CN101948566A - Multifunctional polymer for resisting fungi and cancers and performing cell imaging and preparation method thereof - Google Patents
Multifunctional polymer for resisting fungi and cancers and performing cell imaging and preparation method thereof Download PDFInfo
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- CN101948566A CN101948566A CN 201010217334 CN201010217334A CN101948566A CN 101948566 A CN101948566 A CN 101948566A CN 201010217334 CN201010217334 CN 201010217334 CN 201010217334 A CN201010217334 A CN 201010217334A CN 101948566 A CN101948566 A CN 101948566A
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- 241000233866 Fungi Species 0.000 title claims abstract description 26
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000003384 imaging method Methods 0.000 title claims abstract description 10
- 229920000642 polymer Polymers 0.000 title abstract description 9
- 238000005286 illumination Methods 0.000 claims abstract description 48
- 201000011510 cancer Diseases 0.000 claims abstract description 12
- 239000000178 monomer Substances 0.000 claims abstract description 10
- 150000004032 porphyrins Chemical group 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 6
- 150000003856 quaternary ammonium compounds Chemical class 0.000 claims abstract description 6
- 125000005843 halogen group Chemical group 0.000 claims abstract description 3
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 3
- 239000000126 substance Substances 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 21
- 238000002428 photodynamic therapy Methods 0.000 claims description 21
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- 206010038389 Renal cancer Diseases 0.000 claims description 19
- 201000010982 kidney cancer Diseases 0.000 claims description 19
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 17
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Chemical group BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 12
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- 125000003707 hexyloxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 claims description 5
- 210000005265 lung cell Anatomy 0.000 claims description 5
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- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
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- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 4
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- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 3
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- -1 propoxy- Chemical class 0.000 claims description 3
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- 125000004115 pentoxy group Chemical group [*]OC([H])([H])C([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 claims description 2
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- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical compound [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 description 3
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Abstract
The invention discloses multifunctional polymer for resisting fungi and cancers and performing cell imaging and a preparation method thereof. The structural formula of the polymer is shown as a formula (I), wherein R1 refers to a linear or branch alkyl of which the chain end contains a quaternary ammonium compound; the main chain of the alkyl has 2 to 12 carbon atoms; R2 refers to a gene containing an asymmetrical porphyrin structure; X refers to a halogen group; y is molar fraction of R1 group-containing monomer; and n is the degree of polymerization and is more than 8. The polymer PTP provided by the invention has an amphiphilic structure; the structure can promote close combination of the structure with fungi and cells through electrostatic and hydrophobic effects; the PTP aggregates in a water system into nanoparticles which are easy to enter cancer cells; and during illumination, high-efficiency energy transfer from the thiophene main chain to the porphyrin side chain of the PTP molecules can be generated, and the efficiency of generating singlet oxygen in the system is improved, so the multifunctional polymer has high effect of killing the fungi and the cells. Therefore, the novel high-efficiency photosensitive material provided by the invention has application value in the aspects of resisting cancers and bacteria, performing cell imaging and the like.
Description
Technical field
The present invention relates to a kind of multifunctional polymer that is used for antimycotic, anticancer and cell imaging and preparation method thereof.
Background technology
Cancer becomes the major reason that threatens human health, so the detection of cancer and treatment are extremely important.Because being extensive use of of microbiotic, immunosuppressor clinically, particularly increasing of AIDS patient makes systemic fungal infection increasing in recent years.(Photodynamic Therapy is a kind of cancer therapy new technology that develops rapidly in recent years PDT), and can replaces traditional microbiotic to be used for the treatment of bacterium and fungi infestation photodynamic therapy.Photosensitizers is the core substance of photodynamic therapy (PDT), and the appearance of PDT, development and application all are to improve improved gradually along with the development of photosensitizers research.The new and effective photosensitizers material of designs is being extremely important aspect treatment cancer and the fungi infestation.
At present, the photochromics in photodynamic therapy mainly contains porphyrin class, phthalocyanines and phenothiazines etc.Though these materials can should because fungi is the eukaryote that a class has tough and tensile cell walls, increase the difficulty that photosensitizers enters its cell owing in the antibiotic anticancer field more effectively, therefore need to improve the dosage of photosensitizers and light.
Summary of the invention
The purpose of this invention is to provide a kind of polymkeric substance with anticancer, anti-mycotic activity and cell imaging function.
The structural formula of polymkeric substance provided by the present invention is suc as formula shown in (I):
Formula (I);
R in the formula (I)
1Represent the end of the chain to contain the alkyl of the straight or branched of quaternary ammonium compound, the main chain of described alkyl is long to be 2-12 carbon; R
2Representative contains the group of asymmetric porphyrin structure; X represents halogen group; Y is for containing R
1The molar fraction of group monomer, its value are 0.9-0.99; N is the polymerization degree, n＞8.
But this polymkeric substance called after PTP.
Wherein, R
1Specifically can be selected from the following radicals any one: 6-trimethylamine groups hexyl, 6-triethyamino hexyl, 5-trimethylamine groups amyl group, 5-triethyamino amyl group, 4-trimethylamine groups butyl, 4-triethyamino butyl, 3-trimethylamine groups propyl group and 3-triethyamino propyl group;
R
2Specifically can be selected from the following radicals arbitrarily-kind: 5-[4-(6-(4-phenoxy group) hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, 5-[4-(6-(4-phenoxy group) pentyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, 5-[4-(6-(4-phenoxy group) butoxy) phenyl]-10,15,20-three p-methylphenyl porphyrins and 5-[4-(6-(4-phenoxy group) propoxy-) phenyl]-10,15,20-three p-methylphenyl porphyrins;
X specifically can be chlorine, bromine or iodine.
The preparation method of polymkeric substance shown in the formula (I) comprises the steps: under the condition that FERRIC CHLORIDE ANHYDROUS exists, and makes that compound reacts in nitrogen atmosphere shown in compound shown in the formula (II) and the formula (III), obtains described polymkeric substance;
(formula II) (formula III)
Wherein, the R in the formula (II)
1, in X and the formula (I) R
1, X is identical, the R in the formula (III)
2With in the formula (I) R
2Identical.
Described reaction can be carried out in organic solvent (as chloroform); The temperature of reaction of described reaction is a room temperature, and the reaction times is 2-3 days.
A further object of the present invention provides the application of polymkeric substance shown in the formula (I).
The application of polymkeric substance shown in the formula that provides of the present invention (I) comprises following four aspects:
1) this polymkeric substance is as the application of fluorescent probe in cell imaging;
2) this polymkeric substance is monitored apoptotic application in photodynamic therapy;
3) application of this polymkeric substance in preparation photodynamic therapy usefulness cancer cell-apoptosis inductor;
4) application of this polymkeric substance in preparation photodynamic therapy usefulness anti-mycotic agent.
In the application of described cell imaging, described cell can be cancer cells, as kidney cancer cell, specifically can be kidney cancer cell A498.
In the apoptotic application of monitoring, polymer P TP can dye by pair cell in described photodynamic therapy, before the illumination, mainly concentrates on the cytolemma position of cell by fluorescence microscope PTP, and the nucleus of viable cell can not be aggregated thing dyeing; And when necrocytosis or apoptosis late period, PTP can enter the nucleus of cell, thereby realizes the monitoring of pair cell apoptosis.
In the application in described preparation photodynamic therapy usefulness cancer cell-apoptosis inductor, described cancer cells can be kidney cancer cell and/or lung carcinoma cell, and described kidney cancer cell is preferably kidney cancer cell A498, and described lung carcinoma cell is preferably lung cell A549; Illumination condition can be in the described photodynamic therapy: light source 470nm white light, and illumination 30min, intensity of illumination is 1.4mWcm
-2
In the application in described preparation photodynamic therapy usefulness anti-mycotic agent, described fungi can be black-koji mould; Illumination condition can be in the described photodynamic therapy: light source 470nm white light, and illumination 30min, intensity of illumination is 50mWcm
-2
The main component of fungal cell wall is polysaccharide, protein, lipoid etc., the fungi surface has negative charge, and polymkeric substance shown in the formula (I) all has positive charge, so fungi is combined closely by electrostatic interaction and hydrophobic interaction and parents' molecule PTP, singlet oxygen and fungal cell wall that PTP is produced under illumination condition are enough near, and then destroy its cell walls effectively and suppress the sprouting of spore.Because the high efficiency energy that can take place during illumination from the thiophene main chain of PTP molecule to the side chain porphyrin shifts, can the raising system produce the efficient of singlet oxygen, so the lethal effect of pair cell and fungi is very big.
Polymkeric substance provided by the present invention has following four big advantages, and first: the covalently bound shared molar ratio of porphyrin part very little (about 1-10%) to Compound P TP side chain, therefore can reduce the toxicity of photochromics dark place; Second: parents' structure of PTP can impel it closely to combine with fungi and cell by static and hydrophobic interaction, and PTP is gathered into nano particle and impels and be relatively easy to enter in the cancer cells in aqueous systems; The the 3rd: can reduce to the PTP and the distance of energy donor part energy acceptor molecule is covalently bound, and then the energy transfer efficiency of raising from the donor to the acceptor, therefore improve the generation efficient of singlet oxygen, improved the lethal effect of photochromics pair cell like this; The the 4th: the apoptosis or the death process that can pass through the fluorescence monitoring cell of PTP.The present invention has using value at aspects such as anticancer, antibiotic and cell imagings.
Description of drawings
Fig. 1 is the H-NMR collection of illustrative plates of PTP-1.
Fig. 2 is the linear relationship chart of monomer molar percentage ratio among the PTP.
Fig. 3 is under illumination (excitation wavelength is 470nm) condition, the kidney cancer cell survival rate curve of different PTP concentration correspondences.
Fig. 4 is that polymer P TP-1 is to the colored graph picture of kidney cancer cell before and after the illumination among the embodiment 3, and wherein left side figure is an image before the illumination, and right figure is 30 minutes a back image of illumination.
Fig. 5 be among the embodiment 6 under white light conditions, the fungistatic effect figure of various dose PTP.
Embodiment
Following embodiment is for the ease of understanding the present invention better, but do not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
1,5-[4-(6-bromine hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins synthetic
757mg pyrroles, 805mg 4-(6-bromine hexyloxy) phenyl aldehyde and 1.02g p-tolualdehyde are dissolved in the 154mL chloroform and stir 15min, add the 389mg boron trifluoride diethyl etherate, fully stir 1h, add 1.91g 2,3-two chloro-5,6-dicyan-para benzoquinone (DDQ), mixed solution continue to stir 2h, add triethylamine then, the crude product silica gel column chromatography separating purification, developping agent be sherwood oil/methylene dichloride (1/1, v/v), purple powder (product) 256mg.
The sign of product:
1H-NMR：(400MHz，CDCl
3)δ(ppm)8.85(s，8H)，8.10-8.08(d，8H)，7.56-7.54(d，6H)，7.27-7.25(d，2H)，4.26-4.23(t，2H)，3.52-3.48(t，2H)，2.67(s，9H)，2.01-1.98(m，4H)，1.66-1.65(m，4H)，-2.77(s，2H).。
Characterization data shows: obtained 5-[4-(6-bromine hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins.
2,4-(2-(3-thiophene) oxyethyl group) phenol is synthetic
4.4g Resorcinol and 1.38g salt of wormwood are dissolved in the 150ml acetone, add 30mg hexaoxacyclooctadecane-6-6 and 256mg 2-(3-thiophene)-ethanol p-toluenesulfonic esters, reaction solution is warming up to 70 ℃ under the nitrogen atmosphere, reaction 48h; Reaction stops and being cooled to remove after the room temperature desolvating, and reaction solution is poured in the water, and use dichloromethane extraction, merges organic phase, use distilled water wash, anhydrous magnesium sulfate drying, and filtration, concentrated; Crude product silica gel column chromatography separating purification, developping agent be petrol ether/ethyl acetate (80/1, v/v), obtain light yellow oily liquid (product) 240mg.
The sign of product:
1H-NMR:(400MHz, CDCl
3) δ (ppm) 7.26 (m, 1H), 7.07 (s, 1H), 7.02-7.01 (d 1H), 6.79-6.73 (dd, 4H), 4.75 (s, 1H), 4.13-4.10 (t, 2H), 3.10-3.07 (t, 2H).
Characterization data shows: obtained 4-(2-(3-thiophene) oxyethyl group) phenol.
3,5-[4-(6-(4-phenoxy group-(2-(3-thiophene) oxyethyl group)) hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins synthetic
0.24g 4-(2-(3-thiophene) oxyethyl group) phenol and 0.3g salt of wormwood are dissolved among the 50mL DMF, add 10mg hexaoxacyclooctadecane-6-6 and 0.91g 5-[4-(6-bromine hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, reaction solution is warming up to 70 ℃ under the nitrogen atmosphere, reaction 24h; Reaction stops and being cooled to during room temperature falls back, and use dichloromethane extraction, merges organic phase, use distilled water wash, anhydrous magnesium sulfate drying, and filtration, concentrated; Crude product silica gel column chromatography separating purification, developping agent be petrol ether/ethyl acetate (40/1, v/v), obtain purple powder (product) 0.46g.
The sign of product:
1H-NMR:(400MHz, CDCl
3) δ (ppm) 8.85 (s, 8H), 8.10-8.08 (d, 8H), 7.56-7.54 (d, 6H), and 7.26-7.22 (m, 2H), 7.07-7.02 (m, 2H), 6.86-6.82 (m, 3H), 6.58-6.54 (m, 2H), 4.25-3.99 (t, 6H), 3.10-3.08 (m, 2H), 2.70 (s, 9H), 2.01-1.89 (m, 4H), 1.68 (m, 4H) ,-2.76 (s, 2H).
Characterization data shows: obtained 5-[4-(6-(4-phenoxy group-(2-(3-thiophene) oxyethyl group)) hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins
4,3-(2-(6-bromine hexyloxy) ethyl) thiophene is synthetic
Add 663 μ L 2-(3-thiophene) ethanol, 0.206g sodium hydride, 50mL dry DMF (dimethyl formamide), reaction solution stirring at room 30min under the nitrogen atmosphere in the 100mL single port bottle; Add 4.6mL 1 then, the 6-dibromo-hexane continues stirring and spends the night, and during reaction stops to fall back, and uses dichloromethane extraction, merges organic phase, uses distilled water wash, and anhydrous magnesium sulfate drying filters, and concentrates; Crude product silica gel column chromatography separating purification, developping agent be petrol ether/ethyl acetate (40/1, v/v), obtain white powder (product) 0.54g.
The sign of product:
1H-NMR:(400MHz, CDCl
3) δ (ppm) 7.37 (m, 1H), 7.15-7.10 (m, 2H), 3.76 (t, 2H), 3.57 (t, 2H), 3.53 (t, 2H), 3.04 (t, 2H), 1.99 (m, 2H), 1.72 (m, 2H), 1.61-1.45 (m, 4H).Characterization data shows: obtained 3-(2-(6-bromine hexyloxy) ethyl) thiophene.
5,3-(2-(6-bromination trimethylamine groups hexyloxy) ethyl) thiophene is synthetic
0.29g 3-(2-(6-bromine hexyloxy) ethyl) thiophene is dissolved in the 1mL tetrahydrofuran (THF), adds the methanol solution of 3mL 33% Trimethylamine 99, reaction solution is warming up to 40 ℃ of reactions and spends the night; Reaction stops and being cooled to remove after the room temperature desolvating, and adds the normal hexane washing, and is centrifugal then, dry white solid (product) 0.31g.
The sign of product:
1H NMR (400MHz, CDCl
3) δ (ppm) 7.27 (m, 1H); 7.02-6.98 (m, 2H), 3.65-3.56 (m, 4H), 3.47 (m, 11H), 2.91 (t, 2H, 6.78), 1.75 (br, 2H), 1.58 (br, 2H), 1.42 (br, 4H).
Characterization data shows: obtained 3-(2-(6-bromination trimethylamine groups hexyloxy) ethyl) thiophene.
6, PTP-1's is synthetic
Add the 15mL chloroform in reaction flask successively, the 130mg FERRIC CHLORIDE ANHYDROUS is led to N half an hour
2Drip then and contain 135mg (0.386mmol) 3-(2-(6-bromination trimethylamine groups hexyloxy) ethyl) thiophene and 33mg (0.033mmol) 5-[4-(6-(4-phenoxy group-(2-(3-thiophene) oxyethyl group)) hexyloxy) phenyl]-10,15, the 10mL chloroformic solution of 20-three p-methylphenyl porphyrins, room temperature reaction 2 days.Suction filtration, the filter cake methanol wash, (1/10, v/v) dissolving utilizes the dialysis tubing dialysis (2 day change water 8 time) of molecular weight cut-off for 3500Da with dimethyl sulfoxide (DMSO)/water to obtain solid; Remove and desolvate, drying obtains 11mg dark red solid (product).
The sign of product:
1H-NMR:(400MHz, d
6-DMSO) δ (ppm) 7.31 (br), 3.70 (br), 3.44-3.31 (br), 3.08-2.67 (br), 1.65-1.54 (br), 1.34-1.24 (br)..
Characterization data shows: obtained Compound P TP-1.
Monomer molar fractional checking among the PTP-1:
At the 422nm place uv-absorbing is arranged according to the monomer that contains the porphyrin group among the PTP-1; And with the multipolymer of the monomer preparation that contains quaternary ammonium compound, its Polythiophene main chain all has uv-absorbing at 422nm place and 385nm place.
To contain the monomer of porphyrin group and mix with the different mol ratio example, measure this mixing solutions at 422nm and 385nm ultraviolet absorption value with the multipolymer of the monomer preparation that contains quaternary ammonium compound.The molar fraction that contains the porphyrin group monomer in the above-mentioned mixed solution is designated as (1-y).With A
422nm/ A
385nmFor X-coordinate, (1-y) % are the ordinate zou mapping, see Fig. 2.The typical curve equation of gained be (1-y)=-0.732+0.94x.
Then PTP-1 is mixed with solution and measures A
422nm, A
385nmValue is calculated A
422nm/ A
385nm=2.4.
Should be worth in the substitution typical curve equation, calculate (1-y) %=-0.732+0.94*2.4=1.5.Illustrate that containing the monomeric molar fraction of quaternary ammonium compound among the preparation-obtained PTP-1 is 1.5 (%).
PTP-1 produces the detection of singlet oxygen ability under embodiment 2, the white light
In the aqueous solution (concentration is 10 μ M) of 300 μ L PTP-1, add 6 μ L 9, the aqueous solution (6mM) .D of 10-diphenylanthrancene Sodium Propionate (ADPA)
2O replaces H
2O is as solvent.Use the white light sample, intensity of illumination is 50mW/cm
2, measure sample illumination 0 minute, 1 minute, 2 minutes, 3 minutes, 4 minutes, the ultra-violet absorption spectrum after 5 minutes observed 9, and 10-diphenylanthrancene Sodium Propionate (ADPA) is at the absorption value (A of 378nm
378nm) along with the variation of light application time, the result is as shown in the table:
| T (minute) | 0 | 1.0 | 2.0 | 3.0 | 4.0 | 5.0 |
| A 378nm | 1.00131 | 0.40467 | 0.19133 | 0.12843 | 0.09697 | 0.0787 |
In containing the aqueous solution of PTP-1, after the illumination 9,10-diphenylanthrancene Sodium Propionate (ADPA) significantly reduces in the absorption of the 378nm increase along with light application time, illustrates that PTP-1 can produce singlet oxygen efficiently under the illumination condition.
Embodiment 3, the PTP-1 application in the monitoring apoptosis process
1) cell recovery and cultivation
The frozen pipe of kidney cancer cell A498 is taken out from liquid nitrogen, put in 37 ℃ of waters bath with thermostatic control and melt fast.Change Tissue Culture Flask over to, add the DMEM nutrient solution 10mL that contains 10% new-born calf serum, put CO
2Incubator (5%CO
2, 95% filtrated air, 37 ℃ of constant temperature) and middle the cultivation.
2) cell administration
After kidney cancer cell A498 digestion in good condition, by 8 * 10
4The cell density of individual/mL goes down to posterity and is inoculated in the 35mm culture dish, after incubator is placed 24h, inhales and removes substratum, with cell of 1 * PBS washing, adding 1mL contains the DMEM substratum of 10% serum, adds the PTP-1 of 10 μ M, uses the fluorescence microscope coloration result after continuing to cultivate 12h.
PTP-1 can dye by pair cell, and it mainly concentrates on the cytolemma position (see figure 4) of cell.
3) illumination administration cell
The cell that step 1) is prepared is with the rayed of wavelength 470nm, and intensity of illumination is 1.4mWcm
-2, the time was respectively 0,10 and 30 minute, added ethidium bromide then, after mixing 1-2 minute, used the fluorescence microscope result, saw Fig. 4.
Along with the increase of light application time, the smaller volume of cell, tenuigenin concentrates, chromatin margination, and then lysis and apoptotic body formation.The nucleus of viable cell can not be aggregated thing dyeing, and when necrocytosis or apoptosis late period, ethidium bromide dyestuff and polymkeric substance can enter the nucleus of this cell simultaneously.
Embodiment 4, photodynamic therapy are induced the application of kidney cancer cell A498 apoptosis
Cell survival rate is analyzed: tetramethyl-azo azoles salt colorimetry (MTT method) is the active method of using always of analysis of cells, utilize the viable cell mitochondrial dehydrogenase MTT salt can be reduced into the water-fast first a ceremonial jade-ladle, used in libation of bluish voilet particle, be directly proportional with viable count with the shade reflection cytoactive Jia Za growing amount that presents after the particle dissolving, therefore can infer the number that viable cell according to optical density(OD) OD value, calculate survival rate then.
(1) under the illumination condition, the relation of PTP-1 concentration and cell survival rate
Collect the logarithmic phase cell, adjust kidney cancer cell A498 suspension concentration, every hole adds 100 μ L, and bed board makes cell density to be measured transfer to 4～7 * 10
4/ mL, 5%CO
2, hatch for 37 ℃, be paved with the hole to cell monolayer at the bottom of (96 hole flat underside), add the PTP-1 solution (concentration is followed successively by 0,2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 μ M) of concentration gradient, 5%CO
2, hatched 24 hours for 37 ℃, inhale and remove substratum, with cell of 1 * PBS washing, add the DMEM substratum that 100 μ L contain 10% serum, with the light illumination of 470nm, the time was respectively 30 minutes, and intensity of illumination is 1.4mWcm
-2Cell continues to cultivate 24 hours after the illumination, discards nutrient solution, and every hole adds 100 μ LMTT solution, and (1mg/ml continues to cultivate 4h.Stop cultivating, the careful suction removed nutrient solution in the hole.Every hole adds 150 μ L dimethyl sulfoxide (DMSO), measures OD
520nmLocate the light absorption value in each hole.The ratio of experimental group and control group is the survival rate of cell.
Concentration with PTP-1 is X-coordinate, and cell survival rate is the ordinate zou mapping, sees Fig. 3.As shown in Figure 3, along with the increase of medicine PTP-1 concentration, cell survival rate reduces gradually.The result shows that under the illumination condition, medicine PTP-1 has significant lethal effect to kidney cancer cell A498.
(2) under the illumination condition, the relation of light application time and cell survival rate
Collect the logarithmic phase cell, adjust kidney cancer cell A498 suspension concentration, every hole adds 100 μ L, and bed board makes cell to be measured transfer density to 4～7 * 10
4/ mL, 5%CO
2, hatch for 37 ℃, be paved with the hole to cell monolayer at the bottom of (96 hole flat underside), add PTP-1 (10 μ M), 5%CO
2, hatched 24 hours for 37 ℃, inhale and remove substratum, with cell of 1 * PBS washing, add the DMEM substratum that 100 μ L contain 10% serum, with the light illumination of 470nm, the time was respectively 0 minute, and 10 minutes, 20 minutes, 30 minutes, illumination dose was 2.5Jcm
-2Cell continues to cultivate 24 hours after the illumination, discards nutrient solution, and every hole adds 100 μ L MTT solution, and (1mg/ml continues to cultivate 4h.Stop cultivating, the careful suction removed nutrient solution in the hole.Every hole adds 150 μ L dimethyl sulfoxide (DMSO), measures OD
520nmLocate the light absorption value in each hole.The ratio of experimental group and control group is the survival rate of cell.
Under the illumination condition, medicine PTP-1 has significant lethal effect to kidney cancer cell A498.When PTP-1 concentration was 10 μ M, when light application time was 0,10,20,30 minutes, the survival rate of cell was respectively 100%, 93%, 64%, 34% increase along with the medicine light application time, and cell survival rate reduces gradually.
Embodiment 5, photodynamic therapy are induced the application of lung cell A549 apoptosis
Collect the logarithmic phase cell, adjust lung cell A549 suspension concentration, every hole adds 100 μ L, and bed board makes cell to be measured transfer density to 4～7 * 10
4/ mL, 5%CO
2, hatch for 37 ℃, be paved with the hole to cell monolayer at the bottom of (96 hole flat underside), add the PTP-1 solution (concentration is followed successively by 0,2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 μ M) of concentration gradient, 5%CO
2, hatched 24 hours for 37 ℃, inhale and remove substratum, with cell of 1 * PBS washing, add the DMEM substratum that 100 μ L contain 10% serum, with the light illumination of 470nm, the time was respectively 30 minutes, and intensity of illumination is 1.4mWcm
-2Cell continues to cultivate 24 hours after the illumination, discards nutrient solution, and every hole adds 100 μ LMTT solution, and (1mg/ml continues to cultivate 4h.Stop cultivating, the careful suction removed nutrient solution in the hole.Every hole adds 150 μ L dimethyl sulfoxide (DMSO), measures OD
520nmLocate the light absorption value in each hole.The ratio of experimental group and control group is the survival rate of cell.
Under the illumination condition, medicine PTP-1 has significant lethal effect to lung cell A549.Along with the increase of medicine PTP-1 concentration, cell survival rate reduces gradually, when PTP-1 concentration is 10 μ M, illumination after 30 minutes cell survival rate be reduced to about 32%.
Under embodiment 6, the illumination condition, the antifungal effect of PTP-1
(1) recovery of aspergillus niger strain is cultivated
In Bechtop, scratch the ampere bottle of being bought that aspergillus niger is housed, add a little sterile purified water, gently be stained with transfering loop, streak inoculation is on freshly prepd improvement Martin medium slant then, make it fully to be distributed on the inclined-plane, the room temperature lucifuge is cultivated (about 26 ℃).Approximately 4-5 days, after treating to grow conidium on the slant medium, move on another inclined-plane with transfering loop and to cultivate, pass 3-4 so continuously and be commissioned to train fosterly, obtain stable bacterial strain.
(2) self-assembly of PTP-1 and fungi
(1) fluorescence microscope of PTP-1/ fungi
Get the aspergillus niger that grows spore, add a little aseptic Millipore water, and gently scrape mycelia, the preparation spore suspension with transfering loop.During use, spore concentration is diluted to 5 * 10 with the improvement Martin substratum of liquid
5Individual/mL, add 10 μ MPTP-1, be put in 26 ℃ and cultivate 4h, use fluorescence microscope.
Aspergillus niger is a kind of common filamentous fungus, its cell walls is made up of chitin, dextran, phosphoric acid salt and a small amount of protein and lipid etc., makes that having amphiphatic PTP-1 molecule can assemble in the aspergillus niger surface adsorption by electrostatic attraction and hydrophobic interaction power.The aspergillus niger that can see light field under fluorescent microscope overlaps fully with the fluorescence position of PTP-1, and the fluorescence that aspergillus niger manifested is that PTP-1 is adsorbed on the fluorescence that the fungi surface is sent.
(2) electron microscopic observation of PTP-1/ fungi
Get that handle well, clean fritter silicon chip, denseer mycelia to be checked is directly coated seasoning.Then above-mentioned sample is placed 1%～2% glutaraldehyde phosphoric acid buffer (about pH7.2), in 40 ℃ of refrigerators, fixedly spend the night.Next day, the glutaraldehyde phosphoric acid buffer with 0.15% washed, and dewatered each 15min more successively with 40%, 70%, 90% and 100% ethanol difference.With the sample lyophilize, observe the microtexture of assembly at last.
The hypha,hyphae that has occurred a lot of wire under the low power lens, diameter is at 3～5 μ m, the outside surface of mycelia is very smooth under the high power lens, has added PTP-1 assembly PTP-1/ fungi afterwards and can see that under high power lens the outside surface mycelia has the continuous and coarse membrane structure of one deck.Illustrate that PTP-1 and fungi have very strong binding ability, can well be adsorbed onto the surface of fungi.
(3) antibacterial effect of PTP-1 under the illumination condition
(1) preparation of bacterium inoculation liquid
Inoculated aspergillus niger (CGMCCC, coding: cultivated 4～5 days for 26 ℃ to improveing on Martin's agar slant culture-medium, adds aqua sterilisa, with the spore wash-out by fresh culture thing 3.0808).Then, the sucking-off spore suspension is to sterile test tube.It is 5 * 10 that this spore suspension is diluted to turbidity with the improvement Martin substratum of liquid and aseptic Millipore water
5Individual/mL.
(2) fungi administration
Under aseptic condition, use 96 aseptic micro reaction plates, every row adds the aspergillus niger suspension that 80 μ L steps (1) are prepared, by lower concentration to the order of high density (concentration is followed successively by 0,3.0,5.0,9.0,15.0 μ M) and add the corresponding medicine PTP-1 of 20 μ L, the growth control hole does not add medicine.
(3) under the illumination condition, the anti-mycotic efficiency of PTP-1
The spore suspension that white light step (2) is prepared, intensity of illumination is 50mW/cm
2, light application time is 30 minutes, illumination after 26 ℃ cultivate observations behind the 72h, under the situation of not stirring, compare with control wells.The turbidity of fungi in the visual inspection micropore, photo is seen Fig. 4.The turbidity in each hole of spectrophotometric determination microtest plate compares with control wells.The ratio of experimental group and control group is the survival rate of fungi.
Under the illumination condition, medicine PTP-1 has significant inhibitory effect.Along with the increase of medicine PTP-1 concentration, the fungi turbidity reduces (Fig. 5) gradually, and the minimal inhibitory concentration of PTP-1 is 5.0 μ M.When PTP-1 concentration is 20 μ M, illumination after 30 minutes the fungi surviving rate be reduced to about 10%.
Claims (10)
1. the polymkeric substance shown in the formula (I)
Formula (I);
R in the formula (I)
1Represent the end of the chain to contain the alkyl of the straight or branched of quaternary ammonium compound, the main chain of described alkyl is long to be 2-12 carbon; R
2Representative contains the group of asymmetric porphyrin structure; X represents halogen group; Y is for containing R
1The molar fraction of group monomer, its value are 0.99-0.90; N is the polymerization degree, n＞8.
2. polymkeric substance according to claim 1 is characterized in that: R described in the formula (I)
1Be selected from the following radicals any one: 6-trimethylamine groups hexyl, 6-triethyamino hexyl, 5-trimethylamine groups amyl group, 5-triethyamino amyl group, 4-trimethylamine groups butyl, 4-triethyamino butyl, 3-trimethylamine groups propyl group and 3-triethyamino propyl group;
R
2Be selected from the following radicals any one: 5-[4-(6-(4-phenoxy group) hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, 5-[4-(6-(4-phenoxy group) pentyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, 5-[4-(6-(4-phenoxy group) butoxy) phenyl]-10,15,20-three p-methylphenyl porphyrins and 5-[4-(6-(4-phenoxy group) propoxy-) phenyl]-10,15,20-three p-methylphenyl porphyrins;
X is chlorine, bromine or iodine.
3. polymkeric substance according to claim 1 and 2 is characterized in that: R described in the formula (I)
1Be 6-trimethylamine groups hexyl, described R
2Be 5-[4-(6-(4-methylphenoxy) hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, described X is a bromine, y=0.985, n＞8.
4. polymkeric substance according to claim 3, it is characterized in that: described polymkeric substance is prepared according to following method: under the condition that FERRIC CHLORIDE ANHYDROUS exists, make compound shown in compound shown in the formula (II) and the formula (III) according to mol ratio 0.386: 0.033 in nitrogen atmosphere in room temperature reaction 2 days; After reaction finishes, suction filtration, filter cake methanol wash, the solid volume ratio that obtains are 1: 10 the dimethyl sulfoxide (DMSO) and the mixed solvent dissolving of water, utilize the dialysis tubing dialysis of molecular weight cut-off for 3500Da, remove and desolvate, and promptly obtain described polymkeric substance; Wherein, R
1Be 6-trimethylamine groups hexyl, R
2Be 5-[4-(6-(4-methylphenoxy) hexyloxy) phenyl]-10,15,20-three p-methylphenyl porphyrins, X is a bromine;
(formula II) (formula III).
5. the method for preparing arbitrary described polymkeric substance among the claim 1-3 comprises the steps: under the condition that FERRIC CHLORIDE ANHYDROUS exists, and makes that compound reacts in nitrogen atmosphere shown in compound shown in the formula (II) and the formula (III), obtains described polymkeric substance;
(formula II) (formula III)
Wherein, the R in the formula (II)
1, in X and the formula (I) R
1, X is identical, the R in the formula (III)
2With in the formula (I) R
2Identical.
6. method according to claim 5 is characterized in that: described being reflected in the chloroform carried out; The temperature of reaction of described reaction is a room temperature, and the reaction times is 2-3 days.
Among the claim 1-4 arbitrary described polymkeric substance following 1)-4) and in application in any one:
1) as the application of fluorescent probe in cell imaging;
2) the apoptotic application of monitoring in photodynamic therapy;
3) application in preparation photodynamic therapy usefulness cancer cell-apoptosis inductor;
4) application in preparation photodynamic therapy usefulness anti-mycotic agent.
8. application according to claim 7 is characterized in that: described application 1), described cell is a cancer cells, is preferably kidney cancer cell, more preferably kidney cancer cell A498.
9. application according to claim 7 is characterized in that: described application 3), described cancer cells is kidney cancer cell and/or lung carcinoma cell, and described kidney cancer cell is preferably kidney cancer cell A498, and described lung carcinoma cell is preferably lung cell A549; Illumination condition is in the described photodynamic therapy: light source 470nm white light, and illumination 30min, intensity of illumination is 1.4mWcm
-2
10. application according to claim 7 is characterized in that: described fungi is a black-koji mould; Illumination condition is in the described photodynamic therapy: light source 470nm white light, and illumination 30min, intensity of illumination is 50mWcm
-2
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| CN102276579A (en) * | 2011-06-15 | 2011-12-14 | 中国科学院化学研究所 | Oligothiophene and preparation method and application thereof |
| CN102350277A (en) * | 2011-06-16 | 2012-02-15 | 中国科学院苏州纳米技术与纳米仿生研究所 | Composite microballoon with functions of dual mode imaging and photodynamic activity and preparation method thereof |
| CN102924428A (en) * | 2011-06-15 | 2013-02-13 | 中国科学院化学研究所 | Oligothiophene |
| CN111732718A (en) * | 2020-06-15 | 2020-10-02 | 河北工业大学 | Water-soluble conjugated polymer with antibacterial and antiviral functions and preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102276579A (en) * | 2011-06-15 | 2011-12-14 | 中国科学院化学研究所 | Oligothiophene and preparation method and application thereof |
| CN102924428A (en) * | 2011-06-15 | 2013-02-13 | 中国科学院化学研究所 | Oligothiophene |
| CN102924428B (en) * | 2011-06-15 | 2014-03-12 | 中国科学院化学研究所 | Oligothiophene |
| CN102350277A (en) * | 2011-06-16 | 2012-02-15 | 中国科学院苏州纳米技术与纳米仿生研究所 | Composite microballoon with functions of dual mode imaging and photodynamic activity and preparation method thereof |
| CN102350277B (en) * | 2011-06-16 | 2013-12-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | Composite microballoon with functions of dual mode imaging and photodynamic activity and preparation method thereof |
| CN111732718A (en) * | 2020-06-15 | 2020-10-02 | 河北工业大学 | Water-soluble conjugated polymer with antibacterial and antiviral functions and preparation and application thereof |
| CN111732718B (en) * | 2020-06-15 | 2021-06-18 | 河北凯尔威生物技术有限公司 | Water-soluble conjugated polymer with antibacterial and antiviral functions and preparation and application thereof |
| CN113387961A (en) * | 2021-05-07 | 2021-09-14 | 西安交通大学 | Cationic viologen polymer, preparation method thereof and application thereof in preparing electronic wound dressing |
| CN113387961B (en) * | 2021-05-07 | 2024-03-29 | 西安交通大学 | Cationic viologen polymer, preparation method thereof and application of cationic viologen polymer in preparation of electronic wound dressing |
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