CN105148286B - The preparation method of the compound drug-loading nanoparticles of silk gum/mesoporous silicon of sericin parcel - Google Patents
The preparation method of the compound drug-loading nanoparticles of silk gum/mesoporous silicon of sericin parcel Download PDFInfo
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Abstract
The present invention relates to nanometer pharmaceutical carrier of natural sericin parcel and preparation method thereof and in the application of therapeutic field of tumor, its preparation method includes：Prepare pure silk glue protein solid；Prepare nanometer particle；Prepare amination, aldehyde radical or double vulcanization nanometer particles with regular pore canal；Prepare amination, aldehyde radical or the double vulcanization nanometer particles of carrying medicament；Prepare silk gum/mesoporous silicon composite multi-functional control system of pH/ protease or reduction/protease double responsiveness.The present invention is simple to operate, and raw material acquisition pattern is easy, with low cost, is generally applicable, without Special complex equipment etc..Use the nano composite system prepared by this method, take full advantage of the characteristic of outer layer sericin and internal layer nanometer particle, with pH/ protease or reduction/protease dual responsiveness, possesses suppression tumour cell, the multi-functionals such as nano-particle positioning and insoluble drug release are monitored in real time, and drugloading rate is high, good biocompatibility is particularly in tumour diagnosis and treatment field and possesses wide application prospect in terms of the multi-drug resistant of tumour is solved.
Description
Technical field
The present invention relates to bio-medical composition field, the mesoporous silicon of more particularly to a variety of natural sericin parcels is received
Rice pharmaceutical carrier and preparation method thereof and in the application of therapeutic field of tumor.
Background technology
With developing rapidly for nanoscale science and technology, various organic or inorganic materials, which are made into nanostructured, to be used for
Biomedical sector, more and more extensive pass has been obtained especially with nano material as pharmaceutical carrier for disease treatment
Note.Mesoporous silicon dioxide nano particle（Mesoporous silica nanoparticles, MSN）There is honeybee as one kind
The solid material of nest shape loose structure, with many excellent architectural features, such as great specific surface area and pore volume, it is homogeneous can
The hole of the mesopore size of control, stable physicochemical property, easy surfaces externally and internally modification and good biocompatibility.Cause
This, substantial amounts of research is directed to being used for loading some guest molecules reaching that disease is examined by the use of nanometer particle as main body
The purpose for the treatment of.
In order to realize " zero release " of the medicine before target site is reached, while making drug-loading system that there is environmental stimulus response
Property, nano-particle can be more enriched with target site and reach medicine controlled releasing, researchers design in mesoporous silicon face
Various " valves ", by some inorganic nano-particles, polymer or large biological molecule pass through physical action or one
A little special chemical bonds make it possess pH responses, enzyme response, thermal response, reduction response, photoresponse to mesoporous silicon face
Deng, or even multiple response characteristic.Further, in order to realize the multifunction of controlled release system, many researchers pass through load
Or surface modification some magnetic nano-particles, quantum dot or fluorophor, so as to construct the multi-functional of diagnosis and treatment integration
Intelligent controlled release system.But cumbersome and single " valve " design of most " valve " design process be often difficult to reach it is many
The purpose responded again.In addition, some " valve " raw material such as metallic nanoparticles, due to not biodegradable, be present in and in vivo may be used
Short-term or long term toxicity can be produced, the process being really applied to material in human body is hindered.
Sericin（Silk Sericin）A kind of natural macromolecular viscous protein on fibroin fiber top layer is wrapped around, about
The 20%-30% of silk cocoon content is accounted for, is made up of molecular weight for 24-400 kDa polypeptide, its molecule is by serine, lucid asparagus
18 kinds of amino acid compositions such as propylhomoserin and glycine.For a long time due to people's deficiency recognized sericin and the limitation studied
Property, cause silk gum to be taken as waste to handle in filature industry, waste the natural resources of a large amount of preciousnesses.In recent years it is found that
Sericin has moisturizing, antibacterial, anti-oxidant, anticoagulation and promotes the biological nature such as cell adherence and propagation, meanwhile, silk gum
With hydrophily and degradability, it can be used for biomedical materials field.At present, had research team by sericin with
Other high polymer materials such as gelatin, polyvinyl alcohol, hydroxymethyl cellulose, polyacrylamide etc. are simply mixed, or directly utilize
Pure silk glue protein is prepared into film, biological support etc. applied to field of tissue engineering technology, shows good application prospect.But mesh
It is preceding to prepare drug delivery system using sericin parcel mesoporous silicon without any report.
The present invention successfully extracts sericin in silk cocoon using high temperature alkaline extraction, by electrostatic interaction or utilizes chemical bond
（Imine linkage or cystine linkage etc.）Sericin is wrapped in meso-porous titanium dioxide silicon face by connection first, is successfully prepared for good
Good biocompatibility, compared with high drug load, has the multiple responses such as pH, enzyme and reduction concurrently, and multi-functional with fluorescent characteristic
Intelligent controlled release system, available for antineoplaston, good fragmentation effect is generated especially for resistant tumors cell, and
It can in real time be monitored by fluorescence equipment, can reach the purpose of diagnosis and treatment integration.
The content of the invention
Problem to be solved by this invention be to provide a variety of natural sericins parcels nanometer pharmaceutical carrier and
Its preparation method and application, the nanometer carrier for the natural sericin parcel that this method is prepared have good life
Thing compatibility and higher drugloading rate, and silk gum has cell adhesion in itself, can be swallowed well by tumour cell, and pass through
The pH/ Protease Environments of intracellular lysosome or the reproducibility triggering insoluble drug release of intracellular glutathione, to reach that killing is swollen
The purpose of knurl.Simultaneously as the fluorescent characteristic of sericin causes nano-carrier per se with yellow-green fluorescence, it can supervise in real time
The positioning of nanoparticle and the release of medicine are controlled, the purpose of diagnosis and treatment integration is reached.
It is contemplated that being constructed by the use of sericin as " valve " encapsulating load medicine mesoporous silicon dioxide nano particle simultaneous
Has the multifunctional and composite type controlled drug delivery system of the multiple response such as pH/ protease or reduction/protease.It is real needed for of the invention
Experiment device is simple, and operating process is easy, with low cost.Utilize the silk gum prepared by this method/mesoporous silicon composite Nano material
Material has good biocompatibility and higher drugloading rate, and silk gum has cell adhesion in itself, can be well by tumour
Cell swallows, and pH/ Protease Environments or the reducing property triggering medicine of intracellular glutathione by intracellular lysosome
Release, to reach the purpose of killing tumour.Simultaneously as the fluorescent characteristic of sericin causes nano-carrier per se with yellowish green
Color fluorescence, the positioning of nanoparticle and the release of medicine can be monitored in real time, reach the purpose of diagnosis and treatment integration.
To reach above-mentioned purpose, the preparation for silk gum/mesoporous silicon composite multi-functional controlled drug delivery systems that the present invention is provided
Method, comprises the following steps：
First, the silk gum that pH/ protease double-bang firecracker is answered/mesoporous silicon composite multi-functional control system
（1）Silk cocoon is weighed, sericin is extracted with high temperature alkaline extraction, then pure silk glue egg is can obtain by dialysing, freezing
White solid；
（2）Nanometer particle is prepared using collosol and gel and surfactants' templating；
（3）By step（2）Middle gained nanometer particle is scattered in solvent, adds 3- aminopropyl-triethoxy silicon
Alkane, obtains the nanometer particle of surface amination；
（4）By step（3）In amidized nanometer particle be placed in the mixed liquor of methanol/hydrochloric acid flow back, wash
The amination nanometer particle with regular pore canal is obtained after stripper plate；
（5）By step（4）In amination nanometer particle and glutaraldehyde hybrid reaction, obtain the mesoporous of aldehyde radical
Silicon nano；
（6）By step（4）The amination nanometer particle or step of middle gained（5）The aldehyde radical mesoporous silicon of middle gained
Nano-particle and medicine hybrid reaction, obtain the amination or aldehyde radical nanometer particle of carrying medicament；
（7）Reacted by the amino on the aldehyde radical and sericin on surface in nanometer grain, or directly utilize electrostatic
Sericin is wrapped in nanometer particle surface by effect, that is, is obtained silk gum/mesoporous silicon that pH/ protease double-bang firecrackers answer and be combined
Type multifunctional drug control delivery.
The step（1）The silk cocoon of middle selection is normal silkworm silk cocoon kind（White jade etc.）, bombyx mori silk fibroin deletion form mutation
Kind（185 Nd-s, 140 Nd-s, 139 Nd-sDeng）Or one kind of tussah silk cocoon, ground purchased from Chinese Academy of Agricultural Sciences's sericulture
Study carefully institute.
Further, extracting the detailed process of sericin includes：
1）Appropriate silk cocoon is weighed, 1 cm is cut into2The fragment of left and right；
2）Silk cocoon after shredding is immersed in 0.01-0.05 M sodium carbonate, 100 degrees Celsius dissolve 30-60 minutes；
3）Silk gum liquid after dissolving is transferred in 3500 Da bag filter, is put into distilled water and dialyses 48 hours；
4）Solution in bag filter is transferred in 50 mL centrifuge tubes, collected after centrifugation supernatant is placed in freeze-drying machine
In freeze after can obtain powdered sericin, being placed in -20 degrees Celsius can preserve for a long time.
The step（2）Nanometer particle be the positive silicic acid four using cetyl trimethylammonium bromide as template
Ethyl ester is as organosilicon precursor, and sodium hydroxide is as obtained by base catalyst.Specifically, by cetyl trimethylammonium bromide point
Dissipate in distilled water（Mass volume ratio is 1 g: 480 mL）, add sodium hydroxide（The 28% of template quality）, heating stirring
To 80 degrees Celsius, tetraethyl orthosilicate is slowly added dropwise（5 times of template quality）, after persistently stirring 2-3 hours, obtain white
Color nanometer particle dirty solution, is collected by centrifugation precipitation, is freezed after methanol and distilled water cleaning with vacuum freeze drier, i.e.,
It can obtain the nanometer particle that white powder carries template.
The step（3）Middle selection 3- aminopropyl triethoxysilanes realize the amination of nanometer particle, specifically
Process refers to step（2）The nanometer particle of gained is according to the g of mass volume ratio 1:75 mL are scattered in methanol, so
3- aminopropyl triethoxysilanes are added afterwards, are stirred at room temperature after 24 hours, are collected by centrifugation and obtain amidized nanometer
Particle.The volume ratio of solvent methanol used and 3- aminopropyl triethoxysilanes is 75 in this step: 2.
The step（4）Middle selection methanol/concentrated hydrochloric acid mixed solution elutes the step（3）Template ten in products therefrom
Six alkyl trimethyl ammonium bromides, obtain the amination nanometer particle with regular pore canal.Specifically by step（3）
The amination nanometer particle of middle gained is scattered in methanol/concentrated hydrochloric acid（Volume ratio is 16: 1）In mixed liquor, 80 is Celsius
After being flowed back 48 hours in degree, you can effective eluted template cetyl trimethylammonium bromide, then centrifugation can be obtained with rule
The then amination nanometer particle in duct.
The step（5）Middle selection glutaraldehyde realizes the aldehyde radical of nanometer particle.Specifically, glutaraldehyde used
Concentration of aqueous solution is 2.5wt%, and the mass volume ratio of the amination nanometer particle and 2.5% glutaraldehyde water solution of selection is
100 mg : 10 mL.Further, amination nanometer particle is scattered in glutaraldehyde water solution, room temperature lucifuge
Stirring 24 hours, is then collected by centrifugation product, after then being cleaned repeatedly with distilled water, you can obtain the nanometer of aldehyde radical
Particle.
The step（6）In selected medicine can be tumor Drugs（Such as doxorubicin hydrochloride, cis-platinum, taxol,
Camptothecine etc.）One or more.Further, the medicine of other effects（Such as hormone, antibiotic etc.）It is equally applicable to this hair
It is bright.Preferably, appropriate hydrochloric acid adriamycin is dissolved in PBS by this step（Phosphate buffer, pH 7.4）In, then plus amination or
Person's aldehyde radical nanometer particle, room temperature lucifuge is stirred 24-48 hours.
The step（7）It is to encapsulate nanometer particle with sericin.Detailed process is by the sericin aqueous solution
（5 - 10 mg/mL）Add step（6）Mixed liquor in, 35 degrees Celsius of lucifuge is reacted 6-7 hours；Production is then collected by centrifugation
Thing；Further, product is rinsed repeatedly with distilled water, being subsequently placed in freeze in frozen vacuum dryer can obtain powdered
The mass ratio of the compound drug-loading nanoparticles of silk gum/mesoporous silicon, nanometer grain and sericin is 1: 0.5 – 1 :
2。
2nd, the silk gum of reduction/protease double responsiveness/mesoporous silicon composite multi-functional control system
（1）Nanometer grain containing template is scattered in solvent, mercaptopropyl trimethoxysilane is added, reacts
To the nanometer particle of sulfhydrylation；
（2）By step（1）In the nanometer particle of sulfhydrylation be scattered in solvent, add（2- amino second mercaptos
Base）- 2- mercaptopyridine hybrid reactions, obtain the nanometer particle of double vulcanizations；
（3）By step（2）In double vulcanization nanometer particles be placed in the mixed liquor of methanol/hydrochloric acid flow back, elution
Double vulcanization nanometer particles with regular pore canal are obtained after template；
（4）By step（3）In the nanometer particles of double vulcanizations mixed with medicine, you can obtain the double of carrying medicament
Vulcanize nanometer particle；
（5）By the carboxyl reaction on the amino and sericin of mesoporous silicon face, sericin is wrapped in mesoporous silicon
Nanoparticle surface, you can obtain silk gum/mesoporous silicon composite multi-functional controlled drug delivery systems that reduction/protease double-bang firecracker is answered.
The step（1）Middle selection mercaptopropyl trimethoxysilane realizes the sulfhydrylation of nanometer particle, specific mistake
Journey is by the nanometer particle containing surfactant templates（The silk gum that preparation method is answered with synthesis pH/ protease double-bang firecrackers/
Process in mesoporous silicon composite nano grain is consistent）According to the g of mass volume ratio 1:83 mL are scattered in methanol, are then added
Mercaptopropyl trimethoxysilane, was stirred at room temperature after 24 hours, be collected by centrifugation, clean after obtain sulfhydrylation nanometer grain
Son.The volume ratio of solvent methanol used and mercaptopropyl trimethoxysilane is 36 in this step: 1.
The step（2）In use（2- amino second sulfydryls）- 2- mercaptopyridines（SATH）Realize the double of nanometer grain
Vulcanization.Synthesis（2- amino second sulfydryls）The detailed process of -2- mercaptopyridines includes：1）By the pyridinium dissolution of 2,2 '-two sulphur two in first
Alcohol/glacial acetic acid（Volume ratio 5: 0.2）Mixed solution in；2）Mercaptamine is dissolved in methanol to (mass volume ratio is
1.14 g ：10 mL), step 1 is added dropwise) in mixed liquor in, react at room temperature 48 hours；3）After completion of the reaction, rotation is steamed
Hair falls methanol；4）Obtained product is cleaned after twice with absolute ether, is redissolved in methanol, is added absolute ether and is sunk
Form sediment, can obtain white solid product, be placed in -20 degrees Celsius of refrigerator overnights；5）Next day takes out product, and suction is abandoned after yellow supernatant,
Repeat after precipitating 5 times, be placed in vavuum pump drying, you can obtain powdered（2- amino second sulfydryls）- 2- mercaptopyridines.
The step（3）The mode of middle eluted template with silk gum/mesoporous silicon for answering of synthesis pH/ protease double-bang firecrackers is compound receives
The process of eluted template is consistent in the grain of rice.
The step（4）In selected medicine can be tumor Drugs（Such as doxorubicin hydrochloride, cis-platinum, taxol,
Camptothecine etc.）One or more.Further, the medicine of other effects（Such as hormone, antibiotic etc.）It is equally applicable to this hair
It is bright.Medicine carries the mode that medicine is carried in silk gum/mesoporous silicon composite nano grain that mode is answered with synthesis pH/ protease double-bang firecrackers
Unanimously.
The step（5）It is that nanometer particle is sealed with sericin.The silk cocoon of selection is normal silkworm silk cocoon
Kind（White jade etc.）, bombyx mori silk fibroin deletion form mutating variety（185 Nd-s, 140 Nd-s, 139 Nd-sDeng）Or tussah silk cocoon
One kind, purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences.The silk that sericin extracting method is answered with synthesis pH/ protease double-bang firecrackers
The mode that sericin is extracted in glue/mesoporous silicon composite nano grain is consistent.Sericin encapsulation process is specifically included：1）By silk
Glue protein solid, which is dissolved in appropriate distilled water, is prepared into the sericin aqueous solution（5 - 10 mg/mL）；2）Appropriate add is weighed respectively
Enter 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), N- hydroxysuccinimides (NHS)（With silk gum
The mass ratio of albumen is respectively 6:1, 3:1）, sequentially add in the sericin aqueous solution, react at room temperature 2-3 hours；3）Will be anti-
Solution after answering adds step（4）Mixed solution in, 35 degrees Celsius of lucifuge is reacted 6-24 hours；Production is then collected by centrifugation
Thing；Further, product is rinsed repeatedly with distilled water, being subsequently placed in freeze in frozen vacuum dryer can obtain powdered
The compound drug-loading nanoparticles of silk gum/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 responses.
The beneficial effects of the present invention are：
（1）This method is simple to operate, and raw material acquisition pattern is easy, with low cost, is generally applicable, without Special complex equipment
Deng.
（2）Using the nano composite system prepared by this method, outer layer sericin and internal layer mesoporous silicon are taken full advantage of
The characteristic of nano-particle, with pH/ protease or reduction/protease dual responsiveness, possesses suppression tumour cell, in real time prison
The multi-functionals such as nano-particle positioning and insoluble drug release are surveyed, and drugloading rate is high, good biocompatibility is special in tumour diagnosis and treatment field
It is to possess wide application prospect in terms of the multi-drug resistant of tumour is solved.
Brief description of the drawings
Fig. 1 is the ESEM shape appearance figure of silk gum/mesoporous silicon composite nano particle；
Fig. 2 is the thermogravimetric weight loss curve map of different nanoparticles；
Fig. 3 is the fluorescence spectra and fluorescent microscopy images of silk gum/mesoporous silicon composite nano particle；
Fig. 4 is unloaded silk gum/influence of the mesoporous silicon Nanocomposite Particles to tumor cell activity；
Fig. 5 is the interaction for the macrophage that silk gum/mesoporous silicon composite nano particle is originated with mouse：A is to give
Metamorphosis after the different processing of macrophage；B is to give inflammatory factor tumor necrosis factor after the different processing of macrophage
Son（TNF-α）And interleukin-11（IL-1β）Expression；
Fig. 6 answers drug release patterns figure for the pH/ protease double-bang firecrackers of the compound controlled drug delivery systems of silk gum/mesoporous silicon；
The compound medicine-carried system of silk gum/mesoporous silicon and free Ah mould that Fig. 7 answers for the pH/ protease double-bang firecrackers of load adriamycin
The comparison of influence of the element to drug susceptibility-types and multidrug resistant type tumor cell activity.
Fig. 8 for load adriamycin the compound medicine-carried system of silk gum/mesoporous silicon answered of reduction/protease double-bang firecracker with free Ah
The comparison of influence of the mycin to multidrug resistant type tumor cell activity.
Embodiment
Embodiment 1：The utilization Euplotes woodruffi that the present invention is provided prepares silk gum/mesoporous silicon composite nano particle.
1st, the selection of silk cocoon：
The preferential silk cocoon from bombyx mori silk fibroin deletion mutation kind of the invention（It is purchased from Chinese Academy of Agricultural Sciences's sericulture research
Institute, the bombyx mori silk fibroin deletion mutation kind is stored in state of Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm resource conservation center, protects
Hide numbering185 Nd-s）For raw material, main chemical composition is sericin.
2nd, the extraction of sericin：
（1）2 g bombyx mori silk fibroin deletion form mutating variety silk cocoons are weighed, 1 cm is cut into2The fragment of left and right；
（2）Silk cocoon after shredding is immersed in the M sodium carbonate of 40 mL 0.02,100 degrees Celsius dissolve 30 minutes；
（3）Silk gum solution after dissolving is transferred in 3500 Da bag filter, is put into distilled water and dialyses 48 hours；
（4）Solution in bag filter is transferred in 50 mL centrifuge tubes, collected after centrifugation supernatant is placed in freeze-drying
Solid-state sericin is can obtain after being freezed in machine, -20 degrees Celsius of refrigerators are stored in.
3rd, the preparation of silk gum/mesoporous silicon composite nano particle：
（1）1.0 g cetyl trimethylammonium bromides are scattered in 480 mL distilled waters, 0.28 g hydroxides are added
Sodium, heating stirring is slowly added dropwise the positive silane ethyl esters of 5.0 g to 80 degrees Celsius, after persistently stirring 2 hours, obtains white Jie
Hole silicon nano dirty solution, is collected by centrifugation after precipitation, methanol and distilled water cleaning repeatedly, vacuum freeze drier is freezed, i.e.,
Obtain the nanometer particle that white powder contains template.
（2）By step（1）The nanometer particle of gained（1 g）It is scattered in 75 mL methanol, then adds 2 mL
3- aminopropyl triethoxysilanes, were stirred at room temperature after 24 hours, were collected by centrifugation and obtained amidized nanometer particle.
（3）By step（2）The amination nanometer particle of gained is scattered in 170 mL methanol/concentrated hydrochloric acid（Volume ratio
For 16: 1）In mixed liquor, after being flowed back 48 hours in 80 degrees Celsius, then centrifugation can obtain the amination with regular pore canal
It is lyophilized after nanometer particle, methanol and distilled water cleaning repeatedly.
（4）By step（3）The amination nanometer particle of gained（200 mg）It is scattered in 2.5% glutaraldehyde water solution
（20 mL）In, room temperature lucifuge is stirred 24 hours, and product is then collected by centrifugation, after then being cleaned repeatedly with distilled water, you can obtained
The nanometer particle of aldehyde radical.
（5）By step（4）The aldehyde radical nanometer particle of gained is scattered in 400 mL distilled waters again, adds 20
The mL sericin aqueous solution（5 mg/mL）, after 35 degrees Celsius of lucifuges are stirred 6 hours, precipitation is collected by centrifugation, distilled water cleaning is more
After secondary, freezing vacuum pump is freezed, you can obtain powdered silk gum/mesoporous silicon composite nanometer particle.As shown in Figure 1：By this
Silk gum/mesoporous silicon composite nano grain diameter of method synthesis is 200 nm or so.As shown in Figure 2：When temperature is improved to 770
Degree Celsius when, amination nanometer grain, the weight loss of aldehyde radical nanometer grain and silk gum/nanometer grain
Respectively 14.18%, 26.75% and 42.07%, illustrate that silk gum is successfully modified on nanometer grain.
Experimental example 1：Study the biocompatibility of silk gum/mesoporous silicon composite nano grain.
1st, unloaded silk gum/mesoporous silicon Nanocomposite Particles and kinds of tumor cells were incubated after 48 hours altogether, detected
Cell viability, as shown in Figure 4：When compound system does not carry any medicine, the vigor of cell is not influenceed significantly.
2nd, by unloaded silk gum/mesoporous silicon Nanocomposite Particles and the macrophage in mouse source（Raw264.7
cells）After being incubated 24 hours altogether, cytoskeleton and nucleus are contaminated respectively to observe the form of cell with phalloidine and DAPI,
And detection macrophage inflammatory gene（TNF-α, IL-1 β）Expression.As shown in Figure 5A：When macrophage and silk gum/mesoporous
After silicon composite nano grain is incubated 24 hours altogether, the form of macrophage and and blank cultures（Negative control）It is incubated 24 hours
Cellular morphology afterwards is similar, illustrates that silk gum/mesoporous silicon composite nano grain does not have activating macrophage, and after lipopolysaccharides is stimulated
Macrophage occurs in that obvious polarization, forms substantial amounts of filopodia；As one man, as shown in Figure 5 B, silk gum/mesoporous silicon is multiple
Mould assembly nanoparticle does not promote the expression of macrophage inflammatory gene yet, illustrates silk gum/mesoporous silicon composite nano grain in vitro
Immunogenicity is not shown in research.
Experimental example 2：Detect the fluorescent characteristic of silk gum/mesoporous silicon composite nano particle.
1st, the fluorescence spectrum of silk gum/mesoporous silicon composite nano particle is detected with fluorescence spectrum tester, as shown in Figure 3A：
Under the exciting of appropriate excitation wavelength, transmitting light of the wavelength in 550 nm or so can be produced.
2nd, silk gum/mesoporous silicon composite particle and RAW264.7 macrophages were incubated after 24 hours altogether, use fluorescence microscopy
The fluorescence of sem observation material, as shown in Figure 3 B：In the case where blue light source is excited, material can send green fluorescence.
Experimental example 3：Study the pH/ protease double responsiveness drug release behaviors of the composite drug-loaded system of silk gum/nanometer.
1st, the compound controlled drug delivery systems of silk gum/mesoporous silicon that the pH/ protease double-bang firecrackers of load antineoplastic are answered are prepared.
（1）This research uses broad-spectrum anti-cancer drug doxorubicin hydrochloride.100 mg doxorubicin hydrochlorides are dissolved in 200 first
mL PBS（pH 7.4）In, 100 mg aldehyde radical nanometer particles are then added, room temperature lucifuge is stirred 24 hours.
（2）By the 10 mL sericin aqueous solution（5 mg/mL）Add step（1）Mixed liquor in, 35 degrees Celsius of lucifuge
After reaction 6 hours, product is collected by centrifugation, distilled water rinses product to supernatant close to colourless, is then placed in products therefrom cold
Freeze to freeze in vacuum drier and can obtain the powdered compound drug-loading nanoparticles of silk gum/mesoporous silicon.
2nd, the drugloading rate of the compound drug-loading nanoparticles of silk gum/mesoporous silicon obtained by detection above-mentioned steps.
By 1 mL silk gum/compound drug-loading nanoparticles of mesoporous silicon（1.25 mg）It is scattered in 4 mL hydrofluoric acid, treats thorough
After the dissolving of bottom, by 4 times of liquid diluting, ultraviolet absorptivity of the measurement liquid in 480 nm（0.34871）, line bent further according to standard
Calculate gained liquid doxorubicin concentration be 0.018193 mg/mL；Drugloading rate is 29.1%, i.e. 100 mg silk gum/mesoporous silicon
Nanoparticle can load about 41 mg doxorubicin hydrochloride.
3rd, silk gum/drug release profiles of the mesoporous silicon composite nano medicine-carried system under condition of different pH are detected.
The 2 mg silk gum/nanometer grains for loading adriamycin are divided in 42 mL EP pipes（0.5 mg is often managed）,
In the buffer solution for being separately added into 1.5 mL pH 5.0,6.5, the 7.4 and pH 5.0 containing papain, lucifuge is placed in 37
Degree Celsius incubation is rocked, supernatant is collected at the time point of setting, the light absorption value at 480 nm is determined with ultraviolet specrophotometer,
The adriamycin amount discharged is calculated according to standard curve, then calculates the ratio that the dose discharged accounts for initial drugloading rate
Example, draws release profiles.As shown in Figure 6：The insoluble drug release for having 53.85% in the environment of pH 5.0 after 72 hours comes out, and is adding
Enter after protease, the medicine for having 81.56% is discharged from system, it was demonstrated that the medicine-releasing system is dual to pH and protease quick
Sense.
Experimental example 4：The compound medicine-carried system of silk gum/mesoporous silicon of research load adriamycin is for drug susceptibility-types tumour
The lethal effect of cell and multidrug resistant type tumour cell.
The compound medicine-carried nano particles of silk gum/mesoporous silicon and drug susceptibility-types human liver cancer cell of adriamycin will be loaded
（HepG2 cells）, human cervical carcinoma cell（HeLa cells）, human breast cancer cell（MCF-7 cells）, and multidrug resistant
Type human breast cancer cell（MCF-7/ADR cells）It is incubated 48 hours altogether, cell viability is detected with mtt assay.As shown in Figure 7：Will
Drug susceptibility-types tumour cell is with after free doxorubicin hydrochloride processing, and most tumour cell occurs in that death；Similarly,
Silk gum/mesoporous silicon/doxorubicin hydrochloride composite particles also can effectively inducing death of neoplastic cells, but thin to tumour in low concentration
The lethal effect of born of the same parents is weaker than free hydrochloric acid adriamycin, because doxorubicin hydrochloride is micromolecular water soluble drug, easily passes through diffused sheet
Formula is absorbed by tumour cell and plays GVT in time.And on the contrary, working as multidrug resistant type breast cancer cell with dissociating
After doxorubicin hydrochloride processing, tumour of the cell viability apparently higher than after with silk gum/mesoporous silicon/doxorubicin hydrochloride composite particles processing
Cell, because multidrug resistant type tumour cell has substantial amounts of drug efflux pump, when free adriamycin through cell membrane enters cytoplasm
Soon it has been pumped out afterwards extracellular, thus tumor-killing power is decreased obviously；And compound of silk gum/mesoporous silicon/doxorubicin hydrochloride
Grain enters intracellular by endosome-lysosomal pathway, dramatically avoids the effect of drug efflux pump, " valve " is molten simultaneously
It can be rapidly opened under enzyme body environment, medicine is released in a large amount in an early stage out and is diffused to nucleus, thus it is swollen in multidrug resistant type
Stronger cell killing effect has been played in oncocyte.
Embodiment 2：Silk gum/mesoporous silicon that the utilization cystine linkage that the present invention is provided prepares reduction/enzyme double responsiveness is compound
Controlled drug delivery system.
1st, in the present embodiment, the silk cocoon kind preferentially selected is the silk cocoon of bombyx mori silk fibroin deletion mutation kind（It is purchased from China
Sericulture research institute of Academy of Agricultural Sciences, the bombyx mori silk fibroin deletion mutation kind is stored in country of Inst. of Silkworm, Chinese Academy of Agricultural Sciences
Silkworm resource conservation center, deposit number140Nd-s）；The extracting method of sericin is same as Example 1.
2nd, in the synthesis of sulfhydrylation nanometer particle and embodiment 1 amidized nanometer particle synthesis side
Method is essentially identical, and difference is to change 3- aminopropyl triethoxysilanes into mercapto propyl trimethoxy silicon, the volume of addition
For the 1/36 of solvent methanol.
3rd, the synthesis of the nanometer particle of double vulcanizations：
（1）（2- amino second sulfydryls）The synthesis of -2- mercaptopyridines：1）By the pyridinium dissolution of 4.41 g, 2,2 '-two sulphur two in first
Alcohol（20 mL）/ glacial acetic acid（0.8 mL）Mixed solution in；2）By 1.14 g Mercaptamines, (M=113 are dissolved in 10mL methanol
In, in the mixed liquor in being added dropwise 1), it is stirred at room temperature 48 hours；3）After completion of the reaction, after rotary evaporation of solvent, yellow is obtained
Grease sample material；4）Obtained product absolute ether（50 mL）After twice of cleaning, it is redissolved in 10 mL methanol, then adds
Enter in 200 mL absolute ethers and precipitate, can obtain white solid product；5）Stirring is placed in -20 degrees Celsius of refrigerator mistakes after 10 minutes
Night；6）Next day takes out product, and suction is abandoned after yellow supernatant, obtains yellow crystalline product, repeats after precipitating 2-3 times, is placed in vacuum
Pump is dried, you can obtain powdered（2- amino second sulfydryls）- 2- mercaptopyridines.
（2）The nanometer particle of 200 mg sulfhydrylations is scattered in 30 mL methanol, 200 mg SATH are added,
It is stirred at room temperature after 24 hours, product is collected by centrifugation, is cleaned and freezed afterwards for several times with methanol and distilled water；
（3）Eluting surface activating agent template, method is identical with the process in embodiment 1.
4th, the compound controlled drug delivery systems of silk gum/mesoporous silicon that reduction/enzyme double-bang firecracker of load antineoplastic is answered are prepared：This
The antineoplastic of embodiment selection is doxorubicin hydrochloride, and material preparation method is with preparing pH/ albumen in the experimental example 3 of embodiment 1
The method for the compound controlled drug delivery systems of silk gum/mesoporous silicon that enzyme double-bang firecracker is answered is essentially identical, and difference is：（1）Adriamycin
Consumption is reduced；（2）The sericin used is numbering140 Nd-sBombyx mori silk fibroin deletion mutation kind silk cocoon extract gained；
（3）The nanometer particle used is the nanometer particle of double vulcanizations with regular pore canal；（4）Sericin is wrapped up
First activated before the nanometer particle for carrying the double vulcanizations of medicine, detailed process includes：1）10 mg doxorubicin hydrochlorides are dissolved in
20 mL PBS（pH 7.4）In, the nanometer particle of the double vulcanizations of 20 mg is added, lucifuge is reacted at room temperature 24 hours；2）By 10
Mg sericins are dissolved in 2 mL distilled waters, sequentially add 0.06 g EDC, 30 mg NHS, are reacted at room temperature 3 hours；3）Will step
Rapid 2）Gained solution add step 1）Reaction solution in, room temperature lucifuge react 7 hours after, product, distilled water is collected by centrifugation
Product is rinsed to supernatant close to colourless, then products therefrom, which is placed in frozen vacuum dryer, lyophilized can obtain powdered
The compound drug-loading nanoparticles of silk gum/mesoporous silicon.
Experimental example 1：Detect the drugloading rate of the compound drug-loading nanoparticles of silk gum/mesoporous silicon obtained by above-mentioned steps.
The detection method of this experimental example drugloading rate is identical with the detection method of experimental example 3 in embodiment 1, the load obtained by calculating
Dose is 0.14, i.e., 100 mg silk gum/nanometer particle can load 16.28 mg doxorubicin hydrochlorides.
Experimental example 2：The compound medicine-carried system of silk gum/mesoporous silicon of research load adriamycin is thin for multidrug resistant type tumour
The lethal effect of born of the same parents
The compound medicine-carried nano particles of silk gum/mesoporous silicon and multidrug resistant type human breast cancer cell of adriamycin will be loaded
（MCF-7/ADR cells）It is incubated 48 hours altogether, cell viability is detected with mtt assay.As shown in Figure 8：Silk gum/mesoporous silicon is compound
The fragmentation effect that drug-loading nanoparticles are produced to multidrug resistant type human breast cancer cell line Bcap-37/ADR cells, illustrates nanometer
Grain can be swallowed by tumour cell, and release medicine out by intracellular environment, so as to generate cell killing effect.
Claims (3)
1. the system of the compound drug-loading nanoparticles of silk gum/mesoporous silicon with pH/ protease double-responses of sericin parcel
Preparation Method, it comprises the following steps：
（1）Silk cocoon is weighed, sericin is extracted with high temperature alkaline extraction, the silk gum liquid after dissolving passes through 3500 Da bag filter again
Dialysing, freezing is to obtain pure silk glue protein solid；Wherein described high temperature alkaline extraction is that the silk cocoon after shredding is immersed in into 0.01-
In 0.05M sodium carbonate liquor, 100 degrees Celsius dissolve 30-60 minutes；
（2）Nanometer particle is prepared using collosol and gel and surfactants' templating；
（3）By step（2）Middle gained nanometer particle is scattered in methanol, adds 3- aminopropyl triethoxysilanes, instead
The nanometer particle of surface amination should be obtained；
（4）By step（3）In the nanometer particle of surface amination be placed in the mixed liquor of methanol and hydrochloric acid backflow and wash
Centrifugation obtains the amination nanometer particle with regular pore canal after stripper plate, eluted template；
（5）By step（4）In the amination nanometer particle with regular pore canal be scattered in concentration be 2.5wt% penta
In the dialdehyde aqueous solution, room temperature lucifuge is stirred 24 hours, and product is then collected by centrifugation, and after then being cleaned repeatedly with distilled water, is obtained
The nanometer particle of aldehyde radical；The mass volume ratio of wherein amination nanometer particle and glutaraldehyde water solution is
100 mg : 10 mL；
（6）By step（4）The amination nanometer particle or step of middle gained（5）The aldehyde radical nanometer of middle gained
Particle and medicine hybrid reaction, obtain the amination or aldehyde radical nanometer particle of carrying medicament；
（7）Pass through the aldehyde radical and step on surface in the aldehyde radical nanometer particle of carrying medicament（1）Obtained pure silk glue egg
Amino reaction on white, or sericin is directly wrapped in amination nanometer particle surface using electrostatic interaction, i.e.,
Obtain the compound drug-loading nanoparticles of silk gum/mesoporous silicon with pH/ protease double-responses of sericin parcel；Wherein institute
State the aldehyde radical and step on surface in the aldehyde radical nanometer particle by carrying medicament（1）On obtained pure silk glue protein
What amino reacted comprises the following steps that：By step（1）Obtained pure silk glue protein solid, which adds water, is prepared into 5-10 mg/mL
The glue protein aqueous solution, step is added by the sericin aqueous solution（6）The aldehyde radical nanometer particle of the carrying medicament of gained
In, 35 degrees Celsius of lucifuge is stirred 6-7 hours；Product is then collected by centrifugation；Then product is rinsed with distilled water, is placed in freezing vacuum
Freezed in drying machine and obtain powdered nano particle, the mass ratio of wherein nanometer grain and pure silk glue protein is 1:
0.5 – 1 : 2。
2. the system of the compound drug-loading nanoparticles of silk gum/mesoporous silicon answered with reduction/protease double-bang firecracker of sericin parcel
Preparation Method, it comprises the following steps：
（1）Silk cocoon is weighed, sericin is extracted with high temperature alkaline extraction, the silk gum liquid after dissolving passes through 3500 Da bag filter again
Dialysing, freezing is to obtain pure silk glue protein solid；Wherein described high temperature alkaline extraction is that the silk cocoon after shredding is immersed in into 0.01-
In 0.05M sodium carbonate liquor, 100 degrees Celsius dissolve 30-60 minutes；
（2）Nanometer particle is prepared using collosol and gel and surfactants' templating；
（3）By step（2）Middle gained nanometer particle is according to the g of mass volume ratio 1:83 mL are scattered in methanol, plus
Enter mercaptopropyl trimethoxysilane, that is, obtain the nanometer particle of sulfhydrylation；Wherein methanol and mercapto propyl trimethoxy silicon
The volume ratio of alkane is 36: 1；
（4）By step（3）In the nanometer particles of 200 mg sulfhydrylations be scattered in 30 mL methanol, add 200 mg
（2- amino second sulfydryls）- 2- mercaptopyridines, are stirred at room temperature after 24 hours, product are collected by centrifugation, and number is cleaned with methanol and distilled water
It is lyophilized after secondary, obtain the nanometer particle of double vulcanizations；
（5）By step（4）In double vulcanization nanometer particles be placed in the mixed liquor of methanol and hydrochloric acid flow back, eluted template
Obtain double vulcanization nanometer particles with regular pore canal afterwards；
（6）By step（5）Obtained double vulcanization nanometer particles with regular pore canal and medicine hybrid reaction, you can
To double vulcanization nanometer particles of carrying medicament；
（7）By step（6）The amino and step of double vulcanization nanometer particle surfaces of obtained carrying medicament（1）Obtain
Carboxyl reaction on pure silk glue protein so that sericin is wrapped in nanometer grain surface, that is, obtains sericin parcel
There are the compound drug-loading nanoparticles of silk gum/mesoporous silicon answered of reduction/protease double-bang firecracker；Double vulcanizations by carrying medicament
The amino and step of nanometer particle surface（1）The obtained carboxyl reaction on pure silk glue protein is comprised the following steps that：Will
Step（1）Obtained pure silk glue protein solid, which adds water, is prepared into 5-10 mg/mL sericin aqueous solution, sequentially adds 1-
(3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, N- hydroxysuccinimides, after reacting at room temperature 2-3 hours,
The solution is added into step（6）In double vulcanization nanometer particles of the carrying medicament of gained, 35 degrees Celsius of reactions 6 of lucifuge-
24 hours；Product is then collected by centrifugation；Then product is rinsed with distilled water, is placed in frozen vacuum dryer and freezes i.e. acquisition powder
The compound drug-loading nanoparticles of silk gum/mesoporous silicon answered with reduction/protease double-bang firecracker of the natural sericin parcel of last shape；
The mass ratio of sericin and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides is 1:6, silk gum egg
It is in vain 1 with the mass ratio of N- hydroxysuccinimides:3, nanometer grain is 2 with the mass ratio of pure silk glue protein: 1.
3. preparation method according to claim 1 or 2, it is characterised in that：The medicine is cancer therapy drug, hormone or antibiosis
Element.
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