CN108524944A - A kind of the cancer combination therapy system and construction method of the sensitive type that Biomedia is stablized - Google Patents
A kind of the cancer combination therapy system and construction method of the sensitive type that Biomedia is stablized Download PDFInfo
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Abstract
The present invention relates to cancer pharmaceutical technology fields, and in particular to a kind of the cancer combination therapy system and construction method of the sensitive type that Biomedia is stablized.Sensitive type cancer combination therapy system loads doxorubicin hydrochloride DOXHCl using mesoporous silicon as matrix in mesoporous silicon duct, and Apoptosis peptide is connected by cystine linkage on surface, and bovine serum albumin is coated in outermost layer.In building process, mesoporous silicon particle is carried out to surface sulfhydrylation, amido modified double vulcanizations successively, then alkynyl-modified cystine linkage is introduced on the surface of mesoporous silicon, being reacted with the click chemistry of azido group using alkynyl makes Apoptosis peptide be connected on mesoporous silicon by cystine linkage, then DOXHCl is loaded, and coats bovine serum albumin on the outside.The cancer combination therapy system of the present invention has higher drug loading efficiencies, strong to the targeting of cancer cell, low to the side effect of normal cell, and Apoptosis peptide and anticancer drug collective effect greatly improve therapeutic effect.
Description
Technical field
The present invention relates to cancer pharmaceutical technology fields, and in particular to a kind of cancer connection for the sensitive type that Biomedia is stablized
Close treatment system and construction method.
Background technology
Malignant tumour is considered as threatening one of the three big killers of human health, and the mankind are dead because of caused by malignant tumour
Rate shelter has the second of mortality, is only second to cardiovascular and cerebrovascular disease.Chemotherapy is the main means of clinical treatment tumour,
But because chemotherapeutics is distributed in whole body with blood, causes it to lack selectivity, all have to tumour cell and normal body cell
Lethal effect, caused toxic side effect make patient not be resistant to and its application are made to be severely limited.Therefore structure target tumor
The drug delivery system of tissue is the effective way for solving the problems, such as chemotherapy of tumors.In addition, in the field of current antineoplastic research,
Single treatment means have often been unable to reach and have met Treatment need.And traditional Radiotherapy chemotherapy treatment means are also to just
Often tissue generates prodigious toxicity and injury.Therefore, with a variety of novel treatment means（Such as photo-thermal therapy, photodynamics is treated
Method etc.）, in conjunction with the pharmaceutical carrier of targeting and sensitive release performance, change the administering mode of drug, delay body drug resistance
It generates, and reduces the toxicity of anticancer drug normal tissue, improve the synergistic treatment side of the stability and bioavilability of drug
Formula has become a big hot spot of current research.It is reported that the carrier system for loading two kinds or more anticancer drugs simultaneously is compared
It can be significantly improved in the therapeutic efficiency of drug alone ingredient, multiple medicine drug-loading system.But developed targeting is given at present
Medicine system there are still stability it is poor, biocompatibility and biodegradability are undesirable the problems such as, while load anticancer drug
Between how effectively to play synergistic effect, and fully discharge and play drug effect in vivo, and reduce side effect need into
The research of one step.
Invention content
In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide the Lazers that a kind of Biomedia is stablized
The cancer combination therapy system of sense type, with good stability, biocompatibility, while the drug loaded can be orderly in body
Interior abundant release plays drug effect, plays the role of synergistic treatment cancer, improves oncotherapy effect.
Another object of the present invention is the structures for the cancer combination therapy system for providing the sensitive type that the Biomedia is stablized
Method.
The present invention provides the following technical solution：
A kind of cancer combination therapy system for the sensitive type that Biomedia is stablized, sensitive type cancer combination therapy system is to be situated between
Hole silicon is matrix, loads anticancer drug and Apoptosis peptide simultaneously on mesoporous silicon, and coat ox blood in the outermost layer of mesoporous silicon
Albumin.
As an improvement of the present invention, Apoptosis peptide is connected to the surface of mesoporous silicon by restoring sensitive cystine linkage
On.
As an improvement of the present invention, anticancer drug is doxorubicin hydrochloride DOXHCl, and the duct of loaded mesoporous silicon
It is interior.
As an improvement of the present invention, the grain size of mesoporous silicon is 50~100nm.
The cancer combination therapy system of the present invention, in mesoporous silicon internal load anticancer drug, is being situated between using mesoporous silicon as matrix
The surface of hole silicon connects Apoptosis peptide by restoring sensitive cystine linkage.Large specific surface area, the nanometer of nanometer particle
Particle size and the uniform adjustable, pore volume of mesoporous pore size are big, have good biocompatibility, being capable of payload diagnosis and treatment system
Agent makes the cancer combination therapy system of gained have higher load factor and biocompatibility.Apoptosis peptide KLA is one kind two
Close α spirals promote apoptosis peptide, and amino acid sequence is (KLAKLAK)2, it can destroy mitochondrial membrane and induction relies on mitochondria
Apoptosis, and extracellularly keeping relative nontoxic.Apoptosis peptide is connect with mesoporous silicon through cystine linkage, and cystine linkage has reduction
Sensibility can decompose fracture under the action of glutathione.And containing more glutathione in cancer cell body, therefore when arrival
When cancer cell, cystine linkage fracture makes Apoptosis peptide fall off under the action of glutathione, and is discharged into failure line in cancer cell
Mitochondria Membrane and and induce the apoptosis of cancer cell, play the targeting to cancer cell, and have in not up to cancer cell good
Chemical stability, therefore to the Small side effects of internal normal cell.And the anticancer drug inside mesoporous silicon is supported on also because thin
Born of the same parents' apoptosis peptide falls off and largely discharges, and realizes the total transmission of KLA and anticancer drug, reaches the synergistic treatment of tumour.It is being situated between
The bovine serum albumin of hole silicon outer cladding can enhance the dispersibility and biocompatibility of cancer combination therapy system, make treatment of cancer
System has higher Biomedia stability.It is sensitive by restoring by mesoporous silicon duct internal load anticancer drug, surface
Cystine linkage connect with Apoptosis peptide, and bovine serum albumin is coated on the outside of mesoporous silicon, to make the cancer joint of the present invention
Treatment system can give full play to drug effect inside cancer cell, and Apoptosis peptide and anticancer drug transmit Synergistic anti-cancer, have altogether
Good biological stability, can be kept in 100% serum solution stable dispersion for 24 hours more than, may be in response to reducing environment and enzyme
Environment release anticancer preparation.
The construction method of above-mentioned cancer combination therapy system, includes the following steps：
（1）Prepare mesoporous silicon：It is prepared by raw material of tetraethyl orthosilicate, triethylamine and hexadecyltrimethylammonium chloride CTAC
The mesoporous silicon CTAC@MSN of CTAC modifications；
（2）Prepare the mesoporous silicon of the double vulcanizations in surface：At room temperature by CTAC@MSN and 3-mercaptopropyi trimethoxy silane in methanol
Middle mixing reaction, it is dry after centrifuge washing, then wash CTAC and obtain mercapto-modified mesoporous silicon MSN-SH, then with S-
(2- aminoethanethios) -2- thiopyridines hydrochloride hybrid reaction in methyl alcohol, then centrifuge, wash and be dried to obtain surface connection
There is the mesoporous silicon MSN-SS-NH of cystine linkage2；
（3）Prepare the mesoporous silicon of alkynyl-modified double vulcanizations：Room temperature is by MSN-SS-NH2It is distributed in methanol, bromo third is added dropwise
Alkynes, triethylamine react make alkynyl substitute amino and are connected on cystine linkage, obtain the mesoporous silicon MSN-SS- of alkynyl-modified cystine linkage
alkyne；
（4）Load cells apoptosis peptide：MSN-SS-alkyne is distributed in methanol by room temperature, and sequentially adding end has azido group
Apoptosis peptide N3-KLA、CuSO4·5H2O, sodium ascorbate reacts under nitrogen atmosphere, is then centrifuged for, washs and connected
It is connected to the mesoporous silicon MSN-SS-KLA of Apoptosis peptide；
（5）Load anticancer drug：MSN-SS-KLA is distributed in methanol, vigorous stirring overnight after anticancer drug is added, waits bearing
The anticancer drug of load is DOXHCl, and then dialysis obtains the mesoporous silicon DOX@MSN-SS-KLA of load anticancer drug；
（6）Bovine serum albumin coats：DOX@MSN-SS-KLA are distributed in deionized water, bovine serum albumin BSA is then added dropwise,
It is centrifuged after being stirred overnight at room temperature, washing obtains the mesoporous silicon DOX@MSN-SS-KLA/BSA of BSA claddings.
As a kind of improvement of the method for the present invention, step（2）Middle CTAC@when preparing mercapto-modified mesoporous silicon MSN-SH
MSN, methanol and 3-mercaptopropyi trimethoxy silane mass ratio are 1:100~150:3 ~ 5,24~36h of reaction time.
As a kind of improvement of the method for the present invention, step（2）The middle mesoporous silicon MSN-SS- for preparing amido modified double vulcanizations
NH2When MSN-SH, methanol and S-（2- aminoethanethios）The mass ratio of -2- thiopyridine hydrochlorides is 1:200~300:1 ~ 4, instead
It is 24~36h between seasonable.
As a kind of improvement of the method for the present invention, step（3）Middle MSN-SS-NH2, methanol, bromo propine and triethylamine
Mass ratio is 1:200~300:8.0:0.2 ~ 0.5, the reaction time is 24~36h.
As a kind of improvement of the method for the present invention, step（4）Used in MSN-SS-alkyne, methanol, N3-KLA、
CuSO4·5H2The mass ratio of O and sodium ascorbate is 1:100~200:1~2:1:1.5 ~ 3,3 days reaction time.
As a kind of improvement of the method for the present invention, step（6）The quality of middle DOX@MSN-SS-KLA, deionized water and BSA
Than being 1:1000~15000:1~5.
In the building process of cancer combination therapy system, the mesoporous silicon particle of 50~100nm of grain size is synthesized first, then
By 3-mercaptopropyi trimethoxy silane be modified mesoporous silicon surface introduce sulfydryl-SH, then again with S- (2- amino second sulphur
Base) -2- thiopyridines hydrochloride hybrid reaction in methyl alcohol, the sulfydryl of mesoporous silicon face is by S-（2- aminoethanethios）- 2- sulphur
It is interrupted for the cystine linkage of pyridine hydrochloride and S-（2- aminoethanethios）A part for -2- thiopyridine hydrochlorides, which combines, to be formed
New cystine linkage, S-（2- aminoethanethios）The half structure containing pyridine ring is sloughed in -2- thiopyridine hydrochlorides, makes mesoporous silicon
The double vulcanizations in surface, connect amido modified cystine linkage-SS-NH2, then pass through propinyl substituted-amino-NH2, in mesoporous silicon
Surface forms alkynyl-modified cystine linkage-SS-alkyne, then has the Apoptosis peptide of azido group to be reacted with end,
Sodium ascorbate and CuSO4•5H2Under the action of O, alkynyl occurs click chemistry with azido group and reacts, and carries out nitrine-alkynyl
Then Husigen cycloaddition reactions carry out anticancer drug DOXHCl's again to make Apoptosis peptide be connected on cystine linkage
Load, and bovine serum albumin is coated in mesoporous silicon face finally, improve the stability and dispersibility of cancer combination therapy system.
The cancer combination therapy system built by the above process has higher drug loading rate, Biomedia stability and goes back
Former sensitive and enzyme sensitivity characteristic, and it is strong to the targeting of cancer cell, to the Small side effects of normal cell.
Beneficial effects of the present invention are as follows：
The cancer combination therapy system of the present invention has higher drug first using nano level mesoporous silicon as pharmaceutical carrier
Load efficiency connects Apoptosis peptide secondly by the sensitive cystine linkage of reduction, strong to the targeting of cancer cell, to normal thin
The side effect of born of the same parents is low, while the glutathione in cancer cell makes the cystine linkage of reduction sensitivity be broken, to be discharged in cancer cell
A large amount of Apoptosis peptide, and be coated on the anticancer drug inside the duct of mesoporous silicon and also largely discharge, Apoptosis peptide with it is anti-
Cancer drug collective effect, greatly improves therapeutic effect.With good stability after being coated finally by bovine serum albumin, dispersion
Property and biocompatibility, can be kept in 100% serum solution stable dispersion for 24 hours more than, and may be in response to reducing environment and
Enzyme environment, to discharge anticancer preparation.
Description of the drawings
Fig. 1 is that the Fourier transformation of MSN, MSN-SH, MSN-SS-alkyne and MSN-SS-KLA of 1 gained of embodiment are red
External spectrum figure.
The release that Fig. 2 is DOXs of the 1 gained DOX@MSN-SS-KLA/BSA of embodiment under different trypsin amounts is bent
Line.
Fig. 3 is the release profiles of 1 gained DOX@MSN-SS-KLA/BSA KLA in different DTT contents of embodiment.
Fig. 4 is that the DOX@MSN-SS-KLA/BSA of embodiment 1 co-culture the cytotoxicity figure of 48h with HeLa cells.
Fig. 5 is grain size distributions of the DOX@MSN-SS-KLA/BSA of embodiment 1 in 0% serum solution.
Fig. 6 is grain size distributions of the DOX@MSN-SS-KLA/BSA of embodiment 1 in 100% serum solution.
In Fig. 1：A1 is MSN, B1 MSN-SH, C1 MSN-SS-alkyne, D1 MSN-SS-KLA.
Specific implementation mode
Just the specific implementation mode of the present invention is described further below.
Unless otherwise instructed, the raw material employed in the present invention is commercially available or commonly used in the art, such as
Without special instruction, the method in following embodiments is the conventional method of this field.
Embodiment 1
A kind of cancer combination therapy system for the sensitive type that Biomedia is stablized, using the mesoporous silicon of 50nm as matrix, mesoporous
Anticancer drug and Apoptosis peptide are loaded on silicon simultaneously, and bovine serum albumin is coated in the outermost layer of mesoporous silicon, wherein cell withers
It dies peptide and is connected on the surface of mesoporous silicon by restoring sensitive cystine linkage, anticancer drug is doxorubicin hydrochloride DOXHCl, and
It is supported in the duct of mesoporous silicon.
The construction method of above-mentioned cancer combination therapy system, includes the following steps：
（1）Prepare mesoporous silicon：8.0 g CTAC and 0.24 g triethylamines are dissolved in 80 mL deionized waters, be vigorously stirred and are added
Heat is to 95 DEG C, after condensing reflux 1h, 6.0 mL tetraethyl orthosilicates are slowly added dropwise into above-mentioned solution to be vigorously stirred lower carry out 1
H, then 10000 r/min centrifuge 10 min and obtain white solid object, then are cleaned for several times with deionized water and methanol, vacuum drying
The nanometer particle CTAC@MSN with surfactant CTAC are obtained afterwards；
（2）Prepare the mesoporous silicon of the double vulcanizations in surface：By in 1.2g CTAC@MSN ultrasonic disperses to 150mL methanol, ultrasonic disperse,
4 mL 3-mercaptopropyi trimethoxy silanes are added and are vigorously stirred, for 24 hours, then 10000r/min centrifuges 10min for room temperature reaction
White gummy solid object is obtained, then is cleaned for several times with deionized water and methanol, is obtained containing surfactant after vacuum drying
The nanometer particle CTAC@MSN-SH of sulfhydrylation；Then CTAC@MSN-SH are distributed in methanol, concentrated hydrochloric acid is added simultaneously
48 ~ 60 h of reflux at 60 DEG C are vigorously stirred, surfactant CTAC is extracted after centrifuging, cleaning, is obtained mercapto-modified
Mesoporous silicon MSN-SH；The MSN-SH of 800mg is distributed in 200mL methanol, the S- (2- aminoethanethios)-of 800mg is added
2- thiopyridine hydrochlorides are vigorously stirred 24 h of room temperature reaction, then centrifuge 10 min with 8000 r/min, with deionized water and
Methanol cleans for several times, and MSN-SS-NH is obtained after vacuum drying2；
（3）Prepare the mesoporous silicon of alkynyl-modified double vulcanizations：By 600mg MSN-SS-NH2Methanol of the ultrasonic disperse to 160mL
In, 6mL bromos propine and 6 drop triethylamines is added, is vigorously stirred lower room temperature reaction 24 h, 8000r/min centrifugation 10min, spends
For several times, vacuum drying obtains the mesoporous silicon MSN-SS-alkyne of alkynyl-modified cystine linkage for ionized water and absolute methanol cleaning；
（4）Load cells apoptosis peptide：The MSN-SS-alkyne of 100mg is dissolved in 12.5 mL methanol, then ultrasonic disperse divides
There is the N of azido group in the end for not sequentially adding 100mg3The CuSO of-KLA, 100mg4·5H2The ascorbic acid of O and 158.5mg
Sodium reacts at room temperature 3 days under nitrogen atmosphere protection, and then 5 min of 6000r/min centrifugations, number is cleaned with deionized water and methanol
It is secondary, obtain the MSN-SS-KLA of load cells apoptosis peptide；
Wherein, there is the Apoptosis peptide N of azido group in end3A kind of preparation method of-KLA is as follows：Weigh the 2- chloro- three of 0.7g
Benzyl chlorine resin, is added in the DMF of 10 mL and stirs 15min, is then swollen 30min, then transfers a resin into polypeptide conjunction
Cheng Guanzhong, which is filtered, removes DMF, and the DMF solution of Fmoc-D-Lys (Boc)-OH is then added（I.e.：1.01 g Fmoc-Lys
(Boc)-OH, 8 mL DMF, 2.5 mL DIEA）Then 2 h of room temperature condensation reaction is washed 3 times with 6 ~ 8 mL DMF, is used in combination
DMF:MeOH:DIEA=6:3:The 1 unreacted active cl radical of mixed liquor closing, then washed 3 times with 6 ~ 8 mL DMF, with 20
Piperidines/DMF of mL（1/4）Solution washs 2 times, 20 min remove the Fmoc blocking groups on peptide chain terminal amino group every time, then
It is washed 3 times with DMF.Hereafter when connecting amino acid residue extension peptide chain, it is all made of the fmoc-protected amino acid of 4 equivalents i.e.
Fmoc-D-Leu-OH is 0.766g, and Fmoc-D-Ala-OH is 0.6708 g, and 4 equivalent HBTU i.e. 0.822 g and 6 equivalents are used in combination
DIEA i.e. 1.5 mL are dissolved in activated carboxyl in 8 mL DMF, be then added to it is above-mentioned be connected with peptide chain and removed Fmoc protection
Resin in, carry out condensation reaction, the reaction time be 4 h.Reaction finishes, and is verified with the methanol solution of the ninhydrin of 10 mg/mL
Whether amino is condensed completely, if solution is blue or rubescent expression condensation is incomplete, need to repeat condensation step, if solution does not change colour,
It indicates that this step condensation reaction is completed, continues to remove Fmoc protecting groups, extend peptide chain by amino acid sequence.Wait for all Amino acid synthesis
After, continue to remove Fmoc protecting groups, 0.657g nitrine acetic acid, 2.466g HBTU, 2mLDIEA, 12mLDMF is then added
4 h are reacted, after color passes through, wash resin with DMF, DCM, methanol successively, number is respectively 3 times, 3 times, 4 times, Zhi Houyong
Cut agent containing 95wt%TFA, 2.5wt%TIS and 2.5wt% water cut falling, and 1g resins correspond to 15 mL cut agents, by polypeptide
Cut and fall from resin, while cutting and falling side group protecting groups, cut fall the time be 2 h, cut fall 1 h when stop stirring, standing can
The phenomenon that see layering, upper layer are that black sticks shape object, and lower layer is weak yellow liquid.Backward polypeptide solution will be cut and pass through revolving
Then concentration uses ice ether to precipitate, washing precipitation 3 times, is collected by filtration repeatedly, is dried in vacuo, then passes through the side of freeze-drying
Method removes small molecular weight impurity, obtains final products N3-KLA；
（5）Load anticancer drug：By the MSN-SS-KLA ultrasonic disperses of 50mg in 50 mL absolute methanols, the salt of 8 mg is added
Sour adriamycin DOXHCl, vigorous stirring overnight, dialysis obtain DOX@MSN-SS-KLA after 2 days；
（6）Bovine serum albumin coats：By in the DOX@MSN-SS-KLA ultrasonic disperses to the deionized water of 10mL of 10mg, then drip
The bovine serum albumen solution BSA for adding the 1mg/mL of 10mL, is stirred overnight at room temperature, and by centrifugation, washing obtains bovine serum albumin packet
The mesoporous silicon DOX@MSN-SS-KLA/BSA covered.
Embodiment 2
A kind of cancer combination therapy system for the sensitive type that Biomedia is stablized, using the mesoporous silicon of 75nm as matrix, mesoporous
Anticancer drug and Apoptosis peptide are loaded on silicon simultaneously, and bovine serum albumin is coated in the outermost layer of mesoporous silicon, wherein cell withers
It dies peptide and is connected on the surface of mesoporous silicon by restoring sensitive cystine linkage, anticancer drug is doxorubicin hydrochloride DOXHCl, and
It is supported in the duct of mesoporous silicon.
Its construction method difference from example 1 is that：
Step（2）In, when preparing CTAC@MSN-SH, CTAC@MSN are distributed in the methanol of 190mL, 3- mercaptopropyis used
The volume of trimethoxy silane is 3.6mL, reaction time 30h；Prepare MSN-SS-NH2When, MSN-SH is distributed to 200mL's
In methanol, S- (2- aminoethanethios) -2- thiopyridines hydrochloride 2g, reaction time 30h used；
Step（3）In, when preparing MSN-SS-alkyne, by MSN-SS-NH2In ultrasonic disperse to the methanol of 120mL, it is added dropwise three
Ethamine 8 drips, and reacts 30h；
Step（4）In, MSN-SS-alkyne is distributed in the methanol of 18mL, the N of addition3The quality of-KLA is 150mg, is added
The quality of the sodium ascorbate entered is 200mg；
Step（5）In the middle MSN-SS-KLA ultrasonic disperses to the absolute methanol of 55mL by 75mg, hydrochloric acid Ah mould of 10 mg is added
Plain DOXHCl dialyses 3 days；
Step（6）In the middle@MSN-SS-KLA ultrasonic disperses to the deionized water of 100mL by DOX, the bovine serum albumin of 25mL is added dropwise
Solution.
Embodiment 3
A kind of cancer combination therapy system for the sensitive type that Biomedia is stablized, using the mesoporous silicon of 100nm as matrix, mesoporous
Anticancer drug and Apoptosis peptide are loaded on silicon simultaneously, and bovine serum albumin is coated in the outermost layer of mesoporous silicon, wherein cell withers
It dies peptide and is connected on the surface of mesoporous silicon by restoring sensitive cystine linkage, anticancer drug is doxorubicin hydrochloride DOXHCl, and
It is supported in the duct of mesoporous silicon.
Its construction method difference from example 1 is that：
Step（2）In, when preparing CTAC@MSN-SH, CTAC@MSN are distributed in the methanol of 230mL, 3- mercaptopropyis used
The volume of trimethoxy silane is 6mL, reaction time 36h；Prepare MSN-SS-NH2When, MSN-SH is distributed to the first of 250mL
In alcohol, S- (2- aminoethanethios) -2- thiopyridines hydrochloride 3.2g, reaction time 36h used；
Step（3）In, when preparing MSN-SS-alkyne, by MSN-SS-NH2In ultrasonic disperse to the methanol of 180mL, it is added dropwise three
Ethamine 10 drips, and reacts 36h；
Step（4）In, MSN-SS-alkyne is distributed in the methanol of 25mL, the N of addition3The quality of-KLA is 200mg, is added
The quality of the sodium ascorbate entered is 300mg；
Step（5）In the middle MSN-SS-KLA ultrasonic disperses to the absolute methanol of 60mL by 100mg, hydrochloric acid Ah mould of 12mg is added
Plain DOXHCl dialyses 5 days；
Step（6）In the middle@MSN-SS-KLA ultrasonic disperses to the deionized water of 150mL by DOX, the bovine serum albumin of 50mL is added dropwise
Solution.
Performance test
1, to carry out Fourier transformation respectively to MSN, MSN-SH, MSN-SS-alkyne and MSN-SS-KLA of the gained of embodiment 1 red
Outer test, obtains Fourier transform infrared spectroscopy figure as shown in Figure 1, and wherein A1 is MSN, B1 MSN-SH, C1 MSN-SS-
Alkyne, D1 MSN-SS-KLA.It can be seen from the figure that MSN-SH is in 2550 cm-1The peak of there are one places-SH, MSN-SS-
Alkyne is in 2140cm-1There are one the peaks of alkynyl at place.
2, tablets in vitro is tested
（1）Enzyme sensibility
The DOX@MSN-SS-KLA/BSA 25mg ultrasonic disperses of the gained of embodiment 1 are weighed to 3mL deionized waters, then average mark
It at three groups and is respectively charged into the bag filter of molecule interception 3500, then is separately added into the pancreas egg of 0 μ L, 100 μ L and 200 μ L
White enzyme.Then bag filter is immersed in the PBS buffer solution of 0.1M of 10mL, pH value 7.4, then is placed in shaking table and releases for 37 DEG C
Medicine.Dialyzate is replaced at set time intervals, and DOX is measured in wavelength 592 by sepectrophotofluorometer RF-5301PC
Fluorescence intensity at nm calculates its burst size, wherein accumulation release amount of medicine %=（Mt / M∞）× 100%, wherein MtIt is in the t times
The burst size of DOX, M∞It is the DOX total amounts of mesoporous silicon material package, the results are shown in Figure 2.
As can be seen from Figure 2, the DOX@MSN-SS-KLA/BSA drug release rates of trypsase is not added to be significantly lower than other two groups,
In scope of experiment, trypsinase concentration is bigger, and DOX@MSN-SS-KLA/BSA drug release rates are faster, different tryptoses after 90h
The DOX burst sizes of enzyme content tend towards stability, wherein the accumulative drug of DOX@MSN-SS-KLA/BSA is released when not adding trypsase
It is high-volume 15.1%, the accumulative release amount of medicine of DOX@MSN-SS-KLA/BSA is 22.0% when adding 100 μ L trypsase, addition
The drug release of DOX@MSN-SS-KLA/BSA is 51.1% when 200 μ L trypsase, this, which is primarily due to trypsase, to divide
Solution is coated on the BSA of outer layer, promotes the release of DOX, this shows that DOX MSN-SS-KLA/BSA have good enzyme sensibility.
（2）Reduction-sensitive
Under the conditions of being protected from light, the MSN-SS-KLA 50mg for weighing 1 gained of embodiment are dispersed in the PBS buffer solution 20mL that pH value is 7.4
In, then weigh 2.5mg fluorescein isothiocynates and be dissolved in 5mL deionized waters, and it is slowly dropped to the PBS of above-mentioned MSN-SS-KLA
In buffer solution, it is stirred to react 4h under room temperature, is then centrifuged for, washs, being dried to obtain the MSN- of fluorescein isothiocynate modification
SS-KLA compounds MSN-SS-KLA/FITC.Take MSN-SS-KLA/FITC 25mg ultrasonic disperses to 3mL deionized waters, it is average
Be divided into three groups and be respectively charged into the bag filter of molecule interception 3500, be separately added into a concentration of 0mM of dithiothreitol (DTT) DTT,
The aqueous solution 5mL of 0.5mM and 5mM, bag filter is immersed in the PBS buffer solution of the 0.1M of 10mL, then pH value 7.4 is set
37 DEG C of drug releases in shaking table.Dialyzate is replaced by setting time interval, FITC is measured by sepectrophotofluorometer RF-5301PC
It is the fluorescence intensity at 516 nm in wavelength, is since FITC and KLA has covalent bond effect, the concentration of the FITC of measurement
The concentration of KLA, and then the burst size of KLA is calculated, accumulation release amount of medicine %=（Mt / M∞）× 100%, wherein MtIt is in the t times
The burst size of KLA, M∞It is the KLA total amounts of mesoporous silicon material package, the results are shown in Figure 3.
As can be seen from Figure 3, in scope of experiment, MSN-SS-KLA/FITC increases with DTT concentration, same time KLA releases
Amount also increase.This, which is primarily due to DTT, has reduction, and the cystine linkage of reduction sensitivity can be made to be broken, accelerate releasing for KLA
It puts, that is, shows that DOX@MSN-SS-KLA/BSA have stronger reduction-sensitive, and the glutathione in cancer cell has with DTT
There is similar reduction, and its concentration is far above normal body cell, therefore can promote in the reducing environment of cancer cell
The release of KLA, and burst size in human normal tissue and little, to the Small side effects of human body.
3, vitro cytotoxicity is tested
By HeLa cells respectively with 6 × 103The density in a/hole is inoculated in 96 porocyte culture plates, and 100 μ L are added per hole and contain
The DMEM culture mediums for having 10 % FBS and 1% chain-penicillin, are then placed on containing 5% CO2Incubator in trained at 37 DEG C
Support 24 h.Then the different mesoporous silicon materials of setting concentration gradient are added in every hole respectively（MSN/BSA、MSN-SS-KLA/
NSA, DOX@MSN-SS-KLA/BSA and the DOX@MSN-SS-KLA/BSA, i.e. DOX@MSN-SS-KLA/BSA for loading DTT
with DTT）, pure KLA and doxorubicin hydrochloride Free DOX, continue to cultivate 48 h in above-mentioned culture medium.After culture,
Solution all in hole is removed, 200 μ L fresh cultures and 20 μ L MTT solution are added, continues to cultivate 4 h in the incubator.It is small
The heart removes the solution in hole, and fills into the purple crystals that 100 μ L DMSO dissolvings generate, after rocking mixing, in microplate reader
Absorbance of each hole at 490 nm is recorded on DG5033A, is calculated the relative survival rate of cell, is as a result seen Fig. 4.
The relative survival rate of cell is calculated by formula：
Comparative survival rate of cells %=（OD490(sample)/OD490(control)）× 100；
Wherein, OD490(control)It is that the light absorption value measured when mesoporous silicon material, OD are not added490(sample)It is that mesoporous silicon material is added
The light absorption value measured after material.The measurement of OD values is averaged based on 4 independent parallel samples, is as a result expressed as average value ± standard deviation
Difference.
As can be seen from Figure 4, for MSN/BSA substantially without cytotoxicity, MSN-SS-KLA/BSA only has smaller cytotoxicity, knot
Fig. 2 is closed it is found that in the drug release amount not plus when trypsase being 10% or so when 48 h, i.e. DOX in DOX@MSN-SS-KLA/BSA
When a concentration of 2 μ g/mL, a concentration of 0.2 μ g/mL actually discharged compare the Free DOX of 0.2 μ g/mL and corresponding
The cell survival rate of the MSN-SS-KLA/BSA of concentration：The cell survival rate of Free DOX is 68.2%, MSN-SS-KLA/BSA's
The cell survival rate that cell survival rate is 77.1%, DOX@MSN-SS-KLA/BSA is 9.0%.It these results suggest that DOX@MSN-
SS-KLA/BSA has preferable toxicity, it was demonstrated that the coordinative role of DOX/KLA substantially increases therapeutic effect.
4, dispersion stabilization
By the DOX@MSN-SS-KLA/BSA difference ultrasonic disperse of the gained of embodiment 1 in deionized water with mass concentration 100%
In serum solution, then dispersion concentration 2mg/mL respectively takes 3mL to be placed in sample bottle, and sample is placed in 37 DEG C of insulating boxs,
Per DLS grain size tests are carried out at regular intervals, Fig. 5 and Fig. 6 are as a result seen.From the comparison of two figures it is found that DOX@MSN-SS-KLA/
BSA can be kept in 100% serum solution stable dispersion for 24 hours more than, dispersion stabilization is good.
It should be noted that construction method gained DOX@MSN-SS-KLA/BSA according to the present invention, including 2 He of embodiment
It embodiment 3 and is not limited to the above embodiments, has and above-mentioned properties similar in embodiment 1.
Claims (10)
1. a kind of cancer combination therapy system for the sensitive type that Biomedia is stablized, which is characterized in that sensitive type cancer joins
Treatment system is closed using mesoporous silicon as matrix, loads anticancer drug and Apoptosis peptide simultaneously on mesoporous silicon, and in mesoporous silicon
Outermost layer coats bovine serum albumin.
2. cancer combination therapy system according to claim 1, which is characterized in that Apoptosis peptide is by restoring sensitivity
Cystine linkage is connected on the surface of mesoporous silicon.
3. cancer combination therapy system according to claim 1 or 2, which is characterized in that the anticancer drug be hydrochloric acid Ah
Mycin DOXHCl, and be supported in the duct of mesoporous silicon.
4. cancer combination therapy system according to claim 1 or 2, which is characterized in that the grain size of mesoporous silicon be 50~
100nm。
5. the construction method of the cancer combination therapy system as described in Claims 1-4 is any, includes the following steps：
（1）Prepare mesoporous silicon：It is prepared by raw material of tetraethyl orthosilicate, triethylamine and hexadecyltrimethylammonium chloride CTAC
The mesoporous silicon CTAC@MSN of CTAC modifications；
（2）Prepare the mesoporous silicon of the double vulcanizations in surface：At room temperature by CTAC@MSN and 3-mercaptopropyi trimethoxy silane in methanol
Middle mixing reaction, it is dry after centrifuge washing, then wash CTAC and obtain mercapto-modified mesoporous silicon MSN-SH, then with S-
(2- aminoethanethios) -2- thiopyridines hydrochloride hybrid reaction in methyl alcohol, then centrifuge, wash and be dried to obtain surface connection
There is the mesoporous silicon MSN-SS-NH of cystine linkage2；
（3）Prepare the mesoporous silicon of alkynyl-modified double vulcanizations：Room temperature is by MSN-SS-NH2It is distributed in methanol, dropwise addition bromo propine,
Triethylamine react makes alkynyl substitute amino and is connected on cystine linkage, obtains the mesoporous silicon MSN-SS- of alkynyl-modified cystine linkage
alkyne；
（4）Load cells apoptosis peptide：MSN-SS-alkyne is distributed in methanol by room temperature, and sequentially adding end has azido group
Apoptosis peptide N3-KLA、CuSO4·5H2O and sodium ascorbate react under nitrogen atmosphere, are then centrifuged for, wash and connected
It is connected to the mesoporous silicon MSN-SS-KLA of Apoptosis peptide；
（5）Load anticancer drug：MSN-SS-KLA is distributed in methanol, vigorous stirring overnight after anticancer drug is added, waits bearing
The anticancer drug of load is DOXHCl, and then dialysis obtains the mesoporous silicon DOX@MSN-SS-KLA of load anticancer drug；
（6）Bovine serum albumin coats：DOX@MSN-SS-KLA are distributed in deionized water, bovine serum albumin BSA is then added dropwise,
It is centrifuged after being stirred overnight at room temperature, washing obtains the mesoporous silicon DOX@MSN-SS-KLA/BSA of BSA claddings.
6. construction method according to claim 5, which is characterized in that step（2）It is middle to prepare mercapto-modified mesoporous silicon
CTAC@MSN, methanol and 3-mercaptopropyi trimethoxy silane mass ratio 1 when MSN-SH:100~150:3 ~ 5, the reaction time 24
~36h.
7. construction method according to claim 5 or 6, which is characterized in that step（2）It is middle to prepare amido modified double vulcanizations
Mesoporous silicon MSN-SS-NH2When MSN-SH, methanol and S-（2- aminoethanethios）The mass ratio of -2- thiopyridine hydrochlorides is
1:200~300:1 ~ 4, the reaction time is 24~36h.
8. the construction method stated according to claim 5, which is characterized in that step（3）Middle MSN-SS-NH2, methanol, bromo propine and
The mass ratio of triethylamine is 1:200~300:8.0:0.2 ~ 0.5, the reaction time is 24~36h.
9. construction method according to claim 5, which is characterized in that step（4）Middle MSN-SS-alkyne, methanol, N3-
KLA、CuSO4·5H2The mass ratio of O and sodium ascorbate is 1:100~200:1~2:1:1.5 ~ 3,3 days reaction time.
10. construction method according to claim 5, which is characterized in that step（6）Middle DOX@MSN-SS-KLA, deionization
The mass ratio of water and BSA are 1:1000~15000:1~5.
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|---|---|---|---|---|
| CN104013965A (en) * | 2014-05-06 | 2014-09-03 | 重庆大学 | Method for preparing meso-porous silicon nano medicine carrier with cell specificity target, reduction responsiveness and triple anticancer treatment effects |
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| CN104013965A (en) * | 2014-05-06 | 2014-09-03 | 重庆大学 | Method for preparing meso-porous silicon nano medicine carrier with cell specificity target, reduction responsiveness and triple anticancer treatment effects |
| CN105963702A (en) * | 2016-04-27 | 2016-09-28 | 湖北大学 | Drug delivery controlled-release system having tumor induced targeting capacity and preparation method of drug delivery controlled-release system |
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