CN110152007A - A kind of hectorite polypyrrole nano-carrier and its preparation, modification and methods for using them - Google Patents
A kind of hectorite polypyrrole nano-carrier and its preparation, modification and methods for using them Download PDFInfo
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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
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The present invention provides a kind of hectorite polypyrrole nano-carrier and its preparations, modification and methods for using them.The preparation method of the hectorite polypyrrole nano-carrier, it is characterized in that, it include: that hectorite is added in distilled water, ultrasonic disperse obtains hectorite dispersion liquid, pyrroles is added, it is stirred, initiator solution is added, continue stirring and carry out home position polymerization reaction, after gained mixture centrifuge washing, hectorite polypyrrole nano-carrier is obtained.The LAP-PPy- polymer nanocomposite disk being prepared by means of the present invention has excellent photothermal conversion efficiency, and photosensitizer load effect outstanding is expected to the photo-thermal and photodynamics combination therapy field applied to tumour.
Description
Technical field
The invention belongs to Bio-Nano-Materials fields, are related to a kind of efficient composite optothermal material hectorite polypyrrole nanometer
The research of preparation, modification and its oncotherapy of carrier.
Background technique
Currently, cancer (malignant tumour) is to threaten most dangerous one of the disease of human life and health, this attribution in the world
In the high incidence and the death rate of cancer.Latest data report points out that cancer has become the burden that global country needs to bear, cancer
Disease also becomes a serious public health problem in China, causes government in recent years and the people more and more pay close attention to.Face
Common treatment method includes chemotherapy, operation excision, radiation cure etc. on bed, but these treatment methods exist centainly
Defect.Chemotherapy kills cancer cell using drug, this can generate serious side effect, and be also easy to produce over the course for the treatment of resistance to
Pharmacological property etc..Operation excision can bring serious injury to sufferer, and when range of tumor is wider is difficult to eradicate.Radiation cure is logical
Tumour cell is killed in overshoot, this will bring potentially hazardous and side effect to sufferer.Based on this, find a kind of efficiently and malicious
Evil acts on small treatment means increasingly by the concern of researchers.
A kind of tumor physical therapy method that photo-thermal therapy is mediated as light has minimal invasive, highly selective, it is convenient with it is right
The features such as normal tissue injury is small, thus caused the interest of numerous researchers in recent years.Has stronger near infrared absorbing energy
Power and higher photothermal conversion efficiency are the key that realize good photo-thermal therapy effect.With the continuous development of nanosecond science and technology, more
It is emerged come more nanometer optothermal materials.Polypyrrole (PPy) is a kind of pi-conjugated heterocyclic conducting polymer, is had excellent
Biocompatibility and outstanding photo and thermal stability.It is answered extensively with individual polymer form or the form compound with other materials
For nerve regneration, biosensor, the research of drug conveying and photo-thermal therapy.At present, it has been reported that many is controlled in photo-thermal
PPy the or PPy based composites of the different shape orientation and size of application aspect, such as PPy nano particle (NPs) are treated, PPy receives
Rice/microcapsules, PPy hollow microsphere and the hollow Nano capsule of fusiform PPy etc..But single photo-thermal therapy usually requires higher
Laser power density or higher material concentration usually control photo-thermal therapy with other to reach desired therapeutic effect
Treatment method is implemented in combination with the synergistic treatment of tumour.
Photodynamic therapy (PDT) is to be converted the photon energy of absorption using photodynamic effect i.e. photosensitizer (PSs)
Viability oxygen (ROS) carries out a kind of new technology of oncotherapy.Its generate active oxygen can only move very short distance then with
Neighbouring large biological molecule occurs oxidation reaction and generates cytotoxicity to cause the oxidative damage of target cell, causes Apoptosis
And autophagy.However, photosensitizer have extension delocalization aromatic series Dan electronic system, therefore, they have high hydrophobicity and
Assemble in aqueous environments, limit the generation of active oxygen, above-mentioned limitation excites researchers and finds suitable nano-carrier such as
Polymer micelle, dendritic, liposome and nano particle are to improve the dissolubility of hydrophobic photosensitizer.
Summary of the invention
The object of the present invention is to provide preparation, modification and its cancer therapeutic applications of a kind of composite Nano carrier.
In order to achieve the above object, the present invention provides a kind of preparation methods of hectorite polypyrrole nano-carrier, special
Sign is, comprising: hectorite is added in distilled water, ultrasonic disperse, obtains hectorite dispersion liquid, pyrroles is added, stirring is mixed
It closes, initiator solution is added, continue stirring progress home position polymerization reaction and obtain hectorite after gained mixture centrifuge washing
Polypyrrole nano-carrier (i.e. unmodified LAP-PPy nanometer plate).
Preferably, the initiator is ferric chloride hexahydrate, ammonium persulfate or potassium permanganate, the concentration of initiator solution
For 10-150mg/mL, the volume ratio of initiator solution and distilled water is 1:5-1:10.
Preferably, the concentration of the hectorite dispersion liquid is 1-20mg/mL.
Preferably, the volume ratio of the pyrroles and distilled water are as follows: 1:500-1:1000.
Preferably, the ultrasonic time is 10-60 minutes.
Preferably, the time that is stirred is 10-60 minutes.
Preferably, the home position polymerization reaction temperature is 0-4 DEG C, and the reaction time is 1-8 hours.
Preferably, the centrifuge speed is 5000-12000r/min, and washing is carried out using distilled water, washing times
It is 3-5 times.
The present invention also provides the method for modifying of the hectorite polypyrrole nano-carrier prepared by the above method, feature exists
In, comprising: hectorite polypyrrole nano-carrier is added in ethyl alcohol, polymer solution is added after Ultrasonic Pulverization, again ultrasonic powder
After broken, rotary evaporation removes ethyl alcohol, obtains polymer-modified hectorite polypyrrole nano-carrier (LAP-PPy- polymer nanocomposite
Disk).
Preferably, the polymer is in polyvinylpyrrolidone, polyethylene glycol-400, polyglutamic acid and polyvinyl alcohol
Any one, the concentration of polymer solution is 1-25mg/mL, and the volume ratio of polymer solution and ethyl alcohol is 1:2-1:10.
Preferably, the Ultrasonic Pulverization time is 10-60 minutes.
Preferably, the temperature of the rotary evaporation is 30-50 DEG C, and the time is 10-90 minutes.
The present invention also provides a kind of hectorite polypyrrole nano-carriers, which is characterized in that include hectorite and polypyrrole,
The polypyrrole in hectorite interlayer in-situ polymerization by forming.
The present invention also provides a kind of polymer-modified hectorite polypyrrole nano-carriers, which is characterized in that comprising poly-
Close object and above-mentioned hectorite polypyrrole nano-carrier, table of the polymer-coated in hectorite polypyrrole nano-carrier
Face.
The present invention also provides a kind of optical-thermal conversion material or photosensitizer carrier materials, which is characterized in that comprising above-mentioned
Hectorite polypyrrole nano-carrier, the hectorite polypyrrole nano-carrier load photosensitizer by the method for physical absorption.
Hectorite polypyrrole composite Nano carrier of the invention is loaded after with polymer-modified stabilization by the method for physical absorption
Photosensitizer realizes the combination therapy of photo-thermal-photodynamics.
Compared with prior art, the beneficial effects of the present invention are:
Present invention process is simple, and product is easy to get, and it is good colloid-stabilised that the polymer of physics coating imparts nano material
Property, internal and external biocompatibility etc., the LAP-PPy- polymer nanocomposite disk being prepared by means of the present invention has
Excellent photothermal conversion efficiency, photosensitizer load effect outstanding are expected to combine applied to the photo-thermal and photodynamics of tumour and control
Treatment field.
Detailed description of the invention
Fig. 1 LAP-PPy-PVP nanometer plate is scattered in respectively in (a) distilled water, (b) in physiological saline, (c) 1640 culture medium
In hydration kinetics diameter distribution；It (d-f) is respectively in distilled water, in physiological saline, the dindar in 1640 culture mediums is existing
As；
The SEM of Fig. 2 (a) LAP-PPy nanometer plate schemes, (b) the TEM figure of LAP-PPy-PVP；
The infrared conversion spectrum figure of the Fourier of Fig. 3 (a) LAP, LAP-PPy, LAP-PPy-PVP；(b)LAP,PPy,PVP,
The thermogravimetric analysis map of LAP-PPy, LAP-PPy-PVP；
The Uv-Vis-NIR spectrogram of Fig. 4 (a) various concentration LAP-PPy-PVP nanometer plate；(b) LAP-PPy-PVP nanometers
Disk is in 10 continuous circulation irradiation 980nm, 0.5W/cm2Heating and natural cooling curve under laser；(c) 980nm (0.5W/ is used
cm2) laser irradiation various concentration LAP-PPy-PVP solution and physiological saline heating curve；(d) LAP- corresponding with figure (c)
The thermograph of PPy-PVP nanometer plate；(e) physiology for being 200 μ g/mL with the 980nm laser irradiation concentration of different capacity density
The heating curve of LAP-PPy-PVP nanometer plate in salt water；(f) thermal imaging of LAP-PPy-PVP nanometer plate corresponding with figure (e)
Figure；Cell survival rate of the Fig. 5 (a) after the processing for 24 hours of various concentration LAP-PPy-PVP nanometer plate；(b) various concentration LAP- is used
PPy-PVP nanometer plate handles the hemolysis rate situation after 1h；(c) the cellular morphology figure of control group；(d) with 200 μ g/mL LAP-
Cellular morphology figure after the processing for 24 hours of PPy-PVP nanometer plate；(e) living cells/dead cell of corresponding L929 cell is double in figure (c)
Staining reagent result；(f) the double staining reagent results of living cells/dead cell of corresponding L929 cell in figure (d)；
The Uv-Vis-NIR spectrogram of Fig. 6 (a) free Ce6 and Ce6 load front and back LAP-PPy-PVP；(b) LPP/Ce6 with
The Uv-Vis-NIR spectrogram of different durations is irradiated after DPBF mixing under 660nm laser irradiation；
Fig. 7 (a) survival rate of HT29 cell after LAP-PPy-PVP and 980nm laser treatment；(b) control group and with not
With the double staining reagent results of living cells/dead cell of HT29 cell after concentration LAP-PPy-PVP and laser treatment；(c) it uses
LAP-PPy-PVP, dissociate Ce6, irradiates 980nm, 660nm laser and photograph respectively after LAP-PPy-PVP/Ce6 processing HT29 cell
Cell survival rate after penetrating two kinds of laser, wherein control group is with physiological saline treated cell survival rate；(d) control group and
After being handled with LAP-PPy-PVP+980nm laser irradiation, after free Ce6+660nm laser treatment, LAP-PPy-PVP/Ce6 is used
The double staining reagent results of living cells/dead cell of HT29 cell after+980nm laser+660nm laser treatment；Fig. 8 (a)-
(i) the 7th day and 28 days blood routine test results after the Kunming tail vein injection that physiological saline compares；(j)-(l) raw
The 7th day and 28 days biochemical indicator inspection results after the Kunming tail vein injection that reason salt water compares；
7th day and 28 days tissue after the LAP-PPy-PVP that tail vein injection 200 μ L concentration in the Kunming Fig. 9 is 1mg/mL
Pathological section H&E coloration result (physiological saline compares).
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Used each raw material is commercial product in following embodiment.
Embodiment 1
It weighs 210mg hectorite to be added in 30mL distilled water, makes lithium soap with ultrasonic cleaning instrument ultrasonic disperse 30min
Stone is uniformly dispersed, and obtains hectorite dispersion liquid, and then system is placed in the environment of 4 DEG C of ice-water baths and is stirred, to temperature close to 4 DEG C
When, 42 μ L pyrroles are pipetted with liquid-transfering gun and are added in above-mentioned system, are continued stirring 30min and are mixed them thoroughly, addition has prepared
5mL concentration be 0.124g/mL FeCl3Solution continues magnetic agitation under 4 DEG C of ice-water baths and carries out home position polymerization reaction 4h.Instead
After answering, said mixture is centrifuged 3min at 10000rpm, is washed with distilled water and obtains black product after being centrifuged 3 times
Hectorite polypyrrole nano-carrier (LAP-PPy), polypyrrole is in hectorite interlayer in-situ polymerization.
Embodiment 2
It pipettes LAP-PPy (LP) solution that 1.54mL concentration is 6.5mg/mL to be added in 40mL dehydrated alcohol, uses cell powder
After broken machine Ultrasonic Pulverization 30min, 100mg is added and is dissolved in the PVP (molecular weight 1300KDa) in 10mL dehydrated alcohol, surpasses again
Sound crush 30min after, above-mentioned mixed liquor is transferred in 100mL revolving bottle, with Rotary Evaporators under 40 DEG C of water bath conditions with
The rate rotary evaporation 40min of 120rpm removes ethyl alcohol and PVP is made to be coated in the surface of LP in the process, obtains polymer and repairs
The hectorite polypyrrole nano-carrier (LAP-PPy-PVP (LPP) nanometer plate) of decorations, polymer-coated is in hectorite polypyrrole nanometer
The surface of carrier.LAP-PPy obtains good colloidal stability after PVP is modified.Fig. 1 (a)-(c) shows to be dispersed in distillation
In water, in physiological saline, material in 1640 culture mediums after placing 48h hydration kinetics diameter without significant change.From Fig. 1
(d)-(f) as can be seen that Tyndall phenomenon of the material in three kinds of systems it is obvious, all have good colloidal stability.
Embodiment 3
It takes respectively and is scattered in the embodiment 1 in dehydrated alcohol and LAP-PPy and LAP-PPy-PVP in embodiment 2 a little,
It after ultrasonic disperse is uniform, takes 2-3 drop drop on copper mesh respectively, after infrared etc. be baked to, passes through SEM and tem observation respectively
The configuration of surface and microstructure of LAP-PPy and LAP-PPy-PVP.Resulting materials in embodiment 1 can be seen that by Fig. 2 (a)
Layer structure, and interlayer is observed that particulate material, can be seen that the sheet knot of resulting materials in embodiment 2 by Fig. 2 (b)
Structure.
Embodiment 4
The LAP- prepared in embodiment 2 is tested and analyzed using FTIR (670 infrared spectrometer of Nicolet Nexus)
The Nomenclature Composition and Structure of Complexes of PPy-PVP takes a little LAP, the LAP-PPy synthesized in embodiment 1 and the LAP-PPy- prepared in embodiment 2
PVP is mixed with potassium bromide (KBr) powder 1:100 in mass ratio respectively, and the drying to guarantee sample is ground under infrared lamp, to
Grinding is placed on 670 sample holder of Infrared spectrometer of Nicolet Nexus uniformly and after tabletting, tests 4000-400cm-1Range
FTIR spectrogram.It tests and calculates using TGA (TGA-50 thermogravimetric analyzer) and is each in the LAP-PPy-PVP prepared in embodiment 2
The percentage composition of ingredient, takes LAP respectively, the individual PPy (LAP is not added) synthesized under equal conditions in PVP and embodiment 1, real
The LAP-PPy-PVP for applying the LAP-PPy synthesized in example 1 and synthesizing in embodiment 2, is added to the platinum crucible of thermogravimetric analyzer
In, setting experiment parameter carries out TGA test after reading the quality of sample be added, and test condition is in air atmosphere with 15
DEG C/min from room temperature rises to 650 DEG C.
1,2,3 it is found that 3442cm in analysis chart 3 (a)-1Peak at left and right is the stretching vibration of-Si-O- in LAP, furthermore
1016cm-1The peak at place is the characteristic peak of silicate, be can be observed in 1,2,3.1560cm-1And 1165cm-1Distinguish at the peak at place
It is the stretching vibration peak of C=C and C-N, it was demonstrated that the presence of PPy can be observed in 2,3.In addition, in 1675cm-1Place is
The characteristic absorption peak of ketone carbonyl, 1458cm-1And 1258cm-1Place is curved in the C-H deformation vibration and face of pyrrole ring in PVP respectively
Qu Zhendong, these results prove that PVP has successfully been coated on LAP-PPy.The w residual of LAP is 96.28% in Fig. 3 (b),
It is considered as 100%, thinks that it is not weightless in calculating process, PPy and PVP its w residual are respectively 0.44% He
1.37%, it can be ignored, think that it is weightless completely in calculating process.Based on this, the w residual in LAP-PPy is
The content of LAP, therefore the percentage composition that can calculate LAP and PPy in LAP-PPy is respectively 63.02% and 36.98%.Equally,
W residual in LAP-PPy-PVP is the content of LAP, and in LAP-PPy and LAP-PPy-PVP LAP and PPy content ratio
It is constant, therefore the percentage composition of PPy in LAP-PPy-PVP can be calculated, can be calculated its value is 21.27%, LAP-
Remaining content is the content of PVP in PPy-PVP, i.e., and 42.48%.
Embodiment 5
The Photothermal characterisation of LAP-PPy-PVP is tested.With the UV-3600 type UV-Vis-NIR spectrophotometric of Shimadzu company
Meter analyzes its optical absorption characteristics, test scope 400-1100nm.From Fig. 4 (a) it is found that it is 980nm's that wavelength, which can be absorbed, in material
NIR laser, and with the increase of nano material concentration, near infrared absorption intensity is continuously increased.Various concentration (is followed successively by
25,50,100,200 μ g/mL) LAP-PPy-PVP (solvent is physiological saline) be dispersed in the culture hole of 96 porocyte culture plates
In.It is 0.5w/cm with power2The near-infrared laser of 980nm wavelength irradiate the LAP-PPy-PVP or physiology of various concentration respectively
Salt water (control group) is changed with time situation and corresponding by FLIR E60 thermal infrared imager recording materials dispersion liquid temperature
Infrared thermal imaging photo.One of concentration (200 μ g/mL) is then selected, with the laser irradiation LAP-PPy- of different capacity
PVP, by FLIR E60 thermal infrared imager recording materials dispersion liquid temperature change with time situation and it is corresponding it is infrared heat at
As photo.For detect LAP-PPy-PVP photo and thermal stability, with the material of 100 μ g/mL in 0.5W/cm2Laser 5min is irradiated,
Natural cooling 5min carries out 10 circulations by this, records the solution temperature changing value in each period.
From Fig. 4 (c) it is found that near-infrared laser can be absorbed in the LAP-PPy-PVP of various concentration.In the same time,
With the increase of material concentration, the temperature difference is gradually increased, and system constantly reinforces the photothermal conversion ability of near-infrared laser.From Fig. 4
(e) it is found that the material under the irradiation of different laser power density has a near infrared absorption, and with power density and time interval
Increase, the degree that material absorbs energy constantly enhances, and the temperature difference gradually increases.Fig. 4 (d) and infrared thermal imaging photo in (f) are fresher
The photothermal conversion of LAP-PPy-PVP and the relationship of concentration and laser intensity are illustrated brightly.In short, under experimental conditions, this hair
The LAP-PPy-PVP nanometer plate of bright preparation shows good photothermal conversion ability.In addition, Fig. 4 (b) shows material at 10
The lower heating of circulation is held in 30 DEG C or more, i.e. after irradiation is repeated several times too big variation will not occur for material, has good
Good photo and thermal stability.
Embodiment 6
The cell compatibility and blood compatibility of LAP-PPy-PVP is tested.By L929 cell inoculation to 96 orifice plates, it is added
100 μ L1640 cell culture mediums CO at 37 DEG C2Original cell culture fluid is sucked out in overnight incubation in constant incubator, respectively
The fresh medium that 100 μ L contain 0,50,100,150,200 μ g/mLLAP-PPy-PVP is added, each concentration is arranged four groups and puts down
Row test.Continue culture and culture solution is sucked out afterwards for 24 hours, is tested and observed with CCK-8 solution and the double staining reagents of living cells/dead cell
Cell survival calculates cell survival rate with the OD value that SpectraMax i3 microplate reader is surveyed at 450nm, with Leica DM
The form and survival condition of ILLED inverted phase contrast microscope observation L929 cell.As shown in Fig. 5 (a), even concentration is 200 μ
L929 cell survival rate after the LAP-PPy-PVP and cell of g/mL is co-cultured for 24 hours still has 92.4%, shows material substantially without thin
Cellular toxicity.In addition, observing the form of cell by being inverted poor phase microscope, the LAP-PPy-PVP that concentration is 200 μ g/mL is handled
Cell, cell state and staining conditions and control group (being handled with physiological saline) almost indifference (Fig. 5 (c)-(f)),
Being further illustrated in LAP-PPy-PVP within the scope of experimental concentration has good cell compatibility.
Heart pierces through acquisition KM mouse blood under narcosis, is washed red blood cell 3 times with PBS solution, every time in 3000rpm
Lower centrifugation 3min.LAP-PPy-PVP solution (is guaranteed that the final concentration of LAP-PPy-PVP is followed successively by 50,100,150,200 μ g/
ML it) is mixed with blood, the PBS (pH=7.4) of same volume and distilled water is taken to mix (PBS and distilled water difference with blood respectively
As negative and positive control).After above-mentioned six groups of systems are cultivated 1h at 37 DEG C, it is centrifuged 3min under 3000rpm, measures supernatant
Absorbance of the liquid at 541nm simultaneously calculates hemolysis rate (HP).As shown in Fig. 5 (b), handled with various concentration LAP-PPy-PVP
Its hemolysis rate of cell is not 5% hereinafter, observe apparent haemolysis, it was demonstrated that material has good blood compatibility.
Embodiment 7
With the production of singlet oxygen after the ability and load of Uv-Vis-NIR test material load photosensitizer Ce6.It will
The Ce6 that 5mg/mL is dissolved in dimethyl sulfoxide (DMSO) is mixed (volume ratio 1:50) with the LAP-PPy-PVP of 1mg/mL, and 25
After being protected from light stirring for 24 hours at DEG C.It is centrifuged 10min under 12000rpm and removes free Ce6, supernatant, which retains, to be used to measure free Ce6
Content to determine load efficiency.Precipitating is dispersed in distilled water, measurement load front and back LAP-PPy-PVP is in 300-800nm
Uv-Vis-NIR absorption curve in range.Then by the LAP-PPy- of the DPBF (in ethyl alcohol) of 27.23 μ g/mL and 500 μ g/mL
PVP/Ce6 (in distilled water) is protected from light mixing (volume ratio 7:3), measures its Uv-Vis-NIR absorption curve, then uses 660nm,
0.5W/cm2Laser irradiation 5min, 10min, 15min, 20min, 30min after again respectively survey the UV-Vis-NIR curve of spectrum.Such as
Shown in Fig. 6 (a), after load C e6, can obviously observe the characteristic absorption peak of Ce6 at 404nm and 660nm, show Ce6 at
It has been supported on material to function.Fig. 6's (b) the results show that feature of the DPBF at 405nm is inhaled under the laser irradiation of 660nm
It receives peak to be decreased obviously, and with the passage of irradiation time, absorption peak decline gradually increases but absorption peak downward trend is gradually delayed
Slowly.More obvious in the decline of preceding 5min absorption peak, the decline of absorption peak is slower after 10min.
Embodiment 8
The external photo-thermal therapy of LAP-PPy-PVP and external photo-thermal-photodynamics of LAP-PPy-PVP/Ce6, which are combined, to be controlled
It treats.By in HT29 cell inoculation to 96 orifice plates, 100 μ L, 1640 cell culture medium CO at 37 DEG C is added2It is trained in constant incubator
It supports overnight, original cell culture fluid is sucked out, for external photo-thermal therapy, is separately added into 100 μ L concentration and is followed successively by 0,50,100,
The LAP-PPy-PVP of 150,200 μ g/mL continues every hole 0.5W/cm after culture 6h2980nm laser irradiation 5min, shine
It is further cultured for 6h after penetrating, then tested with CCK-8 solution and the double staining reagents of living cells/dead cell and observes cell survival feelings
Condition.For combination therapy, it is separately added into the LAP-PPy-PVP that 100 μ L concentration are 100 μ g/mL, concentration is the free of 8.9 μ g/mL
Ce6 and Ce6 equivalent concentration is the fresh medium of the LAP-PPy-PVP/Ce6 of 8.9 μ g/mL.Continue to be shone after cultivating 6h with laser
Cell in culture plate is penetrated, laser irradiation situation is shown in Table 1,6h is cultivated after irradiation, then with CCK-8 solution and living cells/dead
The double staining reagents of cell are tested and observe cell survival.By Fig. 7 (a) it is found that after 980nm laser irradiation, cell survival
Situation is significantly inhibited, and with the increase of material concentration, cell survival rate is gradually decreased, when material concentration is 200 μ g/
ML, cell survival rate can be down to 14.01%, and as shown in Fig. 7 (b), cell is dyed to red substantially at this time, shows that cell has withered
It dies.Analysis chart 7 (c) with the cell that LAP-PPy-PVP is handled it is found that only have photo-thermal effect almost without photodynamics effect and connection
Therapeutic effect is closed, there was only photodynamics effect with the cell that free Ce6 is handled, and photo-thermal effect and combination therapy effect can neglect
Slightly disregard.And photo-thermal therapy effect had both been shown with the cell that LAP-PPy-PVP/Ce6 is handled, also show photodynamic therapy
Effect, in addition, the cell that 980nm and 660nm irradiate, cell survival rate more individually irradiates some as shown in Fig. 7 (d)
The cell survival rate of laser is lower, shows apparent combination therapy effect.
Embodiment 9
The internal blood compatibility and histocompatbility of LAP-PPy-PVP is evaluated.Kunming mouse is randomly divided into 4 groups (every group 4
Only), 200 μ L PBS (pH=7.4) of control group tail vein injection, 200 μ L concentration of experimental group tail vein injection are 1mg/mL's
LAP-PPy-PVP.After feeding 7 days, 28 days respectively, heart puncturing extracting blood measures every blood parameters.Blood routine evaluation index
Including leucocyte, red blood cell, hemoglobin, hematocrit value, average volume of red blood cells, MC Hgb, red
Mean corpuscular hemoglobin concentration (MCHC), erythrocyte distribution width, platelet content.By Fig. 8 (a)-(i) it is found that each blood of different groups
Though conventional parameter has fluctuation, all in the normal range, it was demonstrated that material has good blood compatibility within the scope of experimental concentration
Property；By Fig. 8 (j)-(l) it is found that the biochemical parameter of each experimental group is close with control group, without obvious physiological maladies and adverse reaction,
These results further demonstrate LAP-PPy-PVP with good blood compatibility.
Histocompatbility is significant for the chronobiological safety of optothermal material in vivo.Kunming mouse is divided at random
For 4 groups (every group 4), control group and experimental group difference 200 μ L PBS (pH=7.4) and LAP-PPy-PVP of tail vein injection are (dense
Degree is 1mg/mL).After feeding 7 days, 28 days respectively, anesthesia is put to death, and the vital tissues such as the heart, liver, spleen, lung, kidney is obtained, with penta 2
Aldehyde is fixed；With hematoxylin-eosin stains, histotomy situation is observed.As shown in Figure 9, compared with the control group, each master of experimental group
It wants organ without apparent tissue damage and lesion, shows that material has good histocompatbility.
Claims (7)
1. a kind of preparation method of hectorite polypyrrole nano-carrier characterized by comprising hectorite is added to distilled water
In, ultrasonic disperse obtains hectorite dispersion liquid, and pyrroles is added, is stirred, and initiator solution is added, and continues stirring and carries out original
Position polymerization reaction obtains hectorite polypyrrole nano-carrier after gained mixture centrifuge washing.
2. the preparation method of hectorite polypyrrole nano-carrier as described in claim 1, which is characterized in that the initiator
For ferric chloride hexahydrate, ammonium persulfate or potassium permanganate, the concentration of initiator solution is 10-150mg/mL, initiator solution with
The volume ratio of distilled water is 1:5-1:10；The concentration of the hectorite dispersion liquid is 1-20mg/mL；The pyrroles and distillation
The volume ratio of water are as follows: 1:500-1:1000；The ultrasonic time is 10-60 minutes；The time that is stirred is 10-60
Minute；The home position polymerization reaction temperature is 0-4 DEG C, and the reaction time is 1-8 hours；The centrifuge speed is 5000-
12000r/min, washing are carried out using distilled water, and washing times are 3-5 times.
3. the method for modifying of hectorite polypyrrole nano-carrier, feature prepared by preparation method described in claim 1 exist
In, comprising: hectorite polypyrrole nano-carrier is added in ethyl alcohol, polymer solution is added after Ultrasonic Pulverization, again ultrasonic powder
After broken, rotary evaporation removes ethyl alcohol, obtains polymer-modified hectorite polypyrrole nano-carrier.
4. the method for modifying of hectorite polypyrrole nano-carrier as claimed in claim 3, which is characterized in that the polymer
For any one in polyvinylpyrrolidone, polyethylene glycol-400, polyglutamic acid and polyvinyl alcohol, the concentration of polymer solution
For 1-25mg/mL, the volume ratio of polymer solution and ethyl alcohol is 1:2-1:10；The Ultrasonic Pulverization time is 10-60 points
Clock；The temperature of the rotary evaporation is 30-50 DEG C, and the time is 10-90 minutes.
5. a kind of hectorite polypyrrole nano-carrier, which is characterized in that include hectorite and polypyrrole, the polypyrrole passes through
It is formed in hectorite interlayer in-situ polymerization.
6. a kind of polymer-modified hectorite polypyrrole nano-carrier, which is characterized in that include polymer and claim 5
Hectorite polypyrrole prepared by the hectorite polypyrrole nano-carrier or preparation method of any of claims 1 or 2 is received
Meter Zai Ti, the polymer-coated is on the surface of hectorite polypyrrole nano-carrier.
7. a kind of optical-thermal conversion material or photosensitizer carrier material, which is characterized in that poly- comprising the hectorite described in claim 5
Hectorite polypyrrole nano-carrier, the lithium prepared by pyrroles's nano-carrier or preparation method of any of claims 1 or 2
Saponite polypyrrole nano-carrier loads photosensitizer by the method for physical absorption.
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| CN107162011A (en) * | 2017-05-04 | 2017-09-15 | 东华大学 | A kind of preparation method for the hectorite nano particle ICG/LAP for loading indocyanine green |
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