CN106729708A - A kind of preparation method of ZnPc UCNP@SiO2 PEG G nano-complexes - Google Patents
A kind of preparation method of ZnPc UCNP@SiO2 PEG G nano-complexes Download PDFInfo
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Abstract
The present invention discloses a kind of ZnPc UCNP@SiO2The preparation method of PEG G nano-complexes.Its preparation method is as follows：UCNPs up-conversion nanoparticles are synthesized by microemulsion method, one layer of SiO has been wrapped up on its surface2, then in SiO2Surface amination obtains UCNP@SiO2‑NH2, then to UCNP@SiO2‑NH2Surface is modified, and obtains ZnPc UCNP@SiO2PEG COOH nano-complexes, are finally loaded into targeted molecular Gefitinib (G) its surface and obtain ZnPc UCNP@SiO2PEG G nano-complexes.The nano-complex particle prepared by the present invention is evenly distributed, and good crystallinity has good photodynamic activity, is expected to be developed into the new medicine-carried system of high-efficiency low-toxicity.
Description
Technical field
The present invention relates to biology medical material technical field, specifically a kind of ZnPc-UCNP@SiO2- PEG-G is nano combined
The preparation method of thing.
Background technology
Optical dynamic therapy is also referred to as photochemical therapy (Photodynamic therapy, PDT), and it refers to using special
Standing wave light irradiation long locally or systemically injects the medicine (sensitising agent, PS) of body in advance, and sensitising agent is activated and and oxygen therewith
Son effect produces the cytotoxic active oxygen (ROS) of tool, tumor tissues is produced with indirectly-acting by directly irreversible
Damage.Photodynamic therapy is different from traditional treatment tumour means, and it all has optional to target tissue and degree of injury
Property, the damage of normal tissue can be reduced, can be comparatively fast metabolized in human body, it is to avoid other body parts are produced with toxicity, chemistry
The advantages of stability and photostability high.But traditional PDT still suffers from some shortcomings, and such as sensitising agent is to tumor group
The targeting knitted is not enough, simultaneously because sensitiser absorption wavelength is shorter, its tissue penetration is limited, therefore, it is larger to volume
Tumour or deep tumor therapeutic effect it is not good.Nowadays, the fast development of nanometer technology is solution Photodynamic Therapy
The problems such as quick dose of tumor-targeting and treatment depth, provides new way.
Molecular targeted therapy refer on cellular and molecular level by fixed carcinogenic site (i.e. with tumour cell and swell
The related specific molecular target spot of tumor tissue, the target spot is probably a certain protein molecule in tumour cell, a certain nucleic acid fragment
Or a certain gene outcome), design respective ligand or antibody are combined with specific position, so that effective suppressor canceration
Or blocking Cell signal propagation pathways and Tumor angiogenesis, ultimately result in death of neoplastic cells normal thin without injury
A kind of brand-new Therapeutic mode of born of the same parents.Compared with traditional chemotherapy and radiation therapeutic modality, molecular targeted therapy has choosing very high
Selecting property, also ensures relatively low damage normal structure while to tumour cell Efficient killing effect, reaches preferable treatment results.It is lucky
It is non-for Buddhist nun（Gefitinib）Ratified for treating non-small cell lung cancer by U.S. FDA in 2003, be on first getting the Green Light
The oral type small molecule targeted drug for treating non-small cell lung cancer (NSCLC) in city.Gefitinib is a kind of selectivity
EGF-R ELISA (EGFR) tyrosine kinase inhibitor, EGFR-TK is often expressed as being swollen in the entity of epithelial origin
Among knurl, growth and the angiogenesis of tumour cell can be indirectly suppressed by the suppression to EGFR tyrosine kinase activities, and
Increase the apoptosis of tumour cell.Clinical test results show that Gefitinib is to colon cancer, prostate cancer, breast cancer and incidence
The treatment such as tumour confirms effective.
Rare earth up-conversion luminescent material (upconversion luminescence nanoparticles, UCNPs) is
One kind can be excited by near infrared light (NIR) and send the luminescent material of visible ray, i.e. up-conversion nano material can be by light more
Long wave is converted into shortwave by handset system, therefore is referred to as " upper conversion ".Because the luminescence process of up-conversion has run counter to this
Lentor (Stokes) law, therefore the anti-Stokes that is otherwise known as (anti-Stokes) law luminescent material.Upper conversion
Nano material has unique luminosity, and its optical property stabilization, the toxicity to cell is low, and excitation wavelength is near infrared region
(usually 980 nm, the light that the particularly important is this wavelength is placed exactly in the light of biological tissue and passes through window 800-1200 nm
Among), the penetrability ability to biological tissue is strong, and light will not be caused to injure tissue, protein etc., while can effectively drop
The background autofluorescence of low detection, improves fluorescence signal to noise ratio, therefore control in biomarker, multi-modality imaging, medicament transport and light power
The aspects such as treatment have broad application prospects.
Molecular target medicine is introduced in UCNP/ silica/sensitising agent nanosystems, by preparing molecular targeted anticancer nano
Plateform system, makes cellular level of the point of penetration of tumor pharmacother by studying at present improve to molecular level.The UCNP/ of synthesis
In silica/sensitising agent/Gefitinib compound, the FRET effects of UCNP and sensitising agent can solve oncotherapy depth and ask
The synergy of topic, sensitising agent and Gefitinib molecular target medicine can solve targeting not delay of the strong and medicine in tumour cell
Time short problem.These advantages are provided for the design system turns into novel molecular targeting light power carrying platform greatly may be used
Energy.
The content of the invention
It is an object of the invention to provide a kind of ZnPc-UCNP@SiO2The preparation and its application of-PEG-G nano-complexes,
Solve the problems, such as target-oriented drug deficiency and treatment depth as shallow in optical dynamic therapy.
For achieving the above object, the present invention is adopted the following technical scheme that：
A kind of ZnPc-UCNP@SiO2The preparation method of-PEG-G nano-complexes, comprises the following steps：
（1）Upper conversion particles NaYF4:Yb3+,Er3+（UCNPs）Preparation：0.4 g NaOH are weighed in 100 mL reactors,
It is separately added into 20 mL deionized waters again, 30 mL ethanol, the n-hexane of the oleic acid of 7.5 mL and 4 mL, it is solution I to stir and evenly mix；
0.8 mmol rare earth nitrades are dissolved in 5 mL deionized waters, then 20 drop/min obtain solution in being added drop-wise to solution I
Ⅱ；3.2 mmol NaF are weighed again to be dissolved in 5 mL deionized waters, solution II is added to, and are reacted at 180 DEG C are placed in after mixing
6 h；Room temperature is naturally cooled to after reaction, is extracted in three times with 30 mL hexamethylenes, collect hexamethylene layer, add excess ethyl alcohol from
The heart；Alternately washed 3 times with deionized water and ethanol again, obtain NaYF4:Yb3+,Er3+（UCNPs）Particle, is dispersed in hexamethylene
In it is stand-by；
（2）UCNP@SiO2-NH2Preparation：Measure step（1）The mL of cyclohexane solution 1.5 of middle UCNPs, adds 8.5 mL's
Hexamethylene and 0.15 mL polyoxyethylenes (5) nonyl phenyl stir 10 min；Add the NH of 100 μ L mass fractions 28%3·
H2Polyoxyethylenes (5) nonyl phenyl of the O aqueous solution and 0.5 mL, ultrasonic 20 min；It is slowly dropped into 160 μ L thereto again just
Tetraethyl orthosilicate, stirs 6 h at room temperature；30 μ L 3- aminopropyl triethoxysilanes are added, continues to stir 48 h；With ethanol from
The heart is washed 3 times, and product is disperseed into -60 DEG C of freeze-dryings in deionized water, obtains nano-particle UCNP SiO2-NH2, by its point
It is dispersed in 10mL DMF solutions, it is stand-by；
（3）ZnPc-UCNP@SiO2The preparation of-PEG-COOH：Weigh 3 mgβ- ZnPc-COOH and 10 mg HOOC-PEG-
COOH is placed in 25 mL reaction bulbs, sequentially adds 5 mL DMFs, and 5 mg 1- (3- dimethylamino-propyls)-
3- ethyl-carbodiimide hydrochlorides and 3 mg I-hydroxybenzotriazoles, add 50 μ L triethylamines, stirring 30 under ice bath stirring
Min recession ice baths, add step at room temperature（2）Contain UCNP@SiO2-NH2DMF solution continue to stir 12 h, reaction terminates
Afterwards, it is collected by centrifugation, is respectively washed 3 times with DMF and DMSO, finally by product ZnPc-UCNP@SiO2- PEG-COOH is dispersed in
It is stand-by in 12mLDMSO；
（4）ZnPc-UCNP@ SiO2The preparation of-PEG-G：Weigh 7 mgN, N- dicyclohexylcarbodiimides and 5 mg 4- diformazans
Aminopyridine is dissolved in 0.5 mL DMSO respectively, is then added sequentially to step（3）1 h is stirred in reaction mixture for mixed liquor
Ⅰ；Compound G is dissolved in 0.5 mL DMSO, is added in mixed liquor I, 48 h are reacted at room temperature；After reaction terminates, centrifugation is received
Collection, product DMSO washes 3 times, and -60 DEG C lyophilized to obtain ZnPc-UCNP@SiO2-PEG -G。
The rare earth nitrades are Y (NO3)·6H2O and Yb (NO3) ·5H2O and Er (NO3) ·5H2O is with mass ratio
68:30:2 mixing.
Step（1）Middle UCNPs is with hexamethylene with solid-liquid ratio 20：1 mixing, solid-liquid ratio unit is mg/mL.
Step（4）The addition of middle compound G is the 5% ~ 20% of product gross mass.
Described compound G is the miscellaneous octyl group-Gefitinib of the alcoxyl of 8- hydroxyls -3,6.
The beneficial effects of the present invention are：
1）Present invention synthesis ZnPc-UCNP@SiO2- PEG-G nano-complexes, solve optical dynamic therapy in target-oriented drug not
Foot, treats the problem of depth as shallow；
2）The ZnPc-UCNP@SiO of synthesis2- PEG-G nano-complex uniform particle sizes, transferring efficiency of fluorescence resonance energy is high,
980nm excites lower singlet oxygen yield high, solves the problems, such as to treat depth as shallow；
3）The ZnPc-UCNP@SiO of synthesis2- PEG-G nano-complexes show superior photodynamic activity, in vitro light power
Therapeutic process has certain dose dependent to the killing ability of cell, and with the increase of concentration, killing ability strengthens, and
Obvious lethal effect is not shown under non-illuminated conditions to cell；
4) the ZnPc-UCNP@SiO of synthesis2- PEG-G nano-complexes have obvious targeting to cancer cell, and normal
It is distributed less in cell, reduces the side effect in cancer treatment procedure.
Brief description of the drawings
Fig. 1 is (a) up-conversion nanoparticles NaYF4:Yb3+,Er3+(UCNPs) after wrapping up silica with (b)
UCNP@SiO2The X-ray diffractogram of nano-particle；
Fig. 2 is (a) up-conversion nanoparticles NaYF4:Yb3+,Er3+(UCNPs) with (b) UCNP@SiO2TEM figure；
Fig. 3 is UCNPs up-conversion nanoparticles UCNP@SiO2With UCNP@SiO2-NH2Infrared spectrogram；
Fig. 4 is NaYF4:Yb3+,Er3+(UCNPs) up-conversion luminescence figure；
I and II is the corresponding fluorescence emissions of UCNPs in Fig. 5, and III and IV is ZnPc-UCNP@SiO2The corresponding fluorescence hairs of-PEG
Penetrate light figure；
Fig. 6 a are DPBF, UCNPs, UCNP@SiO2, β-ZnPc-COOH, ZnPc-UCNP@SiO2-PEG, ZnPc-UCNP@
SiO2The Ф of-PEG-G∆Compare；Fig. 6 b organize front and rear ZnPc-UCNP@SiO to add2- PEG-G is in 980nm and 670nm light irradiations
Under Ф∆Compare figure；
Fig. 7 is HepG2 cells (circle) and HELF cells (bar shaped) to ZnPc-UCNP@SiO2- PEG-G competitions intake 24
H laser confocal imaging figures；
Fig. 8 is object ZnPc-UCNP@SiO2-PEG-G and tester ZnPc-UCNP@SiO2-PEG to HepG2 cell incubations
Fluorescence statistics block diagram after 24 h；
Fig. 9 a and Fig. 9 b are ZnPc-UCNP@SiO2The positioning figures of-PEG-G in cancer cell HepG2；
Figure 10 is various concentrations ZnPc-UCNP@SiO under no light2- PEG-G and ZnPc-UCNP@SiO2- PEG is incubated lower HepG2
Cell survival rate figure；
Figure 11 is that have various concentrations ZnPc-UCNP@SiO under illumination2- PEG-G and ZnPc-UCNP@SiO2- PEG is incubated lower HepG2
Cell survival rate figure.
Specific embodiment
Technical scheme is described further with specific embodiment below, but protection scope of the present invention is not limited
In this:
Embodiment 1
A kind of ZnPc-UCNP@SiO2The preparation method of-PEG-G nano-complexes, concretely comprises the following steps：
1) 0.4 g NaOH are weighed in 100 mL reactors, then measures 20 mL deionized waters respectively, 30 mL ethanol, 7.5
The n-hexane of the oleic acid of mL and 4 mL, stirring allows it to form uniform solution；Weigh 0.8 mmol rare earth nitrades（0.2390 g
Y(NO3)·6H2O, 0.1065 g Yb (NO3) ·5H2O, 0.0069 g Er (NO3) ·5H2O）It is dissolved in 5 mL deionizations
It is slowly dropped in above-mentioned reaction solution with 20 drop/min in water, then weighs 0.1314 g (3.2 mmol) NaF and is dissolved in 5 mL
It is added in reaction solution in deionized water, takes out stirring magneton encapsulation and be placed in and react 6 h in baking oven at 180 DEG C；After reaction terminates
Room temperature is naturally cooled to, is extracted in three times with 30 mL hexamethylenes, collect hexamethylene layer, add excess ethyl alcohol to be collected by centrifugation；Again
Alternately washed 3 times with deionized water and ethanol, be finally dispersed in stand-by in 10 mL hexamethylenes；
2) mL of cyclohexane solution 1.5 of the UCNPs of 20 mg/mL is measured, the hexamethylene of 8.5 mL is added, 0.15 mL is added
Polyoxyethylenes (5) nonyl phenyl (IGEPAL CO-520) stirs 10 min, adds 100 μ L NH3·H2O (28%) and
IGEPAL CO-520, ultrasonic 20 min of 0.5 mL, are slowly added dropwise 160 μ L tetraethyl orthosilicates (TEOS), stir at room temperature
30 μ L 3- aminopropyl triethoxysilanes (APTES) are added after 6 h, continues to stir 48 h；Add ethanol to be centrifuged 3 times, will produce
Thing dispersion is lyophilized in -60 DEG C in deionized water, obtains in SiO2The nano-particle UCNP@SiO of surface modification amino2-NH2, will
It is dispersed in 10mL DMF solutions, stand-by；
3) 3 mg are weighedβ- ZnPc-COOH and 10 mg HOOC-PEG-COOH are placed in 25 mL reaction bulbs, sequentially add 5
ML DMFs (DMF), 5 mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCI)
With 3 mg I-hydroxybenzotriazoles (HOBt), stirring adds 50 μ L triethylamines, stirring 30 min recession ice under ice bath
Bath, adds the product UCNP@SiO of previous step at room temperature2-NH2DMF solution continue stir 12 h, reaction terminate after, from
The heart is collected, and is respectively washed 3 times with DMF and DMSO, finally product is dispersed in DMSO and obtains ZnPc-UCNP@SiO2- PEG-COOH,
It is stand-by；
4) by the product ZnPc-UCNP@SiO of previous step2- PEG-COOH is dissolved in 12 mL DMSO, weighs 7 mg by N, N- bis-
Carbodicyclo hexylimide (DCC) and 5 mg DMAPs (DMAP) are dissolved in 0.5 mL DMSO respectively, by the solution
Be added in the DMSO solution of product, after 1 h of stirring, then weigh 8 mg compounds G be dissolved in 0.5 mL DMSO add it is anti-
Liquid is answered, 48 h are reacted at room temperature.After reaction terminates, it is collected by centrifugation, product DMSO washes 3 times, -60 DEG C lyophilized to obtain ZnPc-
UCNP@ SiO2-PEG -G。
Step（1）Middle UCNPs is with hexamethylene with solid-liquid ratio 20：1 mixing, solid-liquid ratio unit is mg/mL.
Step（4）The addition of middle compound G is the 5% ~ 20% of product gross mass.
Described compound G is the miscellaneous octyl group-Gefitinib of the alcoxyl of 8- hydroxyls -3,6.
Application Example 1
By ZnPc-UCNP@SiO2The research of-PEG-G nano-complex singlet oxygen yields, studies the tissue penetration of its light
Ability, for research treatment depth provides reference, with more important meaning.
Using the diphenyl isobenzofuran (DPBF) of chemical probe 1,3 to ZnPc-UCNP@SiO2- PEG-G nanometer systems
The generation ability of singlet oxygen is detected under the irradiation of 980 nm laser.Specific experiment operating method is：Weigh a small amount of
DPBF DMSO fully dissolve, and it is 1 then to adjust its absorbance at 420 nm, while adjusting ZnPc-UCNP@SiO2-
Absorbance of the DMSO solution of PEG-G at 670 nm is also 1.Take the DMSO solvents of 1 mL and the ZnPc-UCNP@of 1 mL
SiO2- PEG-G solution is sufficiently mixed in cuvette, has eliminated systematic error as reference, then take DPBF solution and ZnPc-
UCNP@SiO2Each 1 mL of-PEG-G solution is well mixed, and determines the absorbance at 420nm, the suction of DPBF when now for 0s
Shading value, is being then 1W/cm with power density2980 nm laser illumination cuvette in mixed solution, often irradiate
30 s survey absorbance, survey 10 time points.DPBF at 420nm have very strong absorption, when with1O2It is raw after reacting
Into DBB, and DBB shows as the absorbance reduction at 420 nm at 420nm without absorption.Therefore, it is different by measuring
The fluorescence intensity of DPBF can compare produce under different condition total under light application time1O2Amount.
Be can be seen that after the tissue coverage of 1 cm has been added by Fig. 6 a and Fig. 6 b experimental datas, with 980 nm light irradiations
Singlet oxygen yield only have dropped 40.68 %, and have dropped 88.9 % with the singlet oxygen yield of 670 nm light irradiations.Therefore,
Directly excited with the light of 670 nmβ- ZnPc-COOH is compared, and is excited indirectly with the light of 980 nmβ- ZnPc-COOH have penetrate
The advantage of thick tissue, has further highlighted the applicability that ZnPc-UCNP-PEG-G is treated in entity or deep tumor.
Application Example 2
Can medicine effectively kill tumour cell or cause certain damage with tumour cell to medicine to tumour cell
Intake has very big relation, therefore, research cell is necessary to the intake of medicine.
The HepG2 cells in exponential phase are chosen, by adding pancreatin digestion process after PBS washings, culture is added
Base is uniform by cell piping and druming, adds new culture medium to blow and beat cell again uniformly after centrifugation, cell is counted, with culture
Cell is diluted to 300,000 cell/mL by base, is added with 100 μ L per hole in 96 orifice plates, and each medicine sets 6 multiple holes, by cell
It is placed in overnight growth in cell culture incubator.Old culture medium is discarded after adherent, 100 μ L medicines-culture medium solution is added per hole
(mother liquid medicine is 64 mg/ mL, and solvent is DMSO；In order to the final adding consistency of event of DMSO amounts≤1% is in ensureing culture medium
640 μ g/ mL), it is placed in incubator and is incubated 24 h.Upper strata culture medium is discarded, is washed with PBS three times, do not taken the photograph with thoroughly removing
The medicine for taking, then to the SDS lysates for adding 120 μ L concentration to be 1% in every hole, is placed on 37oC shaking tables rock cracking
30 min, are excited with the light of the excitation wavelength of relative medicine, detect that it is glimmering at maximum emission wavelength with multi-function microplate reader
Luminous intensity.
Meanwhile, do the standard curve (8 concentration, 2 multiple holes) of relative medicine：With SDS it is solvent from 325 μ by medicine
Twice dilution obtains eight various concentrations to g/ mL successively, and respective fluorescence intensity is determined with the same terms, soft with Origin 8.5
Part treatment, does fluorescence intensity-concentration rectilinear, obtains final product the standard curve of respective medicine.Passed through by the fluorescence intensity in each hole of experimental group
Pharmaceutical standards curve is that can obtain each hole cellular uptake drug concentration.
Using BCA protein quantification detection methods, (direct result of calculation need to expand 6 times, as to calculate the protein content in each hole
The value of each experimental group).Again with the drug concentration in each hole divided by each PFP total amount, its ratio is then for contained in unit albumen
The amount of sensitising agent.Block diagram (Fig. 8) is done to individual ratio with the statistical analysis softwares of Graph Pad Prism 5.0, as a result with Mean
± SEM represents, parallel laboratory test in triplicate under the same terms.
Be connected to it is polyethyleneglycol modified after the ZnPc-UCNP@SiO that obtain of targeted molecular Gefitinib (G)2-PEG-G
Intake in HepG2 cells is far above the ZnPc-UCNP@SiO without connection Gefitinib2- PEG, illustrates ZnPc-
UCNP-PEG-G has good targeting to hepatocellular carcinoma H22.
Application Example 3
Cytotoxicity test is mainly the security of research Photodynamic Therapy, and medicine is to the toxic action of cell.
The good HepG2 cells for being in logarithmic phase of growth conditions are taken, by after washing, digestion process, being carried out to cell
Count, the cell that will have been counted is diluted to concentration for 100,000 cell/mL, is then inoculated with 100 μ L per hole in 96 orifice plates, will be thin
Incubated overnight in born of the same parents' incubator.ZnPc-UCNP-PEG-G and ZnPc-UCNP-PEG are prepared into 7 concentration gradients with DMSO solution
Mother liquor, then with culture medium by 100 times of each concentration dilution.After washing 3 times through PBS, the drug solution after 100 μ L dilutions is taken
Add into each hole in 96 orifice plates, experimental group sets 6 multiple holes.Blank control group and cell controls group and experimental group are set,
Dosing is placed in be put into cell culture incubator after finishing and is incubated 24 h.
Dark poison experiment：After the h of cell incubation 24 that agent-feeding treatment before is crossed, old culture medium, 3 are washed with PBS before discarding
Time, then to 100 μ L culture mediums are added in every hole, be placed in incubator and be incubated 24 h.
Light poison experiment：After being incubated 24 h, old culture medium before discarding is washed 3 times with PBS, then to adding 100 μ in every hole
The new culture mediums of L, (power density is 1.0 W/cm to irradiate 20 min with 980 nm LEDs2) after, then 96 orifice plates are placed in 37 °
C constant temperature and 5% CO2Continue to be incubated 24 h in culture medium.
OD values are detected：The MTT solution (5 mg/mL) that the complete 10 μ L of cell per well addition of above-mentioned incubation have been prepared, is placed in
4 h are incubated in incubator, original liquid in orifice plate is discarded, then 100 μ L DMSO are added in every hole, be placed in concussion half in shaking table
Hour or so, after first a ceremonial jade-ladle, used in libation is completely dissolved, determine OD value of every hole at 570 nm in 96 orifice plates.By acquired results Graph
Pad Prism 5.0 are processed, and are as a result represented with Mean ± SEM.
Test result indicate that ZnPc-UCNP@SiO2- PEG-G nano-complexes to hepatocellular carcinoma H22 have significantly kill
Wound is acted on, and is killed ability and had certain dose dependent, be will also realize that with the increase of concentration from Figure 10-11, kills energy
Power strengthens, and does not show obvious lethal effect to HepG2 cells under non-illuminated conditions.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to covering scope of the invention.
Claims (6)
1. a kind of ZnPc-UCNP@SiO2The preparation method of-PEG-G nano-complexes, it is characterised in that：Comprise the following steps that：
（1）The preparation of upper conversion particles UCNPs：0.4 g NaOH are weighed in 100 mL reactors, then is separately added into 20 mL and gone
The n-hexane of ionized water, 30 mL ethanol, the oleic acid of 7.5 mL and 4 mL, it is solution I to stir and evenly mix；By 0.8 mmol rare earth nitre
Hydrochlorate is dissolved in 5 mL deionized waters, is then added drop-wise to 20 drop/min and solution II is obtained in solution I；3.2 mmol are weighed again
NaF is dissolved in 5 mL deionized waters, is added to solution II, and 6 h are reacted at 180 DEG C are placed in after mixing；Natural cooling after reaction
To room temperature, extracted in three times with 30 mL hexamethylenes, collect hexamethylene layer, add excess ethyl alcohol centrifugation；Deionized water and second are used again
Alcohol is alternately washed 3 times, obtains UCNPs particles, is dispersed in stand-by in hexamethylene；
（2）UCNP@SiO2-NH2Preparation：Measure step（1）The mL of cyclohexane solution 1.5 of middle UCNPs, adds the ring of 8.5 mL
Hexane and 0.15 mL polyoxyethylenes (5) nonyl phenyl stir 10 min；Add the NH of 100 μ L mass fractions 28%3·H2O
Polyoxyethylenes (5) nonyl phenyl of the aqueous solution and 0.5 mL, ultrasonic 20 min；Instill the positive silicic acid tetrems of 160 μ L thereto again
Ester, stirs 6 h at room temperature；30 μ L 3- aminopropyl triethoxysilanes are added, continues to stir 48 h；With ethanol centrifuge washing 3
It is secondary, by product dispersion in deionized water in -60 DEG C of freeze-dryings, obtain nano-particle UCNP SiO2-NH2, it is dispersed in
It is stand-by in 10 mL DMF solutions；
（3）ZnPc-UCNP@SiO2The preparation of-PEG-COOH：Weigh 3 mgβ- ZnPc-COOH and 10 mg HOOC-PEG-COOH
It is placed in 25 mL reaction bulbs, sequentially adds 5 mL DMFs, 5 mg 1- (3- dimethylamino-propyls) -3- second
Base carbodiimide hydrochloride and 3 mg I-hydroxybenzotriazoles, 50 μ L triethylamines are added under ice bath stirring, stir 30 min
Recession ice bath, adds step at room temperature（2）Contain UCNP@SiO2-NH2DMF solution continue stir 12 h, reaction terminate after, from
The heart is collected, and is respectively washed 3 times with DMF and DMSO, finally by product ZnPc-UCNP@SiO2- PEG-COOH is dispersed in 12mLDMSO,
It is stand-by；
（4）ZnPc-UCNP@ SiO2The preparation of-PEG-G：Weigh 7 mgN, N- dicyclohexylcarbodiimides and 5 mg 4- diformazans
Aminopyridine is dissolved in 0.5 mL DMSO respectively, is then added sequentially to step（3）1 h is stirred in reaction mixture for mixed liquor
Ⅰ；Compound G is dissolved in 0.5 mL DMSO, is added in mixed liquor I, 48 h are reacted at room temperature；After reaction terminates, centrifugation is received
Collection, product DMSO washes 3 times, and -60 DEG C lyophilized to obtain ZnPc-UCNP@SiO2-PEG -G。
2. a kind of ZnPc-UCNP@SiO according to claim 12The preparation method of-PEG-G nano-complexes, its feature
It is：Rare earth nitrades are Y (NO3)·6H2O and Yb (NO3) ·5H2O and Er (NO3) ·5H2O is with mass ratio 68:30:2 mix
Close.
3. a kind of ZnPc-UCNP@SiO according to claim 12The preparation method of-PEG-G nano-complexes, its feature exists
In：Step（1）Middle UCNPs is with hexamethylene with solid-liquid ratio 20：1 mixing, solid-liquid ratio unit is mg/mL.
4. a kind of ZnPc-UCNP@SiO according to claim 12The preparation method of-PEG-G nano-complexes, its feature exists
In：Step（4）The addition of middle compound G is the 5% ~ 20% of product gross mass.
5. a kind of ZnPc-UCNP@SiO according to claim 12The preparation method of-PEG-G nano-complexes, its feature exists
In：Compound G is the miscellaneous octyl group-Gefitinib of the alcoxyl of 8- hydroxyls -3,6.
6. ZnPc-UCNP@SiO of a kind of preparation as claimed in claim 12The application of-PEG-G nano-complexes, its feature exists
In：By described ZnPc-UCNP@SiO2- PEG-G nano-complexes are applied in optical dynamic therapy medicine.
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