CN106866721B - A kind of silicon phthalocyanine derivative and its application for preparing biotin acceptor targeting silicon phthalocyanine photosensitizer - Google Patents
A kind of silicon phthalocyanine derivative and its application for preparing biotin acceptor targeting silicon phthalocyanine photosensitizer Download PDFInfo
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- CN106866721B CN106866721B CN201710141083.XA CN201710141083A CN106866721B CN 106866721 B CN106866721 B CN 106866721B CN 201710141083 A CN201710141083 A CN 201710141083A CN 106866721 B CN106866721 B CN 106866721B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/10—Compounds having one or more C—Si linkages containing nitrogen having a Si-N linkage
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
Abstract
The invention belongs to photo-dynamical medicine and photosensitizer technical fields, and in particular to a kind of silicon phthalocyanine derivative, and its application for being used to prepare biotin acceptor targeting silicon phthalocyanine photosensitizer is further disclosed.Silicon phthalocyanine derivative of the present invention is that biotin is introduced into silicon phthalocyanine axial arrangement by covalent bond on the basis of existing silicon phthalocyanine compounds, has synthesized a kind of new silicon phthalocyanine analog derivative.The silicon phthalocyanine analog derivative can be with target biology element receptor high expression tumour cell, and has excellent photodynamic activity, is a kind of novel biotin acceptor targeting phthalocyanines photosensitizer.Meanwhile the SiPc-biotin derivative will be significantly less than its precursor SiPc-pip to the dark toxicity of human normal cell line, be a kind of potential targeting photosensitizer for photodynamic therapy.
Description
Technical field
The invention belongs to photo-dynamical medicine and photosensitizer technical fields, and in particular to a kind of silicon phthalocyanine derivative goes forward side by side one
Step discloses its application for being used to prepare biotin acceptor targeting silicon phthalocyanine photosensitizer.
Background technique
Optical dynamic therapy (Photodynamic Therapy, abbreviation PDT), also known as photoradiation therapy
(Photoradiation Therapy, abbreviation PRT) or photochemotherapy (Photochemotherapy), are that one kind is based on
The treatment method of the photochemical reaction principle of particular chemicals is a kind of tumor therapeuticing method emerging in recent years.Used
Chemical substance is known as tumour chemistry diagnosis and treatment drug (also referred to as photosensitizer, Photosensitizer, abbreviation PS).PDT therapy processes are
Photosensitizer is injected in vivo and (can also be applied to affected part for skin) by being injected intravenously, by after a certain period of time with specific
The light of wavelength irradiates tumor tissues, and the photosensitizer of tumor tissues is enriched in by illumination excitation, it is made to generate active oxygen species
(Reactive oxygen species, ROS), and then achieve the purpose that kill tumour.Relative to other oncotherapy sides
Method, photodynamic therapy have the advantages that selectivity is good, toxic side effect is small.Therefore, photodynamic therapy is acknowledged as clinically except change
The 4th kind of cancer treatment method except treatment, radiotherapy, operation.Since first photosensitive drugIn 1993~
1997 since the country such as the U.S., Canada, European Union in succession listing, along with the continuous development of laser technology, photodynamic therapy
Enter Rapid development stage.Currently, photodynamic therapy has become most active research neck in the disease prevention and cure subject such as tumour
Domain.
ROS has a very strong oxidisability, high activity can efficiently oxidative biological molecule, such as nucleic acid, albumen and insatiable hunger
With fatty acid etc..Also due to the high activity of ROS also causes its service life very of short duration, light power effect is made to be concentrated mainly on irradiated region
Domain.But since the tumor-targeting of photosensitizer traditional at present is poor, photosensitizer is inevitably enriched in health tissues,
This, which can not only reduce photosensitizer, influences therapeutic effect in the concentration of tumor tissues, and is enriched in the photosensitizer of normal tissue
Some side effects can be brought, bring pain to patient.Therefore, the photosensitizer that there is selectively targeted ability to tumor tissues is developed
It is current photodynamic therapy urgent problem to be solved.
Phthalocyanines photosensitizer is since it in phototherapy window (650-850nm) has strong absorption, and dark toxicity is low, creating singlet oxygen
(1O2) quantum yield height and it is easy to the characteristics of modifying, become photosensitizer most potential in second generation photosensitizer.Silicon phthalocyanine
The one kind of (silicon (IV) phthalocyanine, SiPc) as phthalocyanines photosensitizer, in the research of optical dynamic therapy
Extensive concern is arrived.Have been reported show by introduce have cancer target ability group or molecule, as polyamines, carbohydrate,
Polypeptide, monoclonal antibody and vitamin etc. can contribute to the tumor-targeting of enhancing photosensitizer.A variety of bases have been developed at present
In the novel photosensitive agent of silicon phthalocyanine structure, but it there is a problem that targeting is slightly poor to a certain extent.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in a kind of biotin acceptor targeting silicon phthalocyanine photosensitizer is provided,
To solve the problems, such as that photosensitizer tumor-targeting is not strong in the prior art.
In order to solve the above technical problems, silicon phthalocyanine derivative of the present invention, is the silicon phthalocyanine of biotin axial substituted
Derivative, with structure as follows:
The invention also discloses a kind of methods for preparing the silicon phthalocyanine derivative, include the following steps:
(1) synthesis of SiPc-pip
Under inert gas shielding, with SiPcCl2It is raw material, in the presence of sodium hydride is catalyst with 4- hydroxy piperidine, it is molten
Back flow reaction is carried out in toluene solvant；After reaction, decompression steams solvent, and rinsing residue, is separated with silica gel column chromatography
Navy blue product is obtained, as required SiPc-pip；
(2) synthesis of SiPc-biotin
Using SiPc-pip obtained above as raw material, n-hydroxysuccinimide biotin is added, in the presence of organic amine,
Be dissolved in DMF solvent and being reacted, after evaporated under reduced pressure solvent, navy blue product is obtained with silica gel column chromatography, as needed for
SiPc-biotin；
In the step (1), the SiPcCl2Molar ratio with 4- hydroxy piperidine is 1-4:10.
In the step (1), the specific steps of the silica gel column chromatography separation are as follows: use 200-300 mesh silica gel, use trichlorine
Methane fills column；It takes sample to be dissolved in DMF solvent, is added the silica white adsorption sample of 3 times of quality, after evaporated under reduced pressure DMF, with silica gel
Powder upper prop is eluted for chloroform-methanol solution of 20:1 as eluant, eluent using volume ratio.
In the step (2), the organic amine is n,N-diisopropylethylamine or triethylamine.
In the step (2), the molar ratio of the n-hydroxysuccinimide biotin and SiPc-pip are 4-10:1, and
It is preferred that 6:1.
In the step (2), the specific steps of the silica gel column chromatography are as follows: use 200-300 mesh silica gel, use chloroform
Fill column；It takes sample to be dissolved in DMF solvent, is added the silica white adsorption sample of 3 times of quality, after evaporated under reduced pressure DMF, on silica white
Column is eluted for chloroform-methanol solution of 30:1 as eluant, eluent using volume ratio.
Further, the preparation method further includes the steps that preparing n-hydroxysuccinimide biotin, specifically includes:
Under inert gas protection, using biotin and N- hydroxysuccinimide as raw material, in N, N'- dicyclohexylcarbodiimide or 1-
In the presence of ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, be dissolved in DMF solvent and reacted, after filter
Reaction solution and evaporated under reduced pressure, product are precipitated with ether up to required BNHS；
The molar ratio of the biotin and N- hydroxysuccinimide is 1:1-3, and preferred 1:1.25.
The invention also discloses a kind of biotin acceptors to target silicon phthalocyanine photosensitizer, by the silicon phthalocyanine derivative system
?.
The invention also discloses the silicon phthalocyanine derivatives to be used to prepare photo-dynamical medicine, photosensitive drug or treating cancer
The purposes of drug.
The cancer includes cervical carcinoma.
The invention also discloses a kind of pharmaceutical composition, including the silicon phthalocyanine derivative and pharmaceutically acceptable
Auxiliary material and/or carrier.
The invention also discloses the clinically-acceptable preparations prepared by the pharmaceutical composition according to common process.
Silicon phthalocyanine derivative of the present invention is on the basis of existing silicon phthalocyanine compounds, by covalent bond by biotin
It is introduced into silicon phthalocyanine axial arrangement, has synthesized a kind of new silicon phthalocyanine analog derivative.According to it has been found that in numerous tumours
In targeted molecular, since biotin can promote the growth of cell, relative to normal cell, tumour cell needs more
Biotin is to maintain its fast breeding, so that biotin acceptor (biotin receptor, BR) is in kinds of tumor cells
Middle overexpression.Herein described derivative is targeting group and as swollen using biotin then by means of this advantage of biotin
The target spot of tumor specific drug, synthesized silicon phthalocyanine analog derivative can with target biology element receptor high expression tumour cell, and
It is a kind of novel biotin acceptor targeting phthalocyanines photosensitizer with excellent photodynamic activity.Meanwhile the SiPc-
Biotin derivative will be significantly less than its precursor SiPc-pip to the dark toxicity of human normal cell line, be a kind of potential targeting light
Dynamic therapy photosensitizer.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is compound SiPc-pip and SiPc-biotin UV-vis absorption spectrum in DMF and 0.5%CEL, wherein
Figure (a) is SiPc-pip, figure (b) is SiPc-biotin (10 μM)；The PBS solution of 0.5%CEL:0.5%Cremophor EL
(0.5g is dissolved in 100mL PBS)；
Fig. 2 is the fluorescence spectrum of compound SiPc-pip and SiPc-biotin in DMF and 0.5%CEL, wherein figure (a)
It is SiPc-biotin (5 μM) for SiPc-pip, figure (b)；(0.5g is molten for the PBS solution of 0.5%CEL:0.5%Cremophor EL
In 100mL PBS)；
Fig. 3 is that the creating singlet oxygen that illumination compound SiPc-pip and SiPc-biotin are generated inhales DPBF in 0.5%CEL
Receive the attenuation of spectrum；
Fig. 4 is the amount of Hela intracellular compound SiPc-pip and SiPc-biotin；
Fig. 5 is excess biotin (100 μM) to compound SiPc-pip and SiPc-biotin and Hela cell combination ability
Influence；
Fig. 6 is the fluorescence imaging image of Hela cell；Wherein, figure (a) is (2 μM) culture 1h of SiPc-pip；Scheming (b) is
Hela uses (100 μM) of biotin pretreatments first, then uses (2 μM) culture 1h of SiPc-pip；Scheming (c) is SiPc-biotin (2 μ
M 1h) is cultivated；Figure (d) is that Hela uses (100 μM) of biotin pretreatments first, then uses (2 μM) culture 1h of SiPc-biotin；
Fig. 7 is (λ ≈ 670nm, light intensity 20mWcm under illumination-2, accumulated dose 24Jcm-2), the survival rate of Hela cell；
Fig. 8 is dark and illumination condition (λ ≈ 670nm, light intensity 20mWcm-2, accumulated dose 24Jcm-2) under, compound
Toxicity of the SiPc-pip and SiPc-biotin to Hela；
Fig. 9 is dark toxicity test result of the compound SiPc-pip and SiPc-biotin to LO2.
Specific embodiment
Reagent involved in following embodiment, instrument and method include:
Reagent: phthalocyanine silicon dichloride (SiPcCl2), 4- hydroxy piperidine (4-piperidinol), biotin (biotin), two rings
Hexyl carbodiimide (DCC), n-hydroxysuccinimide (NHS), n,N-diisopropylethylamine (DIPEA), sodium hydride (NaH),
Emulsifier EL-60 (CEL) is purchased from Sigma-Aldrich, Alfa Aesar and J&K company respectively；Dimethylformamide
(DMF) it is steamed again under a nitrogen with toluene (toluene) with sodium hydride；Cell culture reagent is purchased from Bioind (BI) company；
Instrument and method:
Uv-vis spectra and fluorescence spectrum are in Perkin-Elmer Lambda-25 spectrophotometer and Perkin
It is measured on Elmer LS-55 luminoscope；1H NMR is measured on Bruker DMX 400MHz nuclear magnetic resonance chemical analyser；ESI mass spectrum
It is measured on 2 mass spectrograph of Waters SQ Detector；Elemental analysis is surveyed on VarioEL III CHNS elemental analyser
It is fixed；
Fluorescence quantum yield (ΦF) measured in DMF, ZnPc is as reference (Φ in DMFF=0.28)7；Creating singlet oxygen
(1O2) quantum yield measures in DMF, DPBF conduct1O2Capturing agent, ZnPc is as reference (Φ in DMF△=0.56)8。
BNHS used in the following each embodiments of the present invention, can be for existing commercial product or according to method in the prior art
Obtained by synthesis, BNHS is synthesized as follows in the following embodiments of the present invention:
Under nitrogen protection, biotin (0.50g, 2.05mmol) and NHS (0.29g, 2.56mmol) are dissolved in 20mLDMF
In solvent, and the DMF solution (5mL) for containing DCC (0.41g, 2.05mmol) is added, room temperature reaction is overnight.Filtering reacting liquid, and
Evaporated under reduced pressure, product is precipitated to obtain BNHS with ether, and is washed three times with ether.DCC can also be replaced with to 1- ethyl-(3- bis-
Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate reacted.
Embodiment 1
The method that silicon phthalocyanine derivative is prepared described in the present embodiment, includes the following steps:
(1) synthesis of SiPc-pip
Under nitrogen protection, by SiPcCl2(0.10g, 0.16mmol), 4- hydroxy piperidine (0.16g, 1.6mmol) and hydrogenation
Sodium (0.03g, 1.28mmol) is dissolved in toluene solvant (50mL), and back flow reaction 24 hours；Decompression steams solvent, is washed with water residual
Slag；Silica gel column chromatography separation, actual conditions are as follows: use 200-300 mesh silica gel, fill column with chloroform；Sample is taken to be dissolved in 20mL
In DMF, it is added the silica white adsorption sample of 3 times of quality, evaporated under reduced pressure DMF solvent, and with silica white upper prop, with chloroform-
Methanol (volume ratio 20:1) is eluant, eluent, obtains 0.02g navy blue product, and calculating yield is 21%；
It is as follows to detect product structure data:1H NMR(400MHz,CDCl3), δ (ppm): 9.62~9.64 (8H, m, Pc-
Hα), 8.33~8.35 (8H, m, Pc-Hβ), 0.83~0.85 (4H, m, NCH2), 0.64~0.66 (4H, m, NCH2),-1.82
(4H,br,CH2), -2.31~-2.35 (4H, m, CH2), -2.68~-2.71 (2H, m, OCH) .ESI-MS:m/z 763.36
(20%) [M+Na+].Elemental analysis:Found:C,68.2；H,5.1；N, 18.7%；Calc.for
C42H36N10O2Si:C,68.1；H,4.9；N, 18.9%.As it can be seen that product structure is correct.
(2) synthesis of SiPc-biotin
By the BNHS (0.084mmol) of above-mentioned preparation and SiPc-pip (0.01g, 0.014mmol) and DIPEA (10 μ
L, 0.056mmol) it is dissolved in DMF solvent, overnight in room temperature reaction；After evaporated under reduced pressure solvent, silica gel column chromatography, specific steps packet
It includes: using 200-300 mesh silica gel, filling column with chloroform, take sample to be dissolved in 20mL DMF solvent, the silicon of 3 times of quality is added
Rubber powder adsorption sample, evaporated under reduced pressure DMF solvent, and with silica white upper prop, using chloroform-methanol solution as eluant, eluent (volume
Than 30:1), 0.01g navy blue SiPc-biotin product is obtained, calculating products collection efficiency is 79%；
It is as follows to detect product structure data:1H NMR(400MHz,CDCl3),δ(ppm):9.70(8H,br,Pc-Hα),
8.53(8H,br,Pc-Hβ),6.23(2H,s,biotin-NH),6.12(2H,s,biotin-NH),4.13(2H,br,
biotin-CH),3.84(2H,br,biotin-CH),3.09(4H,br,biotin-SCH2),2.72(4H,br,biotin-
SCH),1.99(4H,br,biotin-COCH2), 0.95~0.74 (4H, m, biotin-CH2CH2CH2,CH2NCH2),-1.68
(4H,br,CH2),-2.51(6H,br,CHCH2) .ESI-MS:m/z 1215.78 (87%) [M+Na+]。Elemental
analysis:Found:C,62.6；H,5.5；N,16.2；S, 5.3%；Calc.for C62H64N14O6S2Si:C,62.4；H,
5.4；N,16.4；S, 5.4%.As it can be seen that product structure is correct.
Embodiment 2
The method that silicon phthalocyanine derivative is prepared described in the present embodiment is same as Example 1, and difference is only that, the step
(2) in, n,N-diisopropylethylamine catalyst is replaced with into triethylamine as catalyst.Through detecting, products therefrom structure is correct.
Experimental example
1, lipid (log P)
A certain amount of 1 gained compound of embodiment is taken to be dissolved in n-octyl alcohol-water 1:1 in the mixed solvent, ultrasound 30 minutes
Balance it between the two phases.After centrifugation, with the concentration of absorption spectromtry compound between the two phases.The hydrophilic parent of compound
Lipid can be reacted with its lipid (log P), compound object in concentration/water of compound in P=n-octyl alcohol
Concentration.Log P is bigger, indicates that compound gets over lipophilic, conversely, then more hydrophilic.And the biology of the Lipophilicity of compound and it
Activity is closely related.
Experimental result shows that the lipophilicity that compound SiPc-biotin (log P=1.6) is made in embodiment 1 is significantly stronger than
SiPc-pip (log P=-0.9).Existing research the result shows that, the Lipophilicity of SiPc depends primarily on axial substituted base
Property.In SiPc-pip, since its axial substituted base is hydrophilic piperidines group, show SiPc-pip significantly
Hydrophily.And in SiPc-biotin compound, the introducing of hydrophobic biotin causes the lipophilicity of SiPc-biotin big
It is big to increase.
2, optical physics and spectrochemical property
The spectral property in DMF of detection compound SiPc-pip and SiPc-biotin are as shown in table 1 below.As a result it shows
Show, the fluorescence (Φ of SiPc-biotinF) and creating singlet oxygen (ΦΔ) quantum yield is significantly stronger than SiPc-pip.This result can be with
Explained with intramolecular photo induced electron transfer (PET) effect: the amine groups in compound SiPc-pip are imitated with very strong PET
Answer, the singlet excited of SiPc can be quenched in this, inhibit singlet excited be between pass through (ISC), eventually lead to lower fluorescence
With creating singlet oxygen quantum yield.And in compound SiPc-biotin, amine groups are changed into amido bond, this reduces nitrogen original
The electron donation of son, therefore reduce the PET effect of amine groups.
The photochemical light physical property of table 1.SiPc-pip and SiPc-biotin.
a610nm excitation.bZnPc as reference (in DMF, ΦF=0.28)cZnPc is as reference (DMF
In, ΦΔ=0.56).
Since the application of the photodynamic therapy of SiPc needs to carry out in aqueous solution, we compare compound
Photochemistry and photophysical property of the SiPc-pip and compound SiPc-biotin in PBS (pH=7.4,10mM) solution.
The results show that SiPc-pip and SiPc-biotin all have the characteristic absorption of monomer silicon phthalocyanine: being located at 355nm
B band and positioned at 673nm or so Q band (as shown in Figure 1).610nm wavelength light source excitation under, compound SiPc-pip and
The fluorescent emission of SiPc-biotin is respectively 683nm and 686nm (as shown in Figure 2).
As shown in figure 1 shown in (a), in PBS, the Q band of SiPc-pip is absorbed compared with the about 6nm of red shift in DMF, this mainly by
Caused by solvent effect.Importantly, the Q band absorption band of SiPc-pip is still narrow and strong in PBS, this explanation
SiPc-pip does not occur significantly to assemble in PBS.Meanwhile hyperfluorescence transmitting of the SiPc-pip in PBS also turns out SiPc-pip
Substantially with (such as Fig. 2 (a) is shown) existing for monomeric form in PBS.In addition, fluorescence outline of the SiPc-pip in PBS is strong
In the fluorescence in DMF.This is mainly due to the amidos of piperidines caused by the protonation of PBS, and protonation reduces
The PET effect of amine groups.Different from compound SiPc-pip, the Q band of compound SiPc-biotin is absorbed to be occurred in PBS
Apparent variation, absorption peak broaden, and the absorption peak of two red shifts newly occur.This shows compound SiPc-biotin in PBS
In have the aggregations of some degree, fluorescence SiPc-biotin weaker in PBS also indicates that aggregation has occurred (such as in SiPc-biotin
Shown in Fig. 2 (b)).
Due to assembling the ability decline that will lead to photosensitizer and generate ROSs, and the PDT for reducing photosensitizer is active.Therefore, it is
The comprehensive PDT activity for assessing both photosensitizers, further both measurements generate the efficiency of creating singlet oxygen in PBS.Knot
For fruit as shown in figure 3, in PBS solution, the rate of compound SiPc-pip degradation DPBF is apparently higher than compound SiPc-
Biotin illustrates that SiPc-pip has higher creating singlet oxygen quantum yield.This is with the result in DMF on the contrary, this result
It can be explained in terms of two: firstly, the creating singlet oxygen effect of SiPc-pip can be enhanced in piperidines group protonation in PBS
Rate；Secondly, the aggregation of SiPc-biotin causes its creating singlet oxygen efficiency to reduce.
3, cellular uptake
In order to prove that the binding ability of SiPc Yu biotin acceptor high expression tumour cell can be improved in the introducing of biotin,
The application has carried out cell absorption experiment with extraction, i.e., extracts the photosensitizer that cell absorbs using solvent, determined
Amount analysis.
3.1 cell culture
Biotin acceptor height is used in experiment expresses Hela cell, human cervical carcinoma cell Hela and Human normal hepatocyte
LO2 culture, cultivation temperature in the DMEM (dulbecco's modified eagle medium) containing 10% fetal calf serum
37 DEG C, CO2Concentration is 5%.
Firstly, SiPcs to be dissolved in the mother liquor for being configured to that concentration is 1mM in DMF.Then (contain 0.5% with DMEM culture medium
CEL mother liquor) is diluted to 80 μM mother liquors.Secondary mother liquor is diluted to respective concentration with DMEM further according to requirement of experiment.
Hela cell presses 1 × 106Concentration kind into 6cm culture dish.After growth for 24 hours, former culture medium is discarded, addition contains
The culture medium of SiPc (2 μM), continues 0.5,1,2 and 4h of culture.After culture, culture medium is discarded, PBS is washed 2 times, pancreatin digestion
It collects.The cell of collection is lyophilized, with the SiPc in 0.5mL DMF extraction lyophilized cells.Measure the UV- of SiPc in extract liquor
Vis absorption value compares quantitative with standard curve.In competion experiment, cell is co-cultured with (100 μM) of biotin first
Then 2h is further cultivated in addition SiPc.
Experimental result is as shown in figure 4, under identical condition of culture, and the amount of SiPc-biotin is obviously big in Hela cell
In SiPc-pip, this illustrates that the combination energy of SiPc and biotin acceptor high expression tumour cell has can be improved in the introducing of biotin
Power.
SiPc-biotin is demonstrated to the targeting ability of biotin acceptor using competion experiment.In competitive binding reality
In testing, Hela cell co-cultures to be saturated the biotin acceptor of Hela cell surface first with (100 μM) of excess biotin, then
SiPc is added and carries out one step culture of progress.Shown in Fig. 5, excess biotin is added, greatly reduces SiPc- in Hela cell
The amount (> 50%) of biotin.But little (≈ 5%) is but influenced on SiPc-pip.The result shows, SiPc-biotin with
The combination of Hela cell is mainly to be mediated by biotin acceptor, that is to say, that SiPc-biotin can be targeted in conjunction with biology
The plain highly expressed tumour cell of receptor.
The imaging of 3.2 cell fluorescences
The co-incubation 1h at 37 DEG C by Hela cell and 1 μM of SiPc.Before imaging, cell is rinsed three times with PBS.Cell
It is imaged under Olympus Xcellence cell living cells work station and obtains, excitation light source is 561nm laser.
The fluorescence imaging result of cell further confirms the targeting ability of the biotin acceptor of SiPc-biotin.Such as
Shown in Fig. 6, excess biotin reduces the fluorescence intensity of SiPc-biotin processing cell significantly, handles cell to SiPc-pip
Fluorescence intensity have little effect.This result demonstrates again that compound SiPc-biotin can be targeted and is enriched in biotin
In the tumour cell that receptor is overexpressed.
3.3 Hela cytotoxicity experiments
MTT experiment is used to the photodynamic activity of evaluation SiPc-pip and SiPc-biotin.Firstly, testing independent light
According to the influence to cell survival rate.
Take Hela and LO2 cell with 6 × 103The concentration kind of every sky is in 96 orifice plates, after growth for 24 hours, is added containing difference
Concentration SiPc (10nm-4 μM) culture medium continues after cultivating 2h, discards culture medium containing SiPc, PBS is washed 2 times, rejoins culture
Base.With LED light (λ ≈ 670nm, light intensity 20mWcm-2, total radiation dose 24Jcm-2) irradiating cell.After culture for 24 hours,
It is added MTT (thiazolyl blue), continues to cultivate 4h.Dark contrast tests while carrying out.Culture medium is discarded, DMSO is added.Select 490nm
Wavelength is read at enzyme linked immunological monitor (Bio-Rad microplate reader ader).
If Fig. 7 is shown, the condition of illumination used in this experiment will not cause to damage to cell substantially.Eliminate illumination this
After one influence factor, the application has investigated compound SiPc-pip and SiPc-biotin phototoxicity and dark toxicity.As shown in figure 8,
Almost without dark toxicity under test concentrations (10nm~4 μM), compound SiPc-pip has certain compound SiPc-biotin
Dark toxicity, but dark toxicity is also little.In illumination condition (λ ≈ 670nm, light intensity 20mWcm-2, accumulated dose 24Jcm-2) under, change
It closes object SiPc-pip and SiPc-biotin and shows very strong phototoxicity.
SiPc-biotin is given in the following table 2 under illumination condition, to the half lethal concentration (IC of Hela cell50).It can
See, compound SiPc-biotin of the present invention in addition to the binding ability of Hela cell it is relatively strong other than, while there is preferable light
Toxicity is a kind of effective light power photosensitizer.
Phototoxicity of the table 2.SiPc-pip and SiPc-biotin to Hela.
In upper table 2, since the combination of SiPc-biotin and Hela cell is mainly to be mediated by biotin acceptor, because
This is it is considered that SiPc-biotin also should be in the biotin acceptor expression of cell surface to the phototoxicity of Hela cell
Positive correlation.In order to verify us this it is assumed that referring to competitiveness experiment method, be firstly added excess biotin to full
With the biotin acceptor of cell surface, the detection of photodynamic activity is then carried out.Shown in table 2 as above, the presence of excess biotin
Significantly reduce the phototoxicity of SiPc-biotin.This result also indicates that the phototoxicity of SiPc-biotin and the life of cell surface
Object element receptor expression level correlation, that is to say, that SiPc-biotin can be expressed with target killing biotin acceptor height
Tumour cell.
The dark toxicity of 3.4 pairs of normal cells
Dark toxicity size to normal cell is also to evaluate a major criterion of light power photosensitizer, the small meaning of dark toxicity
Photosensitizer normal tissue damage it is small, safer.Therefore, the application has investigated two kinds of SiPc compounds to people's normal hepatocytes
The dark toxicity of cell LO2.As a result as shown in figure 9, compound SiPc-pip is to the dark toxicity of LO2 cell clearly (IC50≈
3.3μM).Relative to SiPc-pip, compound SiPc-biotin is to the dark toxicity of LO2 with regard to weak more.In fact, in photosensitizer
When concentration is 4 μM, SiPc-pip causes about 80% cell death, and SiPc-biotin only leads to about 20% cell death.
As it can be seen that compound SiPc-biotin of the present invention is used as photosensitizer, with greater advantage.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (12)
1. a kind of silicon phthalocyanine derivative, which is characterized in that the derivative is the silicon phthalocyanine derivative of biotin axial substituted,
With structure as follows:
2. a kind of method for preparing silicon phthalocyanine derivative described in claim 1, which comprises the steps of:
(1) synthesis of SiPc-pip
Under inert gas shielding, with SiPcCl2It is raw material, in the presence of sodium hydride is catalyst with 4- hydroxy piperidine, is dissolved in toluene
Back flow reaction is carried out in solvent；After reaction, decompression steams solvent, and rinsing residue, with the isolated depth of silica gel column chromatography
Blue product, as required SiPc-pip；
(2) synthesis of SiPc-biotin
Using SiPc-pip obtained above as raw material, n-hydroxysuccinimide biotin is added and is dissolved in the presence of organic amine
Reacted in DMF solvent, after evaporated under reduced pressure solvent, navy blue product is obtained with silica gel column chromatography, as needed for SiPc-
biotin；
3. the method according to claim 2 for preparing the silicon phthalocyanine derivative, which is characterized in that in the step (1),
The SiPcCl2Molar ratio with 4- hydroxy piperidine is 1-4:10.
4. the method according to claim 2 or 3 for preparing the silicon phthalocyanine derivative, which is characterized in that the step (1)
In, the specific steps of the silica gel column chromatography separation are as follows: use 200-300 mesh silica gel, fill column with chloroform；Sample is taken to be dissolved in
In DMF solvent, it is added the silica white adsorption sample of 3 times of quality, after evaporated under reduced pressure DMF, with silica white upper prop, is with volume ratio
Chloroform-methanol solution of 20:1 is that eluant, eluent is eluted.
5. the method according to claim 2 for preparing the silicon phthalocyanine derivative, which is characterized in that in the step (2),
The organic amine is n,N-diisopropylethylamine or triethylamine.
6. the method according to claim 2 for preparing the silicon phthalocyanine derivative, which is characterized in that in the step (2),
The molar ratio of the n-hydroxysuccinimide biotin and SiPc-pip are 4-10:1.
7. the method according to claim 2 for preparing the silicon phthalocyanine derivative, which is characterized in that in the step (2),
The specific steps of the silica gel column chromatography are as follows: use 200-300 mesh silica gel, fill column with chloroform；Sample is taken to be dissolved in DMF solvent
In, it is added the silica white adsorption sample of 3 times of quality, is the three of 30:1 with volume ratio with silica white upper prop after evaporated under reduced pressure DMF
Chloromethanes-methanol solution is that eluant, eluent is eluted.
8. the method according to claim 2 for preparing the silicon phthalocyanine derivative, which is characterized in that further include preparation N- hydroxyl
It the step of base succinimide biotin, specifically includes: under inert gas protection, with biotin and N- hydroxysuccinimide
For raw material, in N, N'- dicyclohexylcarbodiimide or 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate exist
Under, be dissolved in DMF solvent and reacted, after filtering reacting liquid and evaporated under reduced pressure, product precipitates with ether up to required
BNHS；
9. silicon phthalocyanine derivative described in claim 1 is used to prepare photo-dynamical medicine, photosensitive drug or treating cancer drug
Purposes.
10. purposes according to claim 9, which is characterized in that the cancer is cervical carcinoma.
11. a kind of pharmaceutical composition, which is characterized in that including silicon phthalocyanine derivative described in claim 1, and pharmaceutically may be used
The auxiliary material and/or carrier of receiving.
12. the clinically-acceptable preparation that the pharmaceutical composition as described in claim 11 is prepared according to common process.
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| CN112566669A (en) * | 2018-08-08 | 2021-03-26 | 国立大学法人东京大学 | Conjugate of biotin-modified dimer and phthalocyanine dye |
| CN108997406B (en) * | 2018-08-31 | 2020-08-25 | 福建师范大学 | Triphenylamine fluoro aryl benzyl ether branch ligand substituted silicon phthalocyanine and preparation method and application thereof |
| CN109908362B (en) * | 2019-04-26 | 2020-09-25 | 山东大学 | Biotin target modified photosensitizer and quercetin nano drug delivery system |
| CN110256313B (en) * | 2019-05-15 | 2021-01-29 | 江苏省原子医学研究所 | Photosensitizer prodrug compound and preparation method and application thereof |
| JPWO2021157655A1 (en) * | 2020-02-05 | 2021-08-12 | ||
| CN111393465A (en) * | 2020-04-08 | 2020-07-10 | 福州大学 | Axial galactose/lactose modified silicon phthalocyanine and preparation method and application thereof |
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