CN105148286A - Natural sericin encapsulated mesoporous silicon nano-carrier and preparation method and application thereof - Google Patents
Natural sericin encapsulated mesoporous silicon nano-carrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a natural sericin encapsulated mesoporous silicon nano-carrier and a preparation method and application thereof to the field of tumor treatment. The preparation method comprises the steps of preparing pure sericin solids; preparing mesoporous silicon nano-particles; preparing aminated, formylated or dual-sulfurated mesoporous silicon nano-particles with regular pore passage; preparing drug-loaded aminated, formylated or dual-sulfurated mesoporous silicon nano-particles; preparing a pH/protease or reduction/protease dual-responsiveness sericin/mesoporous silicon composite multifunctional control system. The natural sericin encapsulated mesoporous silicon nano-carrier and the preparation method and the application thereof have the advantages that the operation is simple to perform, the raw materials are simple and convenient to obtain, the cost is low, the universal applicability is high, no special complex equipment is needed and the like. The nano composite system prepared by adopting the method fully utilizes the characteristics of the sericin on the outer layer and the mesoporous silicon nano-particles on the inner layer, has pH/protease or reduction/protease dual responsiveness, has multiple functions of inhibiting tumor cells, monitoring nano-particle positioning and drug release in real time and the like, is high is drug loading capacity, is good in biocompatibility and has a wide application prospect in the field of tumor diagnosis and treatment, especially in the aspect of solving multi-drug resistance of tumors.
Description
Technical field
The present invention relates to bio-medical composition field, particularly the nanometer pharmaceutical carrier and preparation method thereof of multiple natural silk glue protein parcel and the application at therapeutic field of tumor.
Background technology
Along with the develop rapidly of nanoscale science and technology, various organic or inorganic material is made into nanostructured for biomedical sector, especially utilizes nano material to obtain for disease treatment as pharmaceutical carrier and pays close attention to more and more widely.Mesoporous silicon dioxide nano particle (Mesoporoussilicananoparticles, MSN) as a kind of solid material with cellular structures, there is much excellent architectural feature, as great specific surface area and pore volume, the hole of homogeneous controlled mesopore size, stable physicochemical property, easy surfaces externally and internally is modified and good biocompatibility.Therefore, large quantifier elimination is devoted to utilize nanometer particle to be used for some guest molecules of load to reach the object of disease treatment as main body.
In order to realize " zero release " of medicine before arriving target site, make drug-loading system have environmental stimulus response simultaneously, allow nanoparticle more can reach medicine controlled releasing in target site enrichment, researcheres are in mesoporous silicon surface design various " valve ", by some inorganic nano-particles, polymer or biomacromolecule are incorporated into mesoporous silicon surface by physical action or some special chemical bonded refractories, it is made to possess pH response, enzyme response, thermal response, reduction response, photoresponse etc., even multiple response characteristic.Further, in order to realize the multifunction of controlled release system, many researcheres pass through load or some magnetic nano-particles of finishing, quantum dot or fluorophor, thus the multifunctional intellectual controlled release system constructing diagnosis and treatment integration.But most " valve " design process is loaded down with trivial details, and single " valve " design is often difficult to the object reaching multiple response.In addition, some " valve " raw material ratio, as metallic nanoparticle, due to not biodegradable, be present in body and may produce short-term or long term toxicity, hinder the process be really applied to by material in human body.
Sericin (SilkSericin) is a kind of natural macromolecular viscous protein being wrapped in fibroin fiber top layer, account for the 20%-30% of Bombyx bombycis content, the polypeptide being 24-400kDa by molecular weight forms, and its molecule is made up of 18 seed amino acids such as serine, aspartic acid and glycine.The deficiency be familiar with sericin due to people for a long time and the limitation of research, cause sericin to be taken as refuse process in filature industry, waste a large amount of valuable natural resources.It is found that sericin has moisturizing, antibacterial, antioxidation, anticoagulation and promotes the biological nature such as cell adhesion and propagation in recent years, meanwhile, sericin has hydrophilic and degradability, may be used for biomedical materials field.At present, had research team by sericin with other macromolecular materials as gelatin, polyvinyl alcohol, hydroxy methocel, polyacrylamide etc. simply mix, or directly utilize pure silk glue protein to be prepared into thin film, biological support etc. and be applied to field of tissue engineering technology, show good application prospect.But use sericin parcel mesoporous silicon to prepare drug delivery system without any report at present.
The present invention adopts high-temperature alkali lifting manipulation successfully to extract sericin in Bombyx bombycis, by electrostatic interaction or utilize chemical bond (imine linkage or cystine linkage etc.) connect first sericin is wrapped in meso-porous titanium dioxide silicon face, successfully prepare and there is good biocompatibility, comparatively high drug load, have pH concurrently, the multiple response such as enzyme and reduction, and there is the multifunctional intellectual control delivery of fluorescent characteristic, can be used for antineoplaston, particularly create good fragmentation effect for resistant tumors cell, and monitor in real time by fluorescence equipment, the object of diagnosis and treatment integration can be reached.
Summary of the invention
Problem to be solved by this invention is to provide nanometer pharmaceutical carrier of multiple natural silk glue protein parcel and its preparation method and application, the nanometer carrier of the natural silk glue protein parcel that the method prepares has good biocompatibility and higher drug loading, and sericin itself has cell adhesion, can be engulfed by tumor cell well, and discharged by the reproducibility trigger drug of lysosomal pH/ Protease Environment in cell or born of the same parents' glutathion inside, to reach the object of killing tumor cells.Meanwhile, the fluorescent characteristic due to sericin makes nano-carrier itself with yellow-green fluorescence, can monitor the location of nanoparticle and the release of medicine in real time, reach the object of diagnosis and treatment integration.
The present invention is intended to utilize sericin to encapsulate medicine carrying mesoporous silicon dioxide nano particle as " valve " and constructs the multifunctional and composite type controlled drug delivery system having the multiple response such as pH/ protease or reduction/protease concurrently.Experimental provision required for the present invention is simple, and operating process is easy, with low cost.The sericin prepared by this method/mesoporous silicon composite nano materials is utilized to have good biocompatibility and higher drug loading, and sericin itself has cell adhesion, can be engulfed by tumor cell well, and discharged by the reducing property trigger drug of lysosomal pH/ Protease Environment in cell or born of the same parents' glutathion inside, to reach the object of killing tumor cells.Meanwhile, the fluorescent characteristic due to sericin makes nano-carrier itself with yellow-green fluorescence, can monitor the location of nanoparticle and the release of medicine in real time, reach the object of diagnosis and treatment integration.
For achieving the above object, the preparation method of sericin provided by the invention/mesoporous silicon composite multi-functional controlled drug delivery systems, comprises the following steps:
One, the pH/ protease double-bang firecracker sericin/mesoporous silicon composite multi-functional hierarchy of control of answering
(1) take Bombyx bombycis, extract sericin by high-temperature alkali lifting manipulation, then pure silk glue protein solid can be obtained through dialysis, lyophilizing;
(2) collosol and gel and surfactants' templating is utilized to prepare nanometer granule;
(3) by gained nanometer Granular composite in step (2) in solvent, add 3-aminopropyl triethoxysilane, obtain the nanometer granule of surface amination;
(4) mixed liquor that the amidized nanometer granule in step (3) is placed in methanol/hydrochloric acid is refluxed, after eluted template, namely obtain the amination nanometer granule with regular pore canal;
(5) by the amination nanometer particle in step (4) and glutaraldehyde hybrid reaction, the nanometer particle of aldehyde radical is obtained;
(6) the aldehyde radical nanometer particle of gained in the amination nanometer granule of gained in step (4) or step (5) and medicament mixed are reacted, obtain amination or the aldehyde radical nanometer granule of carrying medicament;
(7) reacted by the amino on aldehyde radical surperficial in nanometer grain and sericin, or directly utilize electrostatic interaction that sericin is wrapped in nanometer particle surface, namely obtain sericin/mesoporous silicon composite multi-functional controlled drug delivery systems that pH/ protease double-bang firecracker is answered.
The Bombyx bombycis selected in described step (1) be normal silkworm Bombyx bombycis kind (white jade etc.), bombyx mori silk fibroin deletion form mutating variety (
185Nd-s, 140Nd-s, 139Nd-sdeng) or the one of Antherea pernyi Guerin-Meneville Bombyx bombycis, purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences.
Further, the detailed process extracting sericin comprises:
1) take appropriate Bombyx bombycis, be cut into 1cm
2the fragment of left and right;
2) be immersed in 0.01-0.05M sodium carbonate by the Bombyx bombycis after shredding, 100 degrees Celsius dissolve 30-60 minute;
3) the sericin liquid after dissolving is transferred in the bag filter of 3500Da, puts into distilled water dialysis 48 hours;
4) be transferred in 50mL centrifuge tube by the solution in bag filter, collected after centrifugation supernatant, can obtain pulverous sericin after being placed in the lyophilizing of freeze-drying machine, being placed in-20 degrees Celsius can preserve for a long time.
The nanometer particle of described step (2) is using cetyl trimethyl ammonium bromide as template, and tetraethyl orthosilicate is as organosilicon precursor, and sodium hydroxide is as obtained by base catalyst.Particularly, cetyl trimethyl ammonium bromide is scattered in (mass volume ratio is 1g:480mL) in distilled water, add sodium hydroxide (28% of template quality) again, heated and stirred to 80 degree Celsius, slowly dropwise add tetraethyl orthosilicate (5 times of template quality), after Keep agitation 2-3 hour, obtain white nanometer particle dirty solution, centrifugal collecting precipitation, use vacuum freeze drier lyophilizing after methanol and distilled water cleaning, the nanometer particle of white powder with template can be obtained.
3-aminopropyl triethoxysilane is selected to realize the amination of nanometer particle in described step (3), detailed process refers to and is scattered in methanol by the nanometer particle of step (2) gained according to mass volume ratio 1g:75mL, then 3-aminopropyl triethoxysilane is added, stirring at room temperature is after 24 hours, and namely collected by centrifugation obtains amidized nanometer particle.The volume ratio of solvent methanol used in this step and 3-aminopropyl triethoxysilane is 75:2.
Select the template cetyl trimethyl ammonium bromide in step (3) products therefrom described in methanol/concentrated hydrochloric acid mixed solution eluting in described step (4), obtain the amination nanometer particle with regular pore canal.Particularly, be by the amination nanometer particle dispersion of gained in step (3) in methanol/concentrated hydrochloric acid (volume ratio is 16:1) mixed liquor, after refluxing 48 hours in 80 degrees Celsius, can effective eluted template cetyl trimethyl ammonium bromide, then centrifugally can obtain the amination nanometer granule with regular pore canal.
Glutaraldehyde is selected to realize the aldehyde radical of nanometer particle in described step (5).Particularly, glutaraldehyde water solution concentration used is 2.5wt%, and the amination nanometer particle selected and the mass volume ratio of 2.5% glutaraldehyde water solution are 100mg:10mL.Further, by amination nanometer particle dispersion in glutaraldehyde water solution, room temperature lucifuge stirs 24 hours, then collected by centrifugation product, then with after distilled water cleaning repeatedly, can obtain the nanometer granule of aldehyde radical.
Medicine selected in described step (6) can be one or more of tumor Drugs (example hydrochloric acid amycin, cisplatin, paclitaxel, camptothecine etc.).Further, the medicine (as hormone, antibiotic etc.) of other effects is equally applicable to the present invention.Preferably, appropriate hydrochloric acid amycin is dissolved in PBS(phosphate buffer by this step, pH7.4) in, then add amination or aldehyde radical nanometer granule, room temperature lucifuge stirs 24-48 hour.
Described step (7) is with sericin encapsulating nanometer granule.Detailed process adds in the mixed liquor of step (6) by sericin aqueous solution (5-10mg/mL), lucifuge 35 degrees Celsius reaction 6-7 hour; Then collected by centrifugation product; Further, rinse product repeatedly with distilled water, be then placed in frozen vacuum dryer lyophilizing and can obtain the compound drug-loading nanoparticles of pulverous sericin/mesoporous silicon, the mass ratio of nanometer grain and sericin is 1:0.5 – 1:2.
Two, the sericin/mesoporous silicon composite multi-functional hierarchy of control of reduction/protease double responsiveness
(1) the nanometer grain containing template is scattered in solvent, adds mercaptopropyl trimethoxysilane, be obtained by reacting the nanometer granule of sulfhydrylation;
(2) by the nanometer Granular composite of the sulfhydrylation in step (1) in solvent, add (2-amino second sulfydryl)-2-mercaptopyridine hybrid reaction, obtain the nanometer granule of two sulfuration;
(3) mixed liquor that the two sulfuration nanometer granules in step (2) are placed in methanol/hydrochloric acid is refluxed, after eluted template, namely obtain two sulfuration nanometer granules with regular pore canal;
(4) by the nanometer granule of the two sulfurations in step (3) and medicament mixed, two sulfuration nanometer granules of carrying medicament can be obtained;
(5) by the carboxyl reaction on the amino on mesoporous silicon surface and sericin, sericin is wrapped in nanometer grain surface, sericin/mesoporous silicon composite multi-functional controlled drug delivery systems that reduction/protease double-bang firecracker is answered can be obtained.
Mercaptopropyl trimethoxysilane is selected to realize the sulfhydrylation of nanometer particle in described step (1), detailed process is scattered in methanol by the nanometer particle (manufacture method is consistent with the process in sericin/mesoporous silicon composite nano grain that synthesis pH/ protease double-bang firecracker is answered) containing surfactant templates according to mass volume ratio 1g:83mL, then mercaptopropyl trimethoxysilane is added, stirring at room temperature, after 24 hours, obtains the nanometer particle of sulfhydrylation after collected by centrifugation, cleaning.Solvent methanol used in this step and the volume ratio of mercaptopropyl trimethoxysilane are 36:1.
(the amino second sulfydryl of 2-)-2-mercaptopyridine (SATH) is used to realize two sulfurations of nanometer grain in described step (2).The detailed process of synthesis (2-amino second sulfydryl)-2-mercaptopyridine comprises: 1) by 2,2 '-two sulfur two pyridinium dissolution in the mixed solution of methanol/glacial acetic acid (volume ratio 5:0.2); 2) Mercaptamine is dissolved in (mass volume ratio is 1.14g:10mL) in methanol, dropwise adds step 1) in mixed liquor in, room temperature reaction 48 hours; 3) after completion of the reaction, rotary evaporation falls methanol; 4) after the product absolute ether cleaning obtained twice, be again dissolved in methanol, then add absolute ether precipitation, can white solid product be obtained, be placed in-20 degrees Celsius of refrigerator overnight; 5) take out product next day, inhale after abandoning yellow supernatant, after repeating precipitation 5 times, be placed in vacuum pump drying, Powdered (the amino second sulfydryl of 2-)-2-mercaptopyridine can be obtained.
In described step (3), the mode of eluted template is consistent with the process of eluted template in sericin/mesoporous silicon composite nano grain that synthesis pH/ protease double-bang firecracker is answered.
Medicine selected in described step (4) can be one or more of tumor Drugs (example hydrochloric acid amycin, cisplatin, paclitaxel, camptothecine etc.).Further, the medicine (as hormone, antibiotic etc.) of other effects is equally applicable to the present invention.Medicine lift-launch mode is consistent with the mode that sericin/mesoporous silicon composite nano grain Chinese medicine carries that synthesis pH/ protease double-bang firecracker is answered.
Described step (5) is sealed nanometer granule with sericin.The Bombyx bombycis selected be normal silkworm Bombyx bombycis kind (white jade etc.), bombyx mori silk fibroin deletion form mutating variety (
185Nd-s, 140Nd-s, 139Nd-sdeng) or the one of Antherea pernyi Guerin-Meneville Bombyx bombycis, purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences.Sericin extracting method is consistent with the mode that sericin in sericin/mesoporous silicon composite nano grain that synthesis pH/ protease double-bang firecracker is answered extracts.Sericin encapsulation process specifically comprises: 1) be dissolved in appropriate distilled water by sericin solid and be prepared into sericin aqueous solution (5-10mg/mL); 2) take respectively and add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) in right amount, N-hydroxysuccinimide (NHS) (is respectively 6:1 with the mass ratio of sericin, 3:1), add in sericin aqueous solution successively, room temperature reaction 2-3 hour; 3) reacted solution is added in the mixed solution of step (4), lucifuge 35 degrees Celsius reaction 6-24 hour; Then collected by centrifugation product; Further, rinse product repeatedly with distilled water, be then placed in frozen vacuum dryer lyophilizing and can obtain the compound drug-loading nanoparticles of pulverous sericin/mesoporous silicon.The mass ratio of nanometer grain and sericin is 2:1.
The invention provides the preparation method of the multifunctional and composite type controlled drug delivery systems of several multiple response.
Beneficial effect of the present invention is:
(1) the method is simple to operate, and raw material acquisition pattern is easy, with low cost, is generally suitable for, without the need to Special complex equipment etc.
(2) nano composite system prepared by the method is used, take full advantage of the characteristic of outer sericin and internal layer nanometer particle, there is pH/ protease or reduction/protease dual responsiveness, possesses inhibition tumor cell, the multi-functionals such as Real-Time Monitoring nanoparticle location and drug release, and drug loading is high, good biocompatibility, particularly possesses wide application prospect in tumor diagnosis and treatment field in the multi-drug resistant solving tumor.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope shape appearance figure of sericin/mesoporous silicon composite nano granule;
Fig. 2 is the thermogravimetric weight loss curve chart of different nanoparticle;
Fig. 3 is fluorescence spectrum figure and the fluorescent microscopy images of sericin/mesoporous silicon composite nano granule;
Fig. 4 is that the sericin/mesoporous silicon Nanocomposite Particles of zero load is on the impact of tumor cell activity;
Fig. 5 metamorphosis that to be sericin/mesoporous silicon composite nano granule from the interaction of the macrophage that mice is originated: A be after giving the different process of macrophage; B is the expression giving inflammatory factor tumor necrosis factor (TNF-α) and interleukin-11 (IL-1 β) after the different process of macrophage;
Fig. 6 is that the pH/ protease double-bang firecracker of the compound controlled drug delivery systems of sericin/mesoporous silicon answers drug release patterns figure;
Fig. 7 is the pH/ protease double-bang firecracker of the load amycin compound medicine-carried system of sericin/mesoporous silicon of answering and free amycin comparing the impact of drug susceptibility-types and multidrug resistant type tumor cell activity.
Fig. 8 is the reduction/protease double-bang firecracker of the load amycin compound medicine-carried system of sericin/mesoporous silicon of answering and free amycin comparing the impact of multidrug resistant type tumor cell activity.
Detailed description of the invention
Embodiment 1: the Euplotes woodruffi that utilizes provided by the invention prepares sericin/mesoporous silicon composite nano granule.
1, the selection of Bombyx bombycis:
The present invention preferentially selects the Bombyx bombycis of bombyx mori silk fibroin deletion mutation kind, and (be purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, this bombyx mori silk fibroin deletion mutation kind is stored in state of Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm resource conservation center, deposit number
185Nd-s) be raw material, main chemical composition is sericin.
2, the extraction of sericin:
(1) take 2g bombyx mori silk fibroin deletion form mutating variety Bombyx bombycis, be cut into 1cm
2the fragment of left and right;
(2) be immersed in 40mL0.02M sodium carbonate by the Bombyx bombycis after shredding, 100 degrees Celsius dissolve 30 minutes;
(3) the sericin solution after dissolving is transferred in the bag filter of 3500Da, puts into distilled water dialysis 48 hours;
(4) solution in bag filter is transferred in 50mL centrifuge tube, collected after centrifugation supernatant, solid-state sericin can be obtained after being placed in the lyophilizing of freeze-drying machine, be stored in-20 degrees Celsius of refrigerators.
The preparation of 3, sericin/mesoporous silicon composite nano granule:
(1) 1.0g cetyl trimethyl ammonium bromide is scattered in 480mL distilled water, add 0.28g sodium hydroxide, heated and stirred to 80 degree Celsius, slowly dropwise add the positive silane ethyl ester of 5.0g, Keep agitation, after 2 hours, obtains white nanometer particle dirty solution, centrifugal collecting precipitation, methanol and distilled water clean repeatedly, and vacuum freeze drier lyophilizing, namely obtains the nanometer particle that white powder contains template.
(2) be scattered in 75mL methanol by the nanometer particle (1g) of step (1) gained, then add 2mL3-aminopropyl triethoxysilane, stirring at room temperature is after 24 hours, and namely collected by centrifugation obtains amidized nanometer particle.
(3) by the amination nanometer particle dispersion of step (2) gained in 170mL methanol/concentrated hydrochloric acid (volume ratio is 16:1) mixed liquor, after refluxing 48 hours in 80 degrees Celsius, then centrifugally can obtain the amination nanometer granule with regular pore canal, lyophilizing after methanol and distilled water cleaning repeatedly.
(4) the amination nanometer granule (200mg) of step (3) gained is scattered in 2.5% glutaraldehyde water solution (20mL), room temperature lucifuge stirs 24 hours, then collected by centrifugation product, then with after distilled water cleaning repeatedly, can obtain the nanometer granule of aldehyde radical.
(5) the aldehyde radical nanometer granule of step (4) gained is scattered in 400mL distilled water again, add 20mL sericin aqueous solution (5mg/mL), after 35 degrees Celsius of lucifuges stir 6 hours, centrifugal collecting precipitation, after distilled water cleaning repeatedly, the lyophilizing of freezing vacuum pump, can obtain pulverous sericin/mesoporous silicon composite nanometer particle.As shown in Figure 1: the sericin synthesized by the method/mesoporous silicon composite nano grain diameter is about 200nm.As shown in Figure 2: when temperature is increased to 770 degrees Celsius, amination nanometer grain, the loss in weight of aldehyde radical nanometer grain and sericin/nanometer grain is respectively 14.18%, 26.75% and 42.07%, describes sericin success and is modified on nanometer grain.
Experimental example 1: the biocompatibility of research sericin/mesoporous silicon composite nano grain.
1, after the sericin of zero load/mesoporous silicon Nanocomposite Particles and kinds of tumor cells being hatched 48 hours altogether, cell viability is detected, as shown in Figure 4: when compound system does not carry any medicine, the vigor of cell is not significantly affected.
2, after the macrophage (Raw264.7cells) that the sericin of zero load/mesoporous silicon Nanocomposite Particles and mice are originated being hatched 24 hours altogether, with phalloidin and DAPI respectively transfect cell skeleton and nucleus with the form of observation of cell, and detect the expression of macrophage inflammatory gene (TNF-α, IL-1 β).As shown in Figure 5A: when macrophage and after sericin/mesoporous silicon composite nano grain hatches 24 hours altogether, the form of macrophage with and blank cultures (negative control) cellular morphology of hatching after 24 hours similar, illustrate that sericin/mesoporous silicon composite nano grain does not have activating macrophage, and lipopolysaccharide stimulates rear macrophage to occur obvious polarization, define a large amount of filopodias; As one man, as shown in Figure 5 B, sericin/mesoporous silicon composite nano grain does not promote the expression of macrophage inflammatory gene yet, illustrates that sericin/mesoporous silicon composite nano grain does not demonstrate immunogenicity in studying in vitro.
Experimental example 2: the fluorescent characteristic detecting sericin/mesoporous silicon composite nano granule.
1, detect the fluorescence spectrum of sericin/mesoporous silicon composite nano granule with fluorescence spectrum tester, as shown in Figure 3A: under the exciting of suitable excitation wavelength, the utilizing emitted light of wavelength at about 550nm can be produced.
2, after sericin/mesoporous silicon composite particle and RAW264.7 macrophage being hatched 24 hours altogether, with the fluorescence of fluorescence microscope material, as shown in Figure 3 B: under blue light source excites, material can send green fluorescence.
Experimental example 3: the pH/ protease double responsiveness drug release behavior of the composite drug-loaded system of research sericin/nanometer.
1, the compound controlled drug delivery systems of sericin/mesoporous silicon that the pH/ protease double-bang firecracker preparing load antitumor drug is answered.
(1) this research uses broad-spectrum anti-cancer drug doxorubicin hydrochloride.First 100mg doxorubicin hydrochloride is dissolved in 200mLPBS(pH7.4) in, then add 100mg aldehyde radical nanometer granule, room temperature lucifuge stirs 24 hours.
(2) the sericin aqueous solution (5mg/mL) of 10mL is added in the mixed liquor of step (1), lucifuge 35 degrees Celsius reaction is after 6 hours, collected by centrifugation product, products therefrom close to colourless to supernatant, be then placed in frozen vacuum dryer lyophilizing and can obtain the compound drug-loading nanoparticles of pulverous sericin/mesoporous silicon by distilled water flushing product.
2, the drug loading of the compound drug-loading nanoparticles of sericin/mesoporous silicon of above-mentioned steps gained is detected.
The compound drug-loading nanoparticles of 1mL sericin/mesoporous silicon (1.25mg) is scattered in 4mL Fluohydric acid., after thoroughly dissolving, by liquid diluting 4 times, measure the ultraviolet absorptivity (0.34871) of liquid at 480nm, bent according to standard again, line is calculated the doxorubicin concentration of gained liquid is 0.018193mg/mL; Drug loading is 29.1%, and namely sericin/nanometer the grain of 100mg load can be about the doxorubicin hydrochloride of 41mg.
3, the sericin/drug release profiles of mesoporous silicon composite nano medicine-carried system under condition of different pH is detected.
The sericin of 2mg load amycin/nanometer grain is divided in 4 2mLEP pipes (0.5mg often manages), add respectively 1.5mLpH5.0,6.5,7.4 and containing papain pH5.0 buffer in, lucifuge is placed in 37 degrees Celsius and rocks and hatch, supernatant is collected at the time point of setting, the light absorption value at 480nm place is measured with ultraviolet spectrophotometer, the amycin amount discharged is calculated according to standard curve, calculate the ratio that the dose discharged accounts for initial drug loading again, draw release profiles.As shown in Figure 6: after 72 hours, under the environment of pH5.0, have the drug release of 53.85% out, after addition of enzymes, there is the medicine of 81.56% to discharge from system, confirm that this medicine-releasing system is to pH and protease Dual Sensitive.
Experimental example 4: the compound medicine-carried system of sericin/mesoporous silicon of research load amycin is for the lethal effect of drug susceptibility-types tumor cell and multidrug resistant type tumor cell.
By the compound medicine-carried nano particles of sericin/mesoporous silicon of load amycin and drug susceptibility-types human liver cancer cell (HepG2cells), human cervical carcinoma cell (HeLacells), human breast cancer cell (MCF-7cells), and multidrug resistant type human breast cancer cell (MCF-7/ADRcells) hatches 48 hours altogether, detects cell viability with mtt assay.As shown in Figure 7: by drug susceptibility-types tumor cell with after free doxorubicin hydrochloride process, death has appearred in most tumor cell; Similarly, sericin/mesoporous silicon/doxorubicin hydrochloride composite particles also can inducing death of neoplastic cells effectively, but when low concentration, free hydrochloric acid amycin is weaker than to the lethal effect of tumor cell, because doxorubicin hydrochloride is micromolecular water soluble drug, is easily absorbed by tumor cell by diffusion way and play Graft Versus Tumor in time.And on the contrary, after by multidrug resistant type breast cancer cell and the process of free hydrochloric acid amycin, cell viability is apparently higher than with the tumor cell after sericin/mesoporous silicon/doxorubicin hydrochloride composite particles process, because there is a large amount of drug efflux pumps in multidrug resistant type tumor cell, after free amycin permeate through cell membranes enters Cytoplasm, be pumped out extracellular soon, thus tumor-killing power obviously declines; And sericin/mesoporous silicon/doxorubicin hydrochloride composite particles enters in cell by endosome-lysosomal pathway, avoid the effect of drug efflux pump dramatically, " valve " can be rapidly opened under lysosome environment simultaneously, make medicine be released in a large amount in an early stage out and diffuse to nucleus, thus in multidrug resistant type tumor cell, play stronger cell killing effect.
Embodiment 2: the compound controlled drug delivery system of sericin/mesoporous silicon utilizing cystine linkage to prepare reduction/enzyme double responsiveness provided by the invention.
1, in the present embodiment, the Bombyx bombycis kind preferentially selected is that the Bombyx bombycis of bombyx mori silk fibroin deletion mutation kind (is purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, this bombyx mori silk fibroin deletion mutation kind is stored in state of Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm resource conservation center, deposit number
140Nd-s); The extracting method of sericin is identical with embodiment 1.
2, the synthesis of sulfhydrylation nanometer particle is substantially identical with the synthetic method of nanometer particle amidized in embodiment 1, difference is to change 3-aminopropyl triethoxysilane into mercapto propyl trimethoxy silicon, and the volume added is 1/36 of solvent methanol.
3, the synthesis of the nanometer particle of two sulfuration:
(1) synthesis of (the amino second sulfydryl of 2-)-2-mercaptopyridine: 1) by 4.41g2,2 '-two sulfur two pyridinium dissolution is in the mixed solution of methanol (20mL)/glacial acetic acid (0.8mL); 2) by the mixed liquor in 1.14g Mercaptamine (M=113 is dissolved in 10mL methanol, dropwise adds 1), stirring at room temperature 48 hours; 3), after completion of the reaction, after rotary evaporation of solvent, yellow grease sample material is obtained; 4) after the product absolute ether (50mL) obtained cleans twice, be again dissolved in 10mL methanol, then add in 200mL absolute ether and precipitate, can white solid product be obtained; 5) stirring is placed on-20 degrees Celsius of refrigerator overnight in 10 minutes; 6) take out product next day, inhale after abandoning yellow supernatant, obtain yellow crystalline product, after repeating to precipitate 2-3 time, be placed in vacuum pump drying, Powdered (the amino second sulfydryl of 2-)-2-mercaptopyridine can be obtained.
(2) by the nanometer particle dispersion of 200mg sulfhydrylation in the methanol of 30mL, add 200mgSATH, stirring at room temperature is after 24 hours, collected by centrifugation product, with lyophilizing after methanol and distilled water cleaning for several times;
(3) eluting surface activating agent template, method is identical with the process in embodiment 1.
4, the compound controlled drug delivery systems of sericin/mesoporous silicon that the reduction/enzyme double-bang firecracker preparing load antitumor drug is answered: the antitumor drug that the present embodiment is selected is doxorubicin hydrochloride, material preparation method is substantially identical with the method preparing the compound controlled drug delivery systems of sericin/mesoporous silicon that pH/ protease double-bang firecracker is answered in embodiment 1 experimental example 3, and difference is: (1) amycin consumption reduces; (2) sericin used is numbering
140Nd-sbombyx mori silk fibroin deletion mutation kind Bombyx bombycis extract gained; (3) the nanometer granule used is the nanometer granule of two sulfurations with regular pore canal; (4) sericin first activates before wrapping up the nanometer granule of the two sulfuration of medicine carrying, detailed process comprises: 1) 10mg doxorubicin hydrochloride is dissolved in 20mLPBS(pH7.4) in, add the nanometer granule of the two sulfuration of 20mg, lucifuge room temperature reaction 24 hours; 2) 10mg sericin is dissolved in 2mL distilled water, adds 0.06gEDC successively, 30mgNHS, room temperature reaction 3 hours; 3) by step 2) the solution of gained add in the reactant liquor of step 1), after room temperature lucifuge reacts 7 hours, collected by centrifugation product, products therefrom close to colourless to supernatant, be then placed in frozen vacuum dryer lyophilizing and can obtain the compound drug-loading nanoparticles of pulverous sericin/mesoporous silicon by distilled water flushing product.
Experimental example 1: the drug loading detecting the compound drug-loading nanoparticles of sericin/mesoporous silicon of above-mentioned steps gained.
The detection method of this experimental example drug loading is identical with the detection method of experimental example 3 in embodiment 1, and the drug loading calculating gained is 0.14, and namely 100mg sericin/nanometer granule can load 16.28mg doxorubicin hydrochloride.
Experimental example 2: the lethal effect of the compound medicine-carried system of sericin/mesoporous silicon for multidrug resistant type tumor cell studying load amycin
The compound medicine-carried nano particles of sericin/mesoporous silicon of load amycin and multidrug resistant type human breast cancer cell (MCF-7/ADRcells) are hatched 48 hours altogether, detects cell viability with mtt assay.As shown in Figure 8: the fragmentation effect that the compound drug-loading nanoparticles of sericin/mesoporous silicon produces multidrug resistant type human breast cancer cell line Bcap-37/ADRcells, illustrate that nano-particle can be engulfed by tumor cell, and by intracellular environment by drug release out, thus create cell killing effect.
Claims (7)
1. the preparation method of the nanometer carrier of natural silk glue protein parcel, the nanometer carrier of described natural silk glue protein parcel be natural silk glue protein parcel there is the nanometer composite multi-functional controlled drug delivery systems that pH/ protease double-bang firecracker answers, its preparation method comprises the following steps:
(1) take Bombyx bombycis, extract sericin by high-temperature alkali lifting manipulation, more namely obtain pure silk glue protein solid through dialysis, lyophilizing;
(2) collosol and gel and surfactants' templating is utilized to prepare nanometer granule;
(3) by gained nanometer Granular composite in step (2) in methanol, add 3-aminopropyl triethoxysilane, be obtained by reacting the nanometer granule of surface amination;
(4) reflux the mixed liquor that the amidized nanometer granule in step (3) is placed in methanol and hydrochloric acid eluted template, centrifugally after eluted template namely obtains the amination nanometer particle with regular pore canal;
(5) by the amination nanometer particle in step (4) and glutaraldehyde water solution hybrid reaction, the nanometer particle of aldehyde radical is obtained;
(6) the aldehyde radical nanometer particle of gained in the amination nanometer granule of gained in step (4) or step (5) and medicament mixed are reacted, obtain amination or the aldehyde radical nanometer granule of carrying medicament;
(7) reacted by the amino on the aldehyde radical on surface in the aldehyde radical nanometer granule nanometer grain of carrying medicament and the pure silk glue protein that obtains of step (1), or directly utilize electrostatic interaction sericin to be wrapped in amination nanometer particle surface, namely obtain the nanometer silicon composite multi-functional controlled drug delivery systems with pH/ protease double-response of sericin parcel.
2. preparation method according to claim 1, is characterized in that: in described step (5), glutaraldehyde water solution mass concentration is 2.5%, and the consumption of amination nanometer particle and glutaraldehyde water solution is 100mg:10mL; Described amination nanometer particle and glutaraldehyde water solution hybrid reaction refer to that room temperature lucifuge stirs collected by centrifugation product after 24 hours, then repeatedly clean with a large amount of distilled water.
3. preparation method according to claim 1, it is characterized in that: reacted by the amino on the aldehyde radical on surface in nanometer grain and pure silk glue protein in described step (7) and refer to, pure silk glue protein solid step (1) obtained adds water and is prepared into 5-10mg/mL sericin aqueous solution, in amination sericin aqueous solution being added the carrying medicament of step (6) gained or aldehyde radical nanometer granule, lucifuge 35 degrees Celsius stirs 6-7 hour; Then collected by centrifugation product; Then rinse product with distilled water, be placed in frozen vacuum dryer lyophilizing and namely obtain the compound drug-loading nanoparticles of pulverous sericin/mesoporous silicon, the mass ratio of nanometer grain and pure silk glue protein is 1:0.5 – 1:2.
4. the preparation method of the nanometer carrier of natural silk glue protein parcel, the nanometer carrier of described natural silk glue protein parcel be natural silk glue protein parcel there is the nanometer silicon composite multi-functional controlled drug delivery systems that reduction/protease double-bang firecracker answers, its preparation method comprises the following steps:
(1) take Bombyx bombycis, extract sericin by high-temperature alkali lifting manipulation, more namely obtain pure silk glue protein solid through dialysis, lyophilizing;
(2) collosol and gel and surfactants' templating is utilized to prepare nanometer granule;
(3) by gained nanometer Granular composite in step (2) in solvent, add mercaptopropyl trimethoxysilane, namely obtain the nanometer granule of sulfhydrylation;
(4) by the nanometer Granular composite of the sulfhydrylation in step (3) in solvent, add (2-amino second sulfydryl)-2-mercaptopyridine hybrid reaction, obtain the nanometer granule of two sulfuration;
(5) mixed liquor that the two sulfuration nanometer granules in step (4) are placed in methanol and hydrochloric acid is refluxed, after eluted template, namely obtain two sulfuration nanometer granules with regular pore canal;
(6) the nanometer granule of the two sulfurations in step (5) and medicament mixed are reacted, two sulfuration nanometer granules of carrying medicament can be obtained;
(7) carboxyl reaction on the pure silk glue protein obtained by amino and the step (1) of two sulfuration nanometer particle surfaces of carrying medicament, sericin is wrapped in nanometer grain surface, what namely obtain sericin parcel has the sericin/mesoporous silicon composite multi-functional controlled drug delivery systems reducing and answer with protease double-bang firecracker.
5. preparation method according to claim 4, it is characterized in that: on the pure silk glue protein obtained by the amino of the nanometer particle surface of carrying medicament and step (1) in described step (7) carboxyl reaction refer to, pure silk glue protein solid step (1) obtained adds water and is prepared into 5-10mg/mL sericin aqueous solution, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride successively, N-hydroxysuccinimide, after room temperature reaction 2-3 hour, this solution is added in two sulfuration nanometer granules of the carrying medicament of step (6) gained, lucifuge 35 degrees Celsius reaction 6-24 hour, then collected by centrifugation product, then rinse product with distilled water, what be placed in that namely frozen vacuum dryer lyophilizing obtain pulverous natural silk glue protein parcel has the compound drug-loading nanoparticles of sericin/mesoporous silicon that reduction answers with protease double-bang firecracker, the mass ratio of sericin and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 1:6, and the mass ratio of sericin and N-hydroxysuccinimide is 1:3, and the mass ratio of nanometer grain and pure silk glue protein is 2:1.
6. the preparation method according to claim 1 or 4, is characterized in that: described medicine is cancer therapy drug, hormone or antibiotic.
7. the application of nanometer silicon composite multi-functional controlled drug delivery systems in preparation diagnosis and treatment integration medicine of the natural silk glue protein parcel obtained by claim 1 or 4.
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| CN111249327A (en) * | 2020-02-24 | 2020-06-09 | 山东大学 | Natural mung bean-based polyphenol nano-drug carrier and application thereof |
| CN111821280A (en) * | 2020-07-22 | 2020-10-27 | 西南大学 | Construction method of pH-responsive sericin-adriamycin nano drug carrier |
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| CN107158481A (en) * | 2017-05-22 | 2017-09-15 | 淮阴工学院 | Prepared in biomaterial surface and carry heparin and Cu2+Nanometer grain coating method |
| CN107158481B (en) * | 2017-05-22 | 2019-12-24 | 淮阴工学院 | Preparation of heparin and Cu on surface of biological material2+Method for coating mesoporous silicon nano particles |
| CN108325482A (en) * | 2018-02-08 | 2018-07-27 | 吉林大学 | A kind of porous organic polymer nanosphere and preparation method thereof with hollow structure |
| CN110664777A (en) * | 2019-10-11 | 2020-01-10 | 杭州协合医疗用品有限公司 | Sericin particle with oxidative stress property and preparation method and application thereof |
| CN111249327A (en) * | 2020-02-24 | 2020-06-09 | 山东大学 | Natural mung bean-based polyphenol nano-drug carrier and application thereof |
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| CN111821280B (en) * | 2020-07-22 | 2022-05-20 | 西南大学 | Construction method of pH-responsive sericin-adriamycin nano drug carrier |
| CN111840570A (en) * | 2020-08-21 | 2020-10-30 | 西南大学 | Preparation method of nano-particles of sericin combined with photosensitizer |
| CN117065089A (en) * | 2023-07-06 | 2023-11-17 | 首都医科大学附属北京口腔医院 | SF/MSN composite drug controlled release coating on surface of drug-loaded TNTs |
| CN117065089B (en) * | 2023-07-06 | 2024-03-05 | 首都医科大学附属北京口腔医院 | SF/MSN composite drug controlled release coating on surface of drug-loaded TNTs |
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