CN104784707B - A kind of hollow nuclear shell structure nano diagnosis and treatment agent of cancer target and its preparation method and application - Google Patents
A kind of hollow nuclear shell structure nano diagnosis and treatment agent of cancer target and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to hollow nuclear shell structure nano diagnosis and treatment agent of a kind of cancer target and preparation method thereof.This nanometer of diagnosis and treatment agent includes upper conversion nano particle kernel, organic inorganic hybridization silica shell and middle cavity structure, can improve the load capacity of sensitising agent, and reduce the reunion of sensitising agent；Surface connection tumor targeted molecular (such as urokinase ammonia dististyle breaks), can specific recognition cancer cell.Under the irradiation of 980nm laser, the VISIBLE LIGHT EMISSION sensitization sensitising agent of this nanometer of diagnosis and treatment agent produces singlet oxygen, can carry out the photodynamic therapy of cancer cell.In addition, the up-conversion luminescence using nanometer diagnosis and treatment agent and the strong absorption characteristic to X-ray, available for up-conversion luminescence and CT double-mode imagings.
Description
Technical field
The invention belongs to nano meter biomaterial technical field, and in particular to a kind of hollow nuclear shell structure nano of cancer target
Diagnosis and treatment agent and its preparation method and application.
Background technology
With biomedical development, the multi-functional combination (abbreviation diagnosis and treatment) of Clinics and Practices turns into new trend.Diagnosis and treatment
Technology can be reduced because multiple dosing or operation bring the risk and pain of patient.In order to realize Precise Diagnosis and efficiently control
Treat, designed diagnosis and treatment agent generally requires to be provided simultaneously with the various functions such as focus targeting, multi-modality imaging and treatment.Rare earth is mixed
Near infrared light can be converted into visible ray by miscellaneous up-conversion nano material, thus have in development Noninvasive diagnosis and treatment agent it is important before
Scape.Due to up-conversion nano material low toxicity, it is anti-light it is Bleachability, without background fluorescence, deeper light penetration depth the features such as can conduct
Bio-imaging fluorescence probe of new generation.Importantly, up-conversion nano material sends visible ray under near infrared light, lead to
Energy transmission is crossed, excites sensitising agent to produce singlet oxygen, toxicity is produced to tumour cell, therefore light can be carried out to tumour cell
Dynamic therapy.In recent years, using the easy modified of the loading functional of silica and surface, development is changed on some
The diagnosis and treatment agent that nano material is combined with silica, for imaging and photodynamic therapy.But curative effect is controlled in these diagnosis and treatment agent
Rate is limited, itself main reason is that the load capacity of sensitising agent is too low, the sensitising agent of load contain conjugation aromatic rings easily reunite and
Self is quenched, and the distance of sensitising agent and upper conversion nano particle have impact on greatly very much energy transfer efficiency.
Therefore, a diagnosis and treatment agent based on up-conversion nano material is designed, above-mentioned sensitising agent loading problem is solved, improved
Photodynamic Therapy is the key of this area.Need to select suitable up-conversion luminescence nano particle and tumour cell simultaneously
Targeting modification molecule, realizes the multi-modality imaging and target function of diagnosis and treatment agent.Such a nanometer of diagnosis and treatment agent is developed in cancer diagnosis and treatment
Using above by with important actual meaning and clinical value.
The content of the invention
The present invention relates to a kind of hollow nuclear shell structure nano diagnosis and treatment agent of cancer target, this nanometer of diagnosis and treatment agent includes upper conversion
Cavity in the middle of nano particle kernel, hybrid inorganic-organic silica shell, kernel and shell and it is supported on the shell
With the sensitising agent in cavity.
According to the present invention, the shell outer surface also has tumor targeted molecular.
According to the present invention, the upper conversion nano particle is hexagonal phase NaLuF4:Gd/Yb/Er is nanocrystalline.
According to the present invention, the hybrid inorganic-organic silica shell is by phenyl bridged bond silica and aminopropyl two
Silica is constituted.We have discovered that, hybrid inorganic-organic silica shell is selected, compared with other silica shells, can be led to
Hydrophobic forces and π-π effect increase sensitising agent load capacity are crossed, and reduce the reunion of sensitising agent.
According to the present invention, the sensitising agent be can absorb that conversion nano particle sends under near infrared light can
See light and produce the sensitising agent of singlet oxygen.Preferably, the sensitising agent is selected from mono carboxylic Phthalocyanine Zinc or Bengal rose red.
According to the present invention, the tumor targeted molecular is one kind in urokinase ammonia dististyle disconnected (ATF), antibody or folic acid etc.
Or it is a variety of.
According to the present invention, the particle diameter of the upper conversion nano particle is 10~100nm, preferably 20~50nm.
According to the present invention, the thickness of the shell is 3~20nm, preferably 5~15nm.
According to the present invention, the thickness of the cavity is 0~10nm, wherein not including 0, preferably 2~8nm.We study hair
Existing, core shell structure of the selection with cavity, compared with without cavity structure, can increase sensitising agent load capacity, while shortening sensitising agent and upper
The distance between conversion nano particle, is conducive to energy transmission.
According to the present invention, the load capacity of the sensitising agent is 10 times of the load capacity of general silica Shell Materials or more
It is high；It is 1.6 times or higher of the load capacity of common core shell structure (no cavity) material.The load of heretofore described sensitising agent
Amount refer to mass percent of the sensitising agent in nanometer diagnosis and treatment agent, by certain wavelength (such as mono carboxylic Phthalocyanine Zinc, can
Selection 699nm) light absorption value calculate and or by ultraviolet-visible absorption spectra calculate and obtain.
According to the present invention, the energy transfer efficiency between upper conversion nano particle and sensitising agent in the diagnosis and treatment agent is general
2 times or higher of the diagnosis and treatment agent of logical silica shell；It is 1.4 times or higher of the diagnosis and treatment agent of common core shell structure.The present invention
Described in energy transfer efficiency from upper swing absorption spectrum change calculate obtain, calculation formula be (I0-I1)/I0, wherein I0、I1
Red light portion (if sensitiser absorption feux rouges) or green portions respectively before and after diagnosis and treatment agent load sensitising agent is (if sensitiser absorption
Green glow) integrated intensity.
The present invention also provides the preparation method of the hollow nuclear shell structure nano diagnosis and treatment agent of above-mentioned cancer target, and it includes following
Step：
(1) the upper conversion nano particle is prepared；
(2) the hollow core shell structure of upper conversion nano particle described in hybrid inorganic-organic coated with silica is prepared；
(3) sensitising agent is adsorbed in the shell and cavity.
It is further comprising the steps of between (2) and step (3) the step of methods described according to the present invention：
(2 ') are in tumor targeted molecular described in the covalent coupling of the shell outer surface.
According to the present invention, that prepared in the step (1) is hexagonal phase NaLuF4:Gd/Yb/Er is nanocrystalline.
According to the present invention, the hexagonal phase NaLuF4:Gd/Yb/Er is nanocrystalline to be prepared by following thermal decomposition method：
(a) sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid gadolinium, trifluoroacetic acid ytterbium and trifluoroacetic acid erbium are weighed at room temperature
Into reaction vessel, solvent is added；It is heated to being continuously heating to 280 under an inert atmosphere after the dissolving of above-mentioned trifluoroacetate~
340 DEG C, reaction naturally cools to room temperature after 0.6~2 hour, precipitates and washs, obtains NaLu (Gd) F4:Yb/Er is nanocrystalline；
(b) by above-mentioned NaLu (Gd) F4:Yb/Er is nanocrystalline to be added to sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid ytterbium
In the oleic acid and octadecylene solvent of trifluoroacetic acid erbium, above-mentioned trifluoroacetate dissolving is heated under an inert atmosphere follow-up of continuing rising
Temperature is to 260~320 DEG C, and reaction naturally cools to room temperature after completing 0.6~2 hour, precipitates and washs, obtains hexagonal phase
NaLuF4:Gd/Yb/Er is nanocrystalline.
According to the present invention, in above-mentioned steps (a), sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid gadolinium, trifluoroacetic acid ytterbium
Molar ratio with each element in trifluoroacetic acid erbium is 1 lutetium：0.3~0.8 gadolinium：2~4Na：0.3~0.5 ytterbium：0.03~0.05
Erbium.
According to the present invention, the solvent in above-mentioned steps (a) is the mixed solvent of oleic acid, oleyl amine and octadecylene.Preferably, institute
The molar ratio for stating in the mixed solvent each component is 1 oleic acid：0.5~1 oleyl amine：0.5~1 octadecylene.
According to the present invention, in above-mentioned steps (b), sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid ytterbium and trifluoroacetic acid erbium
In each element molar ratio be 1 lutetium：0.5~1Na：0.3~0.5 ytterbium：0.03~0.05 erbium.
According to the present invention, the solvent in above-mentioned steps (b) is oleic acid and the mixed solvent of octadecylene solvent.Preferably, institute
The molar ratio for stating in the mixed solvent each component is 1 oleic acid：0.5~1.5 octadecylene.
According to the present invention, the step (2) prepares hybrid inorganic-organic particular by reverse microemulsion process and etch
The hollow core shell structure of upper conversion nano particle described in coated with silica.Its specific steps includes：
NaLuF at room temperature4:Gd/Yb/Er is nanocrystalline to be first scattered in hexamethylene/ammoniacal liquor/CO-520 systems, is first added just
Silester (TEOS), after reacting 3~24 hours, adds Isosorbide-5-Nitrae-bis- (triethoxy silicon substrates) benzene (BTEB) and aminopropyl ethoxy
Base silane (APTES), reacts 6~48 hours, and the molar ratio of three kinds of silicon sources is 1TEOS：0.1~10BETB：0.1~
10APTES；Corrode 3~18 hours in polyvinylpyrrolidone (PVP) aqueous solution, the hollow Core-shell Structure Nanoparticles of gained,
3~20nm of shell thickness, intermediate annular 0~10nm of cavity thickness, but be not 0.
Wherein, the concentration that PVP molecular weight is about 40000, the PVP aqueous solution is 0.05~0.2g/mL, and temperature during corrosion is
90~100 DEG C.
According to the present invention, in the step (2 '), the tumor targeted molecular of covalent coupling is disconnected for urokinase ammonia dististyle
(ATF), the one or more in antibody or folic acid etc..Specifically, covalent coupling ATF method is：
In dimethyl formamide solution, ATF, diisopropylethylamine (DIEA) and BTA-tetramethylurea are added
Hexafluorophosphoric acid ester (HBTU), reacts 10~60 minutes, and ATF is activated rear and hollow Core-shell Structure Nanoparticles at 0~10 DEG C
Reaction 6~24 hours, ATF carboxyl and nano grain surface amino covalence are coupled.
Wherein, reactant quality ratio is 1ATF：10~100DIEA：1~5HBTU：0.1~10 nano particle.
According to the present invention, the absorption sensitising agent in the step (3) is to load sensitising agent by physical absorption.Specifically,
It is described to be adsorbed as：
By the hollow Core-shell Structure Nanoparticles for having connected or being not connected with tumor targeted molecular be added to sensitising agent water or
In dimethyl formamide solution, lucifuge vibrates 6~48 hours, lucifuge Cord blood after centrifuge washing.
Invention additionally discloses following technical scheme：
Application of the above-mentioned nanometer diagnosis and treatment agent in bioanalysis, medical imaging or photodynamic therapy.Specifically diagnosis and treatment agent
Application in treatment medical imaging reagent is prepared, or the application in photodynamic therapy agent is prepared.
In upper conversion imaging application, the nanometer diagnosis and treatment agent is swashed by after cellular uptake using near infrared light (980nm)
Hair, produces feux rouges and green glow, can carry out cell imaging；In CT imaging applications, the nanometer diagnosis and treatment agent has higher CT values, can
It is used as CT contrast agent；In photodynamic therapy application, excited using near infrared light (980nm), pass through upper conversion nano particle
Sensitising agent is sensitized to produce photodynamic therapy application of the singlet oxygen realization in tumour.
Advantages of the present invention：
Sensitising agent is loaded using hollow core shell structure, load capacity had not only been improved but also had shortened sensitising agent and conversion nano on kernel
The distance of grain；Hybrid inorganic-organic silica shell can further increase sensitising agent be born by hydrophobic forces and π-π effects
Carrying capacity, and reduce the reunion of sensitising agent；The urokinase amino moieties (ATF) of surface covalent coupling are overexpressed with cancer cell surfaces
Urokinase receptor (uPA) has high-affinity so that diagnosis and treatment agent has tumour cell target function.Select NaLuF4Matrix it is upper
Conversion nano particle can also realize CT and the double-mode imaging of up-conversion luminescence as the kernel of nanometer diagnosis and treatment agent.Therefore, it is described
The hollow nuclear shell structure nano diagnosis and treatment agent of cancer target will be efficiently applied to imaging and the photodynamic therapy of cancer cell.
Brief description of the drawings
Fig. 1 is hexagonal phase NaLuF in the embodiment of the present invention 14:X-ray powder diffraction figure nanocrystalline Gd/Yb/Er.Instrument
Model MiniFlex2, producer is Rigaku, and copper target radiation wavelength is λ=0.154187nm.
Fig. 2 is the transmission electron microscope picture of nanometer diagnosis and treatment agent in the embodiment of the present invention 1.INSTRUMENT MODEL is JEM-2010, and producer is
JEOL。
Fig. 3 is the upper conversion before and after nanometer diagnosis and treatment agent load mono carboxylic Phthalocyanine Zinc (ZnPc-COOH) in the embodiment of the present invention 1
Launching light spectrogram (excitation wavelength is 980nm).INSTRUMENT MODEL is FSP920-C, and producer is Edinburgh.
Fig. 4 be load ZnPc-COOH in the embodiment of the present invention 1 nanometer diagnosis and treatment agent with ZnPc-COOH in water, in DMF
With the ultraviolet-visible absorption spectroscopy comparison diagram for being carried on general silica material.INSTRUMENT MODEL is Lambda 900, and producer is
Perkin-Elmer。
Fig. 5 is the Up-conversion emission light before and after nanometer diagnosis and treatment agent load Bengal rose red (RB) in the embodiment of the present invention 2
Spectrogram (excitation wavelength is 980nm).INSTRUMENT MODEL is FSP920-C, and producer is Edinburgh.
Fig. 6 be load RB in the embodiment of the present invention 2 nanometer diagnosis and treatment agent with RB in water, DMF neutralizes and is carried on common two
The ultraviolet-visible absorption spectroscopy comparison diagram of silica material.INSTRUMENT MODEL is Lambda 900, and producer is Perkin-Elmer.
Fig. 7 is the singlet oxygen detection of nanometer diagnosis and treatment agent in the embodiment of the present invention 3, by testing DPBF at 417nm
The yield for changing embodiment single line oxygen of absorbance, its result is higher than the common Core-shell structure material for loading ZnPc-COOH and commonly
Earth silicon material.INSTRUMENT MODEL is Lambda 900, and producer is Perkin-Elmer.
Fig. 8 is that nanometer diagnosis and treatment agent is imitated to the efficient photodynamic therapy of human lung carcinoma cell (H1299) in the embodiment of the present invention 3
Really, control experiment shows under the conditions of no light and unsupported sensitising agent without therapeutic effect.INSTRUMENT MODEL is Synergy 4, producer
For BioTek.
Fig. 9 is that nanometer diagnosis and treatment agent is pacified to the biology of normal human embryonic lung fibroblasts (HELF) in the embodiment of the present invention 3
Full property test result.INSTRUMENT MODEL is Synergy 4, and producer is BioTek.
Figure 10 is the cancer target measure of merit of nanometer diagnosis and treatment agent in the embodiment of the present invention 3：Nanometer diagnosis and treatment agent is taken the photograph to H1299
Taken amount is higher than HELF, and not connected ATF nanometer diagnosis and treatment agent is suitable to H1299 and HELF cellular uptake amount.INSTRUMENT MODEL is
Synergy 4, producer is BioTek.
Figure 11 is the nanometer diagnosis and treatment agent in the embodiment of the present invention 3 and the CT imaging effects pair of commercialization contrast agent Iopromide
Than figure.INSTRUMENT MODEL is Inveon MMCT, and producer is SIEMENS.
Figure 12 is upper conversion imaging effect of the nanometer diagnosis and treatment agent to human lung carcinoma cell (H1299) in the embodiment of the present invention 3.Instrument
Device model FV1000, producer is Olympus.
Figure 13 is the structural formula of sensitising agent used in the present invention, (a) mono carboxylic Phthalocyanine Zinc (ZnPc-COOH), (b) Bangladesh rose
Rare red (RB).
The structure of the nanometer diagnosis and treatment agent of Figure 14 present invention and the schematic diagram applied in imaging and photodynamic therapy.
Embodiment
Below in conjunction with drawings and examples, the present invention is described in further detail.But skilled in the art realises that,
Protection scope of the present invention is not limited only to following examples.According to present disclosure, those skilled in the art will recognize that
To in the case where not departing from the technical characteristic and scope given by technical solution of the present invention, embodiment described above is made perhaps
Change and modifications belongs to protection scope of the present invention more.
Embodiment 1：Load the preparation of the nanometer diagnosis and treatment agent of mono carboxylic Phthalocyanine Zinc (ZnPc-COOH)
(1) hexagonal phase NaLuF is prepared based on thermal decomposition method4:Gd/Yb/Er is nanocrystalline
Sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid gadolinium, trifluoroacetic acid ytterbium and trifluoroacetic acid erbium are weighed at room temperature (altogether
2mmoL, the molar ratio of each element is 1 lutetium：0.6 gadolinium：3Na：0.36 ytterbium：0.04 erbium) to three hole flasks, add 6mL oleic acid,
3mL oleyl amines and 3mL octadecylenes are used as solvent；It is continuously heating to after being heated to above-mentioned trifluoroacetate dissolving under an inert atmosphere
300 DEG C, reaction naturally cools to room temperature after 1 hour, precipitates and washs, obtains NaLu (Gd) F4:Yb/Er is nanocrystalline.
By NaLu (Gd) F4:Yb/Er is added in 6mL oleic acid and 6mL octadecylene solution, adds sodium trifluoroacetate, trifluoro
(common 2mmoL, the molar ratio of each element is 1 lutetium for acetic acid lutetium, trifluoroacetic acid ytterbium and trifluoroacetic acid erbium：0.6Na：0.36 ytterbium：0.04
Erbium).It is heated to after above-mentioned trifluoroacetate dissolving being continuously heating to 300 DEG C under an inert atmosphere, reaction is natural after completing 1 hour
Room temperature is cooled to, precipitates and washs, hexagonal phase NaLuF is obtained4:Gd/Yb/Er is nanocrystalline, size about 27nm, its x-ray powder
Diffraction pattern is shown in Fig. 1.
(2) hybrid inorganic-organic coated with silica NaLuF is prepared4:Gd/Yb/Er hollow Core-shell Structure Nanoparticles
20mg NaLuF at room temperature4:Gd/Yb/Er is nanocrystalline to be first scattered in 18mL hexamethylenes/1mL ammoniacal liquor/1mL CO-
In 520 microemulsion systems formed, 150 μ L tetraethyl orthosilicates are added, after reacting 6 hours, 300 μ L Isosorbide-5-Nitrae-bis- (three are added
Ethyl-silicone) benzene and 150 μ L aminopropyl Ethoxysilane, react 18 hours, in polyvinylpyrrolidone (PVP) aqueous solution
6 hours (PVP molecular weight about 40000, solution concentration 0.1g/mL, 96 DEG C of temperature) of middle corrosion, the hollow nuclear shell structure nano of gained
Grain, shell thickness about 7nm, intermediate annular cavity thickness about 3nm (as shown in Figure 2).
(3) covalent coupling ATF
In dimethyl formamide solution, ATF, diisopropylethylamine (DIEA) and BTA-tetramethylurea are added
Hexafluorophosphoric acid ester (HBTU), reacts 30 minutes, and it is small that ATF reacts at 4 DEG C 12 with hollow Core-shell Structure Nanoparticles after being activated
When, ATF carboxyl and nano grain surface amino covalence are coupled.Reactant quality ratio is 1ATF：25DIEA：2.5HBTU：1 receives
Rice grain.
(4) ZnPc-COOH is loaded by physical absorption
5mg connections ATF hollow Core-shell Structure Nanoparticles are added to 1mL, concentration is 5mg/mL ZnPc-COOH's
In dimethyl formamide solution, lucifuge vibrates 12 hours, lucifuge Cord blood after centrifuge washing.Pass through wavelength 699nm extinction
The load capacity that value calculates ZnPc-COOH reaches 7.7wt%, and upper conversion nano particle is calculated from upper swing absorption spectrum change
Energy transfer efficiency between sensitising agent reaches 98% (as shown in Figure 3)；Under other equal conditions, general silica shell
ZnPc-COOH load capacity is 0.7wt%, energy transfer efficiency in layer (non-organic-inorganic hybridization silica shell) material
For 41%；Under other equal conditions, ZnPc-COOH load capacity is 2.7wt% in common core shell structure (no cavity) material,
Energy transfer efficiency is 66%.In addition, ultraviolet-visible absorption spectra (as shown in Figure 4) confirms that described nanometer diagnosis and treatment agent can be reduced
ZnPc-COOH reunion.
Embodiment 2：Load the preparation of the nanometer diagnosis and treatment agent of Bengal rose red (RB)
(1) hexagonal phase NaLuF is prepared based on thermal decomposition method4:Gd/Yb/Er is nanocrystalline
Sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid gadolinium, trifluoroacetic acid ytterbium and trifluoroacetic acid erbium are weighed at room temperature (altogether
2mmoL, the molar ratio of each element is 1 lutetium：0.6 gadolinium：3Na：0.36 ytterbium：0.04 erbium) to three hole flasks, add 6mL oleic acid,
3mL oleyl amines and 3mL octadecylenes are used as solvent；It is continuously heating to after being heated to above-mentioned trifluoroacetate dissolving under an inert atmosphere
300 DEG C, reaction naturally cools to room temperature after 1 hour, precipitates and washs, obtains NaLu (Gd) F4:Yb/Er is nanocrystalline.By NaLu
(Gd)F4:Yb/Er is added in 6mL oleic acid and 6mL octadecylene solution, adds sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid
(common 1.5mmoL, the molar ratio of each element is 1 lutetium for ytterbium and trifluoroacetic acid erbium：0.6Na：0.36 ytterbium：0.04 erbium).In indifferent gas
280 DEG C are continuously heating to after above-mentioned trifluoroacetate dissolving is heated under atmosphere, reaction naturally cools to room temperature after completing 1 hour,
Precipitate and wash, obtain hexagonal phase NaLuF4:Gd/Yb/Er is nanocrystalline, size about 22nm.
(2) hybrid inorganic-organic coated with silica NaLuF is prepared4:Gd/Yb/Er hollow Core-shell Structure Nanoparticles
20mg NaLuF at room temperature4:Gd/Yb/Er is nanocrystalline to be first scattered in 10mL hexamethylenes/1mL ammoniacal liquor/1mL CO-
In 520 microemulsion systems formed, 100 μ L tetraethyl orthosilicates are added, after reacting 8 hours, 200 μ L Isosorbide-5-Nitrae-bis- (three are added
Ethyl-silicone) benzene and 100 μ L aminopropyl Ethoxysilane, react 24 hours, in polyvinylpyrrolidone (PVP) aqueous solution
9 hours (PVP molecular weight about 40000, solution concentration 0.1g/mL, 96 DEG C of temperature) of middle corrosion, the hollow nuclear shell structure nano of gained
Grain, shell thickness about 5nm, intermediate annular cavity thickness about 5nm.
(3) covalent coupling ATF
In dimethyl formamide solution, ATF, diisopropylethylamine (DIEA) and BTA-tetramethylurea are added
Hexafluorophosphoric acid ester (HBTU), reacts 30 minutes, and it is small that ATF reacts at 4 DEG C 12 with hollow Core-shell Structure Nanoparticles after being activated
When, ATF carboxyl and nano grain surface amino covalence are coupled.Reactant quality ratio is 1ATF：25DIEA：2.5HBTU：1 receives
Rice grain.
(4) RB is loaded by physical absorption
5mg connections ATF hollow Core-shell Structure Nanoparticles are added to 1mL, concentration is 5mg/mL RB dimethyl
In formamide solution, lucifuge vibrates 12 hours, lucifuge Cord blood after centrifuge washing.RB is calculated by ultraviolet-visible absorption spectra
Load capacity reach 3.2wt%, calculate the energy between upper conversion nano particle and sensitising agent from upper swing absorption spectrum change
Amount transmission efficiency reaches 53% (as shown in Figure 5).Under other equal conditions, general silica shell (non-organic-inorganic miscellaneous
Change silica shell) RB load capacity is 0.3wt% in material, energy transfer efficiency is 12%；Under other equal conditions,
RB load capacity is 1.9wt% in common core shell structure (no cavity) material, and energy transfer efficiency is 28%.In addition, ultraviolet can
See that absorption spectra (as shown in Figure 6) confirms that described nanometer diagnosis and treatment agent can reduce RB reunion.
Embodiment 3：Load the application performance detection of ZnPc-COOH nanometer diagnosis and treatment agent
(1) detection of singlet oxygen is produced under 980 illumination：As shown in fig. 7, the nanometer diagnosis and treatment agent prepared by embodiment 1,
Power is 1W/cm2980nm laser irradiate 26 minutes, the singlet oxygen of generation can make probe 1,3- diphenyl isobenzofurans
(DPBF) 93% is declined, and other contrast materials (including general silica shell and common core shell structure) only decline about
50%, illustrate that this nanometer of diagnosis and treatment agent can produce more singlet oxygens.
(2) the ex-vivo photodynamic therapeutic effect detection of human lung carcinoma cell (H1299)：As shown in figure 8, embodiment 1 is made
Standby nanometer diagnosis and treatment agent, PBS is washed after human lung carcinoma cell (H1299) is incubated 4 hours, carries out photodynamic therapy test, carefully
Born of the same parents' survival rate is calculated using mtt assay.Power is 0.5W/cm2980nm laser irradiate 10 minutes, nanometer diagnosis and treatment agent concentration is higher than
During 0.05mg/mL, the H1299 death rates reach 90%, illustrate that there is efficient photodynamics to control to cancer cell for this nanometer of diagnosis and treatment agent
Therapeutic effect.
(3) biological safety is tested：As shown in figure 9, the nanometer diagnosis and treatment agent prepared by embodiment 1, under non-illumination condition,
In 0~0.1mg/mL concentration ranges, normal human embryonic lung fibroblasts (HELF) are incubated 24 hours, HELF cell survival rates
More than 80%, illustrate that this nanometer of diagnosis and treatment agent has biological safety.
(4) cancer target performance is tested：As shown in Figure 10, the nanometer diagnosis and treatment agent prepared by embodiment 1, to H1299 cells
Intake is higher than HELF, and is not connected with ATF nanometer diagnosis and treatment agent, and the cellular uptake amount to H1299 and HELF is suitable, illustrates connection
ATF nanometer diagnosis and treatment agent has targeting to tumour cell.
(5) CT imaging effects are tested：As shown in figure 11, the nanometer diagnosis and treatment of the load ZnPc-COOH prepared by embodiment 1
Agent, in 1.25~10mg/mL concentration ranges, as concentration increases, CT signal enhancings.CT values are under 10mg/mL concentration
165HU, and there was only 140HU with the Iopromide CT values of concentration commercialization, illustrate that this nanometer of diagnosis and treatment agent is expected to turn into new class CT
Contrast agent.
(5) transition cell imaging effect is tested on：As shown in figure 12, load ZnPc-COOH prepared by embodiment 1 receives
Rice diagnosis and treatment agent can be applied to cell imaging.Concentration is 0.1mg/mL nanometer diagnosis and treatment agent, is incubated for 37 DEG C in human lung carcinoma cell (H1299)
PBS after 4 hours is educated to wash.Under 980nm laser excitations, the green emission in cell can be observed under confocal fluorescent microscope
(520~560nm) and red emission (640~680nm).
Claims (25)
1. the hollow nuclear shell structure nano diagnosis and treatment agent of a kind of cancer target, it is characterised in that this nanometer of diagnosis and treatment agent includes upper conversion
Cavity in the middle of nano particle kernel, hybrid inorganic-organic silica shell, kernel and shell and it is supported on the shell
With the sensitising agent in cavity；
The hybrid inorganic-organic silica shell is made up of phenyl bridged bond silica and aminopropyl silica；
The sensitising agent is selected from mono carboxylic Phthalocyanine Zinc or Bengal rose red；
The sensitising agent is loaded by physical absorption；
The thickness of the shell is 3~20nm.
2. according to claim 1 nanometer of diagnosis and treatment agent, it is characterised in that the shell outer surface is also with cancer target point
Son.
3. according to claim 1 nanometer of diagnosis and treatment agent, it is characterised in that the upper conversion nano particle is hexagonal phase
NaLuF4:Gd/Yb/Er is nanocrystalline.
4. according to claim 2 nanometer of diagnosis and treatment agent, it is characterised in that the tumor targeted molecular is urokinase ammonia dististyle
One or more in disconnected ATF, antibody or folic acid.
5. the nanometer diagnosis and treatment agent according to claim any one of 1-4, it is characterised in that the grain of the upper conversion nano particle
Footpath is 10~100nm, and the thickness of the shell is 5~15nm, and the thickness of the cavity is 0~10nm and is not 0.
6. according to claim 5 nanometer of diagnosis and treatment agent, it is characterised in that the particle diameter of the upper conversion nano particle is 20~
50nm, the thickness of the cavity is 2~8nm.
7. the preparation method of the hollow nuclear shell structure nano diagnosis and treatment agent of the cancer target any one of claim 1 to 6, its
It is characterised by, the described method comprises the following steps：
(1) the upper conversion nano particle is prepared；
(2) the hollow core shell structure of upper conversion nano particle described in hybrid inorganic-organic coated with silica is prepared；
(3) sensitising agent is adsorbed in the shell and cavity, and the absorption sensitising agent is to load light by physical absorption
Quick dose.
8. method according to claim 7, it is characterised in that also include between (2) and step (3) the step of methods described
Following steps：
(2 ') are in tumor targeted molecular described in the covalent coupling of the shell outer surface.
9. the method according to claim 7 or 8, it is characterised in that prepared in the step (1) is hexagonal phase NaLuF4:
Gd/Yb/Er is nanocrystalline.
10. method according to claim 9, it is characterised in that the hexagonal phase NaLuF4:Gd/Yb/Er is nanocrystalline to be passed through
It is prepared by following thermal decomposition method：
(a) sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid gadolinium, trifluoroacetic acid ytterbium and trifluoroacetic acid erbium are weighed at room temperature to anti-
Answer in container, add solvent；280~340 DEG C are continuously heating to after being heated to above-mentioned trifluoroacetate dissolving under an inert atmosphere,
Reaction naturally cools to room temperature after 0.6~2 hour, precipitates and washs, obtains NaLu (Gd) F4:Yb/Er is nanocrystalline；
(b) by above-mentioned NaLu (Gd) F4:Yb/Er is nanocrystalline to be added to sodium trifluoroacetate, trifluoroacetic acid lutetium, trifluoroacetic acid ytterbium and three
In the oleic acid and octadecylene solvent of fluoroacetic acid erbium, it is continuously heating to after above-mentioned trifluoroacetate dissolving is heated under an inert atmosphere
260~320 DEG C, reaction naturally cools to room temperature after completing 0.6~2 hour, precipitates and washs, obtains hexagonal phase NaLuF4:Gd/
Yb/Er is nanocrystalline.
11. method according to claim 10, it is characterised in that in above-mentioned steps (a), sodium trifluoroacetate, trifluoroacetic acid
Lutetium, trifluoroacetic acid gadolinium, the molar ratio of trifluoroacetic acid ytterbium and each element in trifluoroacetic acid erbium are 1 lutetium：0.3~0.8 gadolinium：2~
4Na：0.3~0.5 ytterbium：0.03~0.05 erbium.
12. method according to claim 10, it is characterised in that the solvent in above-mentioned steps (a) is oleic acid, oleyl amine and ten
The mixed solvent of eight alkene.
13. method according to claim 12, it is characterised in that the molar ratio of the in the mixed solvent each component is 1
Oleic acid：0.5~1 oleyl amine：0.5~1 octadecylene.
14. method according to claim 10, it is characterised in that in above-mentioned steps (b), sodium trifluoroacetate, trifluoroacetic acid
The molar ratio of lutetium, trifluoroacetic acid ytterbium and each element in trifluoroacetic acid erbium is 1 lutetium：0.5~1Na：0.3~0.5 ytterbium：0.03~
0.05 erbium.
15. method according to claim 10, it is characterised in that the solvent in above-mentioned steps (b) is oleic acid and octadecylene
The mixed solvent of solvent.
16. method according to claim 15, it is characterised in that the molar ratio of the in the mixed solvent each component is 1
Oleic acid：0.5~1.5 octadecylene.
17. method according to claim 7, it is characterised in that the step (2) is by reverse microemulsion process and corrosion
Method prepares the hollow core shell structure of upper conversion nano particle described in hybrid inorganic-organic coated with silica.
18. method according to claim 17, it is characterised in that step (2) comprises the following steps：
NaLuF at room temperature4:Gd/Yb/Er is nanocrystalline to be first scattered in hexamethylene/ammoniacal liquor/CO-520 systems, first adds positive silicic acid second
Ester TEOS, after reacting 3~24 hours, adds Isosorbide-5-Nitrae-bis- (triethoxy silicon substrate) benzene BTEB and aminopropyl Ethoxysilane
APTES, reacts 6~48 hours, and the molar ratio of three kinds of silicon sources is 1TEOS：0.1~10BETB：0.1~10APTES；In poly- second
Corrode 3~18 hours in the alkene pyrrolidone aqueous solution, the hollow Core-shell Structure Nanoparticles of gained, 3~20nm of shell thickness, it is middle
Toroidal cavity 0~10nm of thickness, but be not 0.
19. method according to claim 18, it is characterised in that PVP molecular weight is that the concentration of 40000, the PVP aqueous solution is
0.05~0.2g/mL, temperature during corrosion is 90~100 DEG C.
20. method according to claim 8, it is characterised in that in the step (2 '), the cancer target point of covalent coupling
Son is the one or more in urokinase ammonia dististyle disconnected ATF, antibody or folic acid.
21. method according to claim 20, it is characterised in that covalent coupling ATF method is：
In dimethyl formamide solution, ATF, diisopropylethylamine DIEA and BTA-tetramethylurea hexafluoro phosphorus are added
Acid esters HBTU, reacts 10~60 minutes, and ATF reacts 6~24 with hollow Core-shell Structure Nanoparticles after being activated at 0~10 DEG C
Hour, ATF carboxyl and nano grain surface amino covalence are coupled.
22. method according to claim 21, it is characterised in that reactant quality ratio is 1ATF：10~100DIEA：1~
5HBTU：0.1~10 nano particle.
23. the method according to claim 7 or 8, it is characterised in that step is adsorbed as described in (3)：
The hollow Core-shell Structure Nanoparticles for having connected or being not connected with tumor targeted molecular are added to the water or diformazan of sensitising agent
In base formamide solution, lucifuge vibrates 6~48 hours, lucifuge Cord blood after centrifuge washing.
24. the preparation-obtained nanometer of any one of any one of claim 1 to 6 or claim 7 to 23 is examined
Agent is treated in the application in preparing medical imaging reagent and the application in photodynamic therapy agent is prepared.
25. application according to claim 24, it is characterised in that the imaging includes upper conversion imaging or CT imagings.
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