CN103990124A - Graphene nanocomposite particle used for multimodal imaging/photodynamic therapy, and preparation method and application thereof - Google Patents
Graphene nanocomposite particle used for multimodal imaging/photodynamic therapy, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a graphene nanocomposite particle used for multimodal imaging/photodynamic therapy and a preparation method and application thereof. The nanocomposite particle contains a nuclide-labeled photosensitizer used for photodynamic therapy, graphene nanocomposite particles and a surface modified hydrophilic polymer. The invention further relates to application of the graphene nanocomposite particle in photodynamic/nuclear medical imaging and fluorescence imaging cooperative treatment monitoring. As a drug delivery vector of photodynamic therapy, graphene can obviously improve drug effects; and the graphene nanocomposite particle has the advantages of simple preparation, a cheap price and substantial curative effects.
Description
Technical field
The present invention relates to a kind of graphene nano compound particle with multi-modality imaging/optical dynamic therapy, its preparation method and institute's metal nanometer material, for photodynamics/multi-modality imaging Synergistic treatment application of the diseases such as tumor, belong to biological medicine material and nanosecond medical science field.
Background technology
According to the incidence trend of current tumor, the year two thousand twenty whole world tumor incidence will be than present increase by 50%, and the annual newly-increased tumor patient number in the whole world will reach 1,500 ten thousand people (World Health Organization (WHO) (WHO) delivers < < world cancer report > > in the recent period).Malignant tumor has become China resident's underlying cause of death, serious harm China people ' s health not only, and the efficient specific diagnostic, treatment that have increased considerably government and individual medical expense burden China tumor are not only to extensive patients, and even the development of whole national medical and health care system and social harmony are stable, and the realization of well-off society is all extremely important and very urgent demand.Aspect treatment, operation, radiation and chemotherapy are three kinds of Main Means for the treatment of malignant tumor.Yet operative treatment causes a large amount of normal structures or organ structure and merit loss of energy, and cannot thoroughly effect a radical cure, and more seriously may promote sending out of tumor cell.Chemotherapy and radiation often produces the toxic and side effects such as cardiac toxicity, hepatic and renal function injure, bone marrow depression, jeopardizes patient's life or causes life quality seriously to reduce.Therefore, how to improve therapeutic effect of malignant tumour, reduce toxic and side effects and become the target that people pursue for a long time.
Photodynamic therapy (PDT) is a kind of pattern of carrying out topical therapeutic for various tumors, cardiovascular disease, dermatosis, ophthalmic diseases of FDA approval.PDT produces active oxygen (ROS) by photosensitive photosensitizer (PS) under suitable rayed, thus inducing cytotoxic compare with chemotherapy and radiation, PDT has the specific cell fragmentation effect of minimum side effect and Geng Gao.Therefore thereby the high efficiency tumor sites that is transported to of photosensitizer is realized to the focus that better tumor response becomes this field.There are many methods, as photosensitizer molecule and receptor coordination compound or antibodies are improved to its specificity, but poor effect, as conventionally needed quantitatively far more than the part of receptor in order to obtain optimal dose, has limited the application of targeting photosensitizer molecule in PDT treatment mostly.The Potential Vector that has the nanometer platform of high capacity and transportation performance to be used as many photosensitizer is studied, as phthalocyanine, and porphyrin etc.Nanoparticle has following advantage: surface area is large; Can carry out easily surface chemical modification according to different carrying medicaments; Its size of scalable makes it by EPR effect, be easier to be gathered in tumor sites.Graphene has been subject to paying close attention to widely in biomedical applications since within 2004, finding, it can be used as the carrier of medicine and gene transportation its specific surface area ambassador; Its intrinsic near-infrared region high absorbance makes it can be used for the photo-thermal therapy in treatment of cancer.
Summary of the invention
Primary and foremost purpose of the present invention is to provide the graphene nano compound particle with multi-modality imaging/optical dynamic therapy performance that can load photosensitizer of a kind of good biocompatibility, good water solubility, no cytotoxicity;
Another object of the present invention is to provide a kind of simple and practical preparation method that can this particle of large-scale production of the new graphene nano compound particle with multi-modality imaging/optical dynamic therapy performance;
A further object of the present invention is to provide a kind of described graphene composite particle as the nuclear medicine of the diseases such as tumor, the application of fluorescent imaging diagnosis/optical dynamic therapy.
Object of the present invention can be achieved through the following technical solutions:
A new graphene nano compound particle with multi-modality imaging/optical dynamic therapy, the graphene composite particle of modifying containing the hydrophilic polymer that is useful on photo-thermal therapy in its structure is, the photosensitizer of photodynamic therapy and at the nucleic of its surface markers.
Wherein, described Graphene granule comprises at least one in the graphene oxide particle (rGO) after graphene oxide particle (GO), reduction.
Wherein, described nucleic comprises
64at least one in Cu or other nuclear medicine nucleic.
Wherein, described photosensitizer comprise chlorin, methyl pyrophaeophorbide alkyl ether derivative ((2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, HPPH), the many derivatives of porphyrin of carboxyl, temoporfin, four (4-aminophenyl) porphyrin, hematoporphyrin monomethyl ether, chlorin, at least one in 5-ALA etc.
Wherein, described finishing hydrophilic polymer is Polyethylene Glycol (PEG), and the molecular weight ranges of PEG is 5-20kDa, and the unit of molecular weight is dalton.
Graphene nano compound particle (GO-PEG-[of the present invention
64cu] HPPH) can produce as follows
1) by paddling process, take graphite powder as the synthetic graphene oxide of original material, in 0.01-1M NaOH being joined to graphene oxide suspension at 40-60 ℃, stir 1-10 hour, with HCL, solution being adjusted to pH is that 0.5-2. passes through rinsing repeatedly and centrifugal gained solution neutralized, then by 1-10mg/mL PEG-NH
2solution joins in 0.1-10mg/mL graphene oxide solution, ultrasonotomography 1-10 minute, then 1-10mg EDC is joined in the mixture of two deciles, and stirred overnight at room temperature, obtains GO-PEG solution, further dialysed purification saving backup at 4 ℃ of this solution;
2) first 10-100 μ g HPPH is dissolved in 10-100 μ L DMSO, with 100-500 μ L0.01-0.5M, the sodium acetate of pH5-6 is cooked buffer, then add 100-300 μ L ethanol, add again and contain the isotopically labeled sodium-acetate buffer (pH5-6 of 0.1-10mCi, 50-100 μ L), add solution, mixture is heated to 100 ℃, be 20-40 minute heat time heating time, high effective liquid chromatography for measuring labeling effciency, after labelling, with 10 * PBS, neutralize and mix with low temperature HPPH, according to the process of foregoing description, half product will be at next step for the preparation of GO-PEG-[isotope labeling] HPPH,
3) isotope labeling-HPPH is dissolved in DMSO, 1-10mg isotope labeling-HPPH mixes and stirred overnight at room temperature with 1-10mg GO – PEG in 0.1%-10%Tween-20/ aqueous solution 1mL, the excessive HPPH centrifugal filtration that by centrifuge and ultrafilter membrane, molecular weight is less than to 100KD is again fallen, then use distilled water flushing 4-5 time, finally by GO-PEG-[isotope labeling] HPPH complex saves backup at 4 ℃.
More preferably adopt following steps to produce:
1) by paddling process, take graphite powder as the synthetic graphene oxide of original material, stir 4 hours in NaOH (0.05M) being joined to graphene oxide (GO) suspension at 50 ℃.With Hcl, solution is adjusted to PH=1.By rinsing repeatedly with centrifugal gained solution is neutralized, then by PEG-NH
2(5mg/mL) solution joins in (1mg/mL) GO solution, ultrasonotomography 5 minutes, then 5mg N-(3-dimethylaminopropyl-N-ethylcarbodiimide) hydrochloride (EDC) is joined in the mixture of two deciles.Stirred overnight at room temperature, obtains GO-PEG solution.Further dialysed purification saving backup at 4 degrees Celsius of this solution.
2) in order to prepare isotopically labeled HPPH, as
64cu-HPPH, is first dissolved in HPPH (50 μ g) in DMSO (50 μ L), with sodium acetate (200 μ l, 0.1M, pH5.5), cooks buffer, then adds ethanol (200 μ L), then adds and contain
64cu (1mCi) sodium-acetate buffer (pH5.5,75 μ L) adds solution, mixture to be heated to 100 ℃, and be 30 minutes heat time heating time.High effective liquid chromatography for measuring labeling effciency.After labelling, with 10 * PBS, neutralize and mix with low temperature HPPH.According to the process of foregoing description, half product will be at next step for the preparation of GO-PEG-[
64cu] HPPH.
3) will
64the HPPH of Cu labelling is dissolved in dimethyl sulfoxine, 4mg
64cu-HPPH mixes in 1%Tween-20/ aqueous solution (1mL) with GO – PEG (2mg) and stirred overnight at room temperature.The excessive HPPH centrifugal filtration that by centrifuge and ultrafilter membrane, molecular weight is less than to 100KD is again fallen, and then uses distilled water flushing 4-5 time, finally by GO-PEG-[
64cu] HPPH complex saves backup at 4 ℃.
Beneficial effect of the present invention:
1. the present invention can load photosensitizer as PHHP on graphene nanometer composite surface, makes it have optical dynamic therapy performance.
The surface coverage of graphene nanometer composite of the present invention biocompatible materials as after PEG and other polymer, in multiple importing approach, all there is no showed cell toxicity, and nanoparticle can be removed by liver and two kinds of approach of kidney.
3. the photosensitizer in graphene nanometer composite of the present invention has isotope labeling as the surface of PHHP, as
64cu, can be used for the multi-modality imaging of PET imaging and fluorescence, optoacoustic.
4. graphene nanometer composite (the GO-PEG-[of loading of the present invention PHHP
64cu] HPPH) to compare with free HPPH, the cell marking efficiency of GO-PEG-HPPH obviously increases.Photoacoustic imaging and right in body
64the HPPH of Cu labelling carries out nuclear medicine, has shown after intravenous injection, and GO-PEG-HPPH has higher tumor uptake, by the low-energy laser irradiation tumor of 671nm, can destroy tumor.This graphene nano structure carrier has better HPPH transportation and imaging potential, can effectively instruct photodynamic therapy.
Accompanying drawing explanation
Fig. 1 graphene nano compound particle Electronic Speculum and UV Vis Spectroscopic Characterization result
A) GO-PEG-HHPH structural representation .b) GO-PEG-HPPH is dispersed in the AFM imaging in ultra-pure water.C) GO-PEG, the conventional ultraviolet-visible spectrum of HPPH and GO-PEG-HPPH, GO-PEG-HPPH has two characteristic absworption peaks at 414nm and 665nm place.D) free HPPH, GO-PEG and GO-PEG-HPPH is in the fluorescence spectrum of 1 μ M of HPPH and 0.49 μ g GO-PEG.E) with 671nm laser (75mW/cm
2) irradiate the time that HPPH is different with GO-PEG-HPPH (1 μ M) and produce singlet oxygen.
Fig. 2 tumor cell is engulfed the external fluorescent effect of graphene nanometer composite
A) 0.49 μ g/mL GO-PEG for tumor cell, 1 μ M free HPPH, the fluorescence imaging of equivalent GO-PEG-HPPH (1 μ M HPPH and 0.49 μ g/mL GO-PEG) after 24 hours.B) after cytophagy nano-particle with the average fluorescence intensity of flow cytometry analysis of cells, with PBS, 0.49 μ g/ml GO-PEG first peak), 1 μ M HPPH (the second peak), and GO-PEG-HPPH (the 3rd peak) cultivation was carried out Flow Cytometry Analysis average fluorescent strength after 24 hours.C) the external fluorescence imaging of cell, with 1 μ M free HPPH, 0.49 μ g/mL GO-PEG or GO-PEG-HPPH cultivate laser irradiation (671nm, 2-8mW/cm after 24 hours
2, 3 minutes) and the fluorescence imaging that carries out Calcein AM/Ethidium homodimer-1 dyeing.D) use the free HPPH of variable concentrations, GO-PEG, the 4T1 cell that GO-PEG-HPPH cultivates is used 671nm laser irradiation (2-8mW/cm
2, 3 minutes), the relative survival activity in later stage.
Fig. 3 is the distribution of graphene nanometer composite in tumor tumor-bearing mice body and near-infrared fluorescence imaging and the PET video picture of tumor transportation evaluation.
A) distribution of different time points in 4T1 tumor-bearing mice body and the near-infrared fluorescence imaging of tumor transportation evaluation after HPPH and GO-PEG-HPPH injection.B) irradiate medicine and after 24 hours, collect the external near-infrared fluorescence imaging of in vitro tumor and major organs.C) inject 3.7MBq's (100 μ Ci)
64cu-HPPH and HPPH mixture or
64the GO-PEG-HPPH living animal PET image that Cu-HPPH connects.D) tumor-bearing mice tumor uptake
64after Cu-HPPH 15,30,60,120 minutes, its tumor PET imaging quantitative data analysis.
Tumor growth situation after the treatment of Fig. 4 tumor-bearing mice.The tumor growth curve after the treatment of tumor-bearing mice on the same group not.B) Kaplan-Meier survival curve after the treatment of tumor-bearing mice on the same group not.
Fig. 5 is the representative picture of tumor-bearing mice on the same group treatment after 14 days not a), and circle represents knub position.B) collect not the H & E dyeing in tumor cross section after tumor-bearing mice treatment 24h on the same group, GO-PEG-HPPH processed group, tumor is badly damaged after laser irradiation.
Fig. 6 a) 4T1 tumor-bearing mice at 671nm laser (75mW/cm
2, 20min) pre-irradiation and the photoacoustic imaging of irradiation after 24 hours.Injection PBS, GO-PEG, HPPH, or after GO-PEG-HPPH24 hour, carry out laser irradiation.Circled is tumor tissues.B) optoacoustic data cross section oxygen saturation quantitative analysis.
The specific embodiment
By concrete preparation example and embodiment, can make the present invention be illustrated more clearly in below:
One, preparation example: the chemical substance of using in following synthesis step is commercial goods.
The preparation method of graphene nanometer composite, comprises the following steps:
1) by paddling process, take graphite powder as the synthetic graphene oxide of original material
2) stir 4 hours in NaOH (ultimate density after adding is 0.05M) being joined to graphene oxide (GO) suspension at 50 ℃.With HCL, solution is adjusted to pH=1, by rinsing repeatedly with centrifugal gained solution is neutralized.
3) by 1ml PEG-NH
2(5mg/mL) solution joins in 1ml GO solution (1mg/mL), ultrasonotomography 5 minutes, again 5mg N-(3-dimethylaminopropyl-N-ethylcarbodiimide) hydrochloride (EDC) is joined in the mixture of two deciles, stirred overnight at room temperature, obtains GO-PEG solution.
4) make 50 μ g HPPH) be dissolved in 50 μ L DMSO (dimethyl sulfoxide), with sodium acetate (200 μ l, 0.1M, pH5.5), cook buffer, then add ethanol (200 μ L), then add and contain
64cu (1mCi) sodium-acetate buffer (pH5.5,75 μ L) adds solution, mixture to be heated to 100 ℃, and be 30 minutes heat time heating time.
5) will
64the HPPH of Cu labelling is dissolved in DMSO, 4mg
64cu-HPPH mixes in 1%Tween-20/ aqueous solution (1mL) with GO – PEG (2mg) and stirred overnight at room temperature.
6) the excessive HPPH centrifugal filtration that by centrifugal and ultrafilter membrane, molecular weight is less than to 100KD is fallen, and then uses distilled water flushing 4-5 time, finally by GO-PEG-[
64cu] HPPH complex saves backup at 4 ℃.
Two, embodiment:
1. graphene nano compound particle physics characteristic characterizes:
Application atomic force microscope is carried out morphology characterization to gold nano compound particle.As shown in Fig. 1 b atomic force microscope, graphene nano compound particle can singlely disperse in aqueous phase solvent, and granule size is comparatively even.Ultraviolet-visible absorption spectroscopy shows that GO-PEG-HPPH has two characteristic absworption peaks (Fig. 1 d) at 414nm and 665nm place, shows that photosensitizer is successfully modified on graphene nano particle.
Graphene nano compound particle is further transferred to ultra-pure water, PBS, cell culture medium, in serum, cultivates and does not observe precipitation after 24 hours, and water solublity and stability that this nanoparticle is good are described.
2. the extracorporeal biology performance evaluation of graphene nano compound particle:
In order to carry out cellular uptake experiment, cell is proceeded in Lab Tek II8 orifice plate, cell density is 1 * 10
4individual/mL also makes degree of converging reach 60-80%, then uses the GO-PEG of same concentrations HPPH (1 μ M), and GO-PEG-HPPH and free HPPH cultivate respectively cell 24 hours, finally uses 1 * phosphate buffer to rinse 3 times.By fluorescence microscope, observe (Fig. 2 a-c), can find out that tumor cell can be engulfed this graphene nanometer composite effectively in vitro.
Graphene nano-photosensitizer complex is processed after cell, produces singlet oxygen successful under laser irradiation.Once HPPH has been connected to GO-PEG surface, because HPPH and Graphene are directly had an effect and caused its fluorescence to be quenched immediately.The singlet oxygen that 671nm exciting light singlet oxygen sensor detects GO-PEG-HPPH and free HPPH produces.SOSG ' the s fluorescence intensity of GO-PEG-HPPH and free HPPH all strengthens along with the time, has shown the generation of singlet oxygen.Although HPPH fluorescence is obviously by GO-PEG cancellation, the ability of GO-PEG-HPPH generation singlet oxygen only has the 60-70% (Fig. 1 e) of free HPPH, is enough to carry out the PDT treatment of tumor.Its nanometer size effect will promote to carry in tumor tissues passive target and cell, can compensate the singlet oxygen of minimizing.
In order to measure the cytotoxicity of graphene nano material, tumor cell inoculation is entered in 96 orifice plates, every hole density is 1 * 10
4/ hole.After 24 hours with the GO-PEG-HPPH of a series of variable concentrations (maximum concentration is 20 μ M), free HPPH, and GO-PEG processes.Then every hole adds 20 μ l MTT (5.0mg/mL) solution, after 4 hours, remove and cultivate medium, finally add 100ul dimethyl sulfoxine (DMSO) to dissolve crystal, by microplate reader, measure cytotoxicity, result shows GO-PEG, the equal no cytotoxicity of GO-PEG-HPPH nano-complex.
For the cell model of the outer photodynamic therapy of detection bodies, we inoculate 4T1 cell in 96 orifice plates, and every hole density is 5 * 10
3/ hole, culture medium is RPMI-1640 complete medium.The GO-PEG-HPPH of same concentrations, free HPPH, and GO-PEG cultivates cell 24 hours.PBS rinses 3 times.Every hole adds 100 μ L fresh cultures, then with 671nm laser instrument, with different power, irradiates (2-8mW/cm immediately
2) 3 minutes.Flat board is put into incubator incubated overnight.By above-mentioned standard MTT experimental evaluation cytoactive.After laser irradiation, GO-PEG-HPPH nano-complex killing off tumor cells successful is better than other groups (Fig. 2 d).
6. build 4T1 breast cancer tumour bearing mouse model, when gross tumor volume reaches 100mm
3time, tail vein injection GO-PEG-HPPH (1.0mg/kg HPPH and 0.77mg/kg GO-PEG), GO-PEG (0.77mg/kg) or free HPPH (1.0mg/kg), laser after 24 hours (671nm, 90J/cm
2, 75mW/cm
2) irradiate 20 minutes.Matched group is accepted medicine still without laser irradiation.After treatment, observe mouse tumor growth 60 days.Every other day detect tumor size, and calculate gross tumor volume.All mices are every other day measured body weight one time, can find that the growth of tumor has been subject to obvious inhibition (Fig. 4 a-b).
After PDT treatment, tumor-bearing mice major organs 4% formalin is at room temperature fixing, dyeing, microscopic examination.The gross tumor volume of graphene nano-photosensitizer complex processed group and matched group (free photosensitizer HPPH processed group and nano-particle GO processed group) have significant significant difference.Tumor tissues HE dyeing is presented at PBS matched group tumor biopsy and does not observe obvious neoplasm necrosis.Collect not the HE dyeing in tumor cross section after tumor-bearing mice treatment on the same group.After the laser irradiation of GO-PEG-HPPH processed group tumor, be badly damaged (Fig. 5 b), show that graphene nano-photosensitizer complex has better inhibition tumor effect.
7. the bimodal video picture of the near-infrared fluorescent of pair mouse tumor model and PET, mouse mainline GO-PEG-HPPH, free HPPH or GO-PEG (200 μ L, 1mg/kg HPPH, 0.77mg/kg GO-PEG).In injection 0.5,1,2,6,24,36, and after 72 hours, use Maestro II optical imaging system to carry out imaging.By Maestro II software, HPPH spectrum and automatic fluorescence spectrum are separated, near-infrared fluorescence imaging is as Fig. 3 a and Fig. 3 b.Intravenous injection 3.7MBq (100 μ Ci)
64the HPPH of Cu labelling and unlabelled HPPH (200 μ g) or GO-PEG-HPPH, carries out the dynamic living imaging of PET, image data acquiring and reconstruction after 24 hours.After imaging, mice is put to death immediately, its blood, tumor and major organs are weighed and gamma counter measurement.
Graphene nanometer composite is a kind of new graphene nano-photosensitizer compound particle of developing of the present invention for oncotherapy, compare with existing conventional optical dynamic therapy, realized the integrated of diagnosis and treatment, cheap practicality is conducive to apply clinically more simultaneously.Loading photosensitizer of the present invention is as the graphene nanometer composite (GO-PEG-[of HPPH
64cu] HPPH) to compare with free HPPH, in the cell of GO-PEG-HPPH, conevying efficiency obviously increases.Photoacoustic imaging and right in body
64the HPPH of Cu labelling carries out PET imaging, has shown after intravenous injection, and GO-PEG-HPPH has higher tumor uptake, by the low-energy laser irradiation tumor of 671nm, can destroy tumor.Graphene nano carrier after PEG modification is simultaneously less than the Graphene carrier organism toxicity of unmodified, has strengthened water solublity, the stability of material.This graphene nano structure carrier has better photosensitizer transportation and imaging potential, can effectively instruct photodynamic therapy.
The present invention's graphene nano granule used can also comprise: the graphene oxide of oxidized form Graphene and reduction, other how optional photodynamic therapy comprises chlorin with photosensitizer, methyl pyrophaeophorbide alkyl ether derivative (HPPH), the many derivatives of porphyrin of carboxyl, temoporfin, four (4-aminophenyl) porphyrin, hematoporphyrin monomethyl ether, chlorin, 5-ALAs etc. are not limited to HPPH etc., polyethyleneglycol modified and the nano level distribution of sizes of graphene nano particle surface realizes its tumor passive target function, also can carry out the peptide modified tumor-targeting function that realizes simultaneously, preparation method is similar.
Claims (8)
1. the graphene nano compound particle for multi-modality imaging/optical dynamic therapy, it is characterized in that, this nano-complex particle is containing the hydrophilic polymer that is useful on isotopically labeled photosensitizer, graphene nano compound particle and the finishing of photodynamic therapy.
2. a kind of graphene nano compound particle for multi-modality imaging/optical dynamic therapy as claimed in claim 1, it is characterized in that, described graphene nano compound particle comprises at least one in the graphene oxide particle after graphene oxide particle or reduction.
3. the graphene nano compound particle of a kind of multi-modality imaging/optical dynamic therapy as claimed in claim 1, is characterized in that, described photosensitizer is isotopically labeled photosensitizer, comprises
64cu-HPPH, the many derivatives of porphyrin of carboxyl, temoporfin, four (4-aminophenyl) porphyrin, hematoporphyrin monomethyl ether, chlorin, at least one in 5-ALA.
4. a kind of graphene nano compound particle for multi-modality imaging/optical dynamic therapy as claimed in claim 1, is characterized in that, described finishing hydrophilic polymer comprises Polyethylene Glycol.
5. the preparation method of the graphene nano compound particle as described in claim 1 to 4 any one, comprises the steps:
1) by paddling process, take graphite powder as the synthetic graphene oxide of original material, in 0.01-1M NaOH being joined to graphene oxide suspension at 40-60 ℃, stir 1-10 hour, with HCL, solution being adjusted to pH is 0.5-2, by rinsing repeatedly with centrifugal gained solution is neutralized, then by 1-10mg/mL PEG-NH
2solution joins in 0.1-10mg/mL graphene oxide solution, ultrasonotomography 1-10 minute, then 1-10mg EDC is joined in the mixture of two deciles, and stirred overnight at room temperature, obtains GO-PEG solution, further dialysed purification saving backup at 4 ℃ of this solution;
2) first 10-100 μ g HPPH is dissolved in 10-100 μ L DMSO, with 100-500 μ L0.01-0.5M, the sodium acetate of pH5-6 is cooked buffer, then add 100-300 μ L ethanol, add again and contain the isotopically labeled pH5-6 of 0.1-10mCi, 50-100 μ L sodium-acetate buffer, add solution, mixture is heated to 100 ℃, be 20-40 minute heat time heating time, high effective liquid chromatography for measuring labeling effciency, after labelling, with 10 * PBS, neutralize and mix with low temperature HPPH, according to the process of foregoing description, half product will be at next step for the preparation of GO-PEG-[isotope labeling] HPPH,
3) isotope labeling-HPPH is dissolved in DMSO, 1-10mg isotope labeling-HPPH mixes and stirred overnight at room temperature with 1-10mg GO – PEG in 0.1%-10%Tween-20/ aqueous solution 1mL, the excessive HPPH centrifugal filtration that by centrifuge and ultrafilter membrane, molecular weight is less than to 100KD is again fallen, then use distilled water flushing 4-5 time, finally by GO-PEG-[isotope labeling] HPPH complex saves backup at 4 ℃.
6. the preparation method of stannic oxide/graphene nano compound particle as claimed in claim 1, comprises the steps:
By paddling process, take graphite powder as the synthetic graphene oxide of original material, stir 4 hours in 0.05M NaOH being joined to graphene oxide suspension at 50 ℃; With HCL, solution is adjusted to pH=1; By rinsing repeatedly with centrifugal gained solution is neutralized, then 5mg/mL mPEG-NH2 solution is joined in 1mg/mL graphene oxide solution, ultrasonotomography 5 minutes, then 5mg EDC is joined in the mixture of two deciles and be used for preparing PEG-GO; 50 μ g HPPH are dissolved in the DMSO of 50 μ L, and with 200 μ L, 0.1M, pH5.5 sodium acetate is cooked buffer, then adds 200 μ L ethanol, adds and contains 1mCi
64cu, pH5.5,75 μ L sodium-acetate buffers, add solution, and mixture is heated to 100 ℃, and be 30 minutes heat time heating time, to prepare
64the HPPH of Cu labelling; By 4mg
64cu-HPPH and 2mg GO – PEG mix and stirred overnight at room temperature in 1%Tween-20/ aqueous solution 1mL, and the excessive HPPH centrifugal filtration that molecular weight is less than to 100KD by centrifuge and ultrafilter membrane falls, and then use distilled water flushing 4-5 time, finally by GO-PEG-[
64cu] HPPH complex saves backup at 4 ℃.
7. the purposes of graphene nano compound particle as claimed in claim 1, its photodynamics for the preparation of tumor/multi-modal video picture Synergistic treatment reagent.
8. graphene nano compound particle as claimed in claim 1, in the purposes of preparing the tumor imaging diagnostic reagent of nuclear medicine, fluorescence and optoacoustic.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105286803A (en) * | 2015-12-03 | 2016-02-03 | 华南师范大学 | Method for enhancing complex probe photoacoustic signals on basis of fluorescence quenching effect |
| CN105582541A (en) * | 2014-10-21 | 2016-05-18 | 张绍良 | PEGylated graphene oxide-porphyrin dimer salt complex and use thereof |
| CN106139242A (en) * | 2015-04-27 | 2016-11-23 | 中国科学院上海硅酸盐研究所 | Bioceramic scaffold material of graphene modified and its preparation method and application |
| CN110662748A (en) * | 2017-02-03 | 2020-01-07 | 尼尔瓦纳科学股份有限公司 | Hydroporphyrins for photoacoustic imaging |
-
2014
- 2014-04-25 CN CN201410171196.0A patent/CN103990124A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| PENGFEI RONG 等: "Photosensitizer Loaded Nano-Graphene for Multimodality Imaging Guided Tumor Photodynamic Therapy", 《THERANOSTICS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105582541A (en) * | 2014-10-21 | 2016-05-18 | 张绍良 | PEGylated graphene oxide-porphyrin dimer salt complex and use thereof |
| CN105582541B (en) * | 2014-10-21 | 2018-09-07 | 张绍良 | The graphene oxide of Pegylation-Porphyrin dimer salt composite and application thereof |
| CN106139242A (en) * | 2015-04-27 | 2016-11-23 | 中国科学院上海硅酸盐研究所 | Bioceramic scaffold material of graphene modified and its preparation method and application |
| CN105286803A (en) * | 2015-12-03 | 2016-02-03 | 华南师范大学 | Method for enhancing complex probe photoacoustic signals on basis of fluorescence quenching effect |
| CN110662748A (en) * | 2017-02-03 | 2020-01-07 | 尼尔瓦纳科学股份有限公司 | Hydroporphyrins for photoacoustic imaging |
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