) bionical corpusculum is prepared；
(3) nano enzyme and ABTS complex contrast agent are packed into bionical corpusculum and prepare the bionical corpusculum of nano enzyme.
2. method according to claim 1, wherein the nano enzyme is carbon dots nano enzyme (GQDzyme).
3. according to the method described in claim 2, it is characterized in that,
The method of contrast agent is prepared described in step (1) are as follows:
1) polyacrylonitrile carbon fiber is added in the mixed solution of the concentrated sulfuric acid and nitric acid；Ultrasonic mixing dissolution, is warming up to 100 DEG C
And it stirs abundant；
2) it molecular cut off 3KD ultrafiltration solution and is lyophilized, GQDzyme is made；
3) GQDzyme is mixed and is stirred overnight in aqueous solution with azine di-ammonium salts (ABTS), GQDzyme/ is lyophilized to obtain in ultrafiltration
ABTS contrast agent.
4. method according to claim 1 or 2, which is characterized in that
The method of bionical corpusculum is prepared described in step (2) are as follows:
1) red blood cell is added in the hypotonic buffer liquid of pre-cooling, mixing is placed on 1~2h in 4 DEG C, is allowed to complete hemolysis；
2) the centrifugation removal hemoglobin supernatant at 4 DEG C, precipitating are erythrocyte membrane；
3) repeated centrifugation is washed 3~5 times, and the biggish bionical corpusculum of partial size is made；
4) bionical corpusculum is ultrasonically treated, the bionical corpusculum that obtained partial size is about 400nm.
5. according to the method in claim 2 or 3, which is characterized in that by GQDzyme/ABTS contrast agent described in step (3)
The method for being packed into bionical corpusculum are as follows: GQDzyme/ABTS contrast agent is loaded into bionical corpusculum using the mode of physical impact and is obtained
Obtain the bionical corpusculum of nano enzyme.
6. the contrast agent prepared using any the method for claim 1-5 or the nano enzyme comprising the contrast agent are bionical
Corpusculum.
7. contrast agent according to claim 6, bionical corpusculum or the bionical corpusculum of nano enzyme, which is characterized in that described makes
The particle size of shadow agent is about 10nm, and the particle size of the bionical corpusculum is about 100nm, the bionical corpusculum of the nano enzyme
Particle size be about 50nm.
8. contrast agent described in claim 6 or 7 or the bionical corpusculum of the nano enzyme comprising the contrast agent are preparing cancer target
Application in preparation,
The cancer target preparation are as follows: oncotherapy preparation is loaded into the bionical corpusculum of nano enzyme,
The oncotherapy preparation is antibody, polypeptide, aptamer or functional form albumen.Preferably, the functional form
Albumen is folic acid.
9. contrast agent described in claim 6 or 7 or the bionical corpusculum of the nano enzyme comprising the contrast agent preparation tumour light at
As the application in contrast agent, the tumour is solid tumor.
10. application according to claim 9, which is characterized in that the tumour light image-forming contrast medium is to be given by vein
The mode of medicine marks the contrast agent of the lesions position of solid tumor.
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