CN104651405A - Pullulan-based drug and gene-loaded carrier, and preparation method and applications thereof - Google Patents
Pullulan-based drug and gene-loaded carrier, and preparation method and applications thereof Download PDFInfo
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- CN104651405A CN104651405A CN201510066829.6A CN201510066829A CN104651405A CN 104651405 A CN104651405 A CN 104651405A CN 201510066829 A CN201510066829 A CN 201510066829A CN 104651405 A CN104651405 A CN 104651405A
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Abstract
The invention discloses a pullulan-based drug and/or gene-loaded double-function carrier Pullulan-SA-PEI. The Pullulan-SA-PEI is prepared by comprising the following steps: sequentially grafting stearic acid and low-molecular-weight polyethyleneimine onto a pullulan main chain through an esterification and amidation reaction. The invention further discloses applications of the double-function carrier, the Pullulan-SA-PEI can be self-assembled to form a micellar structure in a water-soluble environment, a core is used for coating anti-cancer drug, a shell is used for adsorbing therapeutic gene, so as to further realize the co-transmission of the drug and gene. The co-transmission system can be effectively bonded with and transport pDNA so as to slowly release drug, and the better killing effect on cancer cells can be achieved after the therapeutic gene is adsorbed. The double-function carrier Pullulan-SA-PEI has potential application values in the field of drug sustained release and gene therapy.
Description
Technical field
The invention belongs to technical field of biological materials, be specifically related to a kind of carrying medicament based on pulullan polysaccharide or/and gene bifunctional vector, and preparation method thereof, also relate to the application of this carrier in drug-carrier transfer system, gene-carrier transfer system and medicine-gene-carrier transfer system.
Background technology
At present, chemotherapy is one of important cancer treatment method, but is used alone chemotherapeutics and needs very large dosage just to have antitumor efficacy, brings certain toxic side effect, as the cardiac toxic of Zorubicin simultaneously.Therefore, chemotherapeutics has become a new research direction with the conbined usage of other treatment technology.Research in recent years shows: cancer is a kind of genopathy, is under the effect of outside environmental elements, and the inherent proto-oncogene of human body cell is activated and the deactivated long-term evolution process gradually of cancer suppressor gene.As can be seen here, gene therapy has extremely important meaning for the radical cure of tumour.
Gene therapy refers to and utilizes suitable carrier that foreign gene is imported target cell; correct gene unconventionality or defect; and then reaching the object of disease therapy, the key of gene therapy is that therapeutic gene (as Suppressor p53 etc.) is imported target cell by the carrier that whether there is the high and low poison of transfection efficiency.
Common genes carrier comprises virus vector and non-virus carrier two class.Although virus vector has good gene delivery effect, easily cause immunological response, and production cost is high.The various shortcoming of virus vector makes people that research emphasis is turned to non-virus carrier gradually, and current non-viral gene vector mainly comprises liposome, polymer carrier and natural polysaecharides material.Such as, polymine is a kind of common efficient polymer carrier, a large amount of amino because containing in its structure, electrostatic interaction can be utilized to form mixture in conjunction with electronegative DNA, have again " proton sponge effect " simultaneously, be beneficial to mixture and carry out endosome escape, and then the DNA of delivery is effectively transported in nucleus.But there is the larger shortcoming of cytotoxicity in polymine, for addressing this problem, investigator is had to use polyoxyethylene glycol to modify polymine, reduce the interaction between protein in mixture and blood, improve blood circulation time, but single gene therapy is also difficult to the growth of anticancer long-term effectively.Therefore, the conbined usage of the homogenic treatment of chemotherapeutics becomes an important research direction in oncotherapy.
Pulullan polysaccharide is a kind of safety non-toxic, biocompatibility is good, have the soluble in water and dimethyl sulfoxide (DMSO) of the natural polysaccharide of liver target, be rich in hydroxyl, chemical property is more active, hydrophobic compound can be connected on the main chain of pulullan polysaccharide by esterification, amidate action and carboxymethylation reaction, give it amphipathic, then nano-micelle is formed, as the controllable release carrier of cancer therapy drug by hydrophobic interaction self-assembly in aqueous; Also can connect the compound with positively charged on the main chain of pulullan polysaccharide, obtain the carrier that can deliver therapeutic gene, therapeutic gene be imported to correct self-defect in cell, recover patient health.Because the biocompatibility of pulullan polysaccharide itself is good, by the genophore that obtains of modification there is lower cytotoxicity.But, existing research is that pulullan polysaccharide is modified mostly, for single pharmaceutical carrier or genophore research, the present invention uses stearic acid and branched chain type polymine to modify pulullan polysaccharide simultaneously, prepare the nano-carrier Pullulan-SA-PEI having useful for drug delivery and gene delivery two kinds of functions concurrently, there is not yet the report of such carrier, the research of this novel nano carrier will provide new approaches for administration nano-drug administration system antineoplaston.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of carrying medicament based on pulullan polysaccharide or/and gene bifunctional vector, and its preparation method and application.
First object of the present invention be to provide a kind of based on pulullan polysaccharide for carrying medicament or/and the carrier of gene, described carrier take stearic acid as hydrophobic core, with pulullan polysaccharide and low molecular weight polyethylene imines for hydrophilic outer shell, this carrier has the structure of formula I:
In formula I, m is the integer between 5 to 24, and n is the integer between 11 to 47; In formula II, n is the integer between 9 to 45.
Further, described low molecular weight polyethylene imines is branched chain type polymine.
Further, the weight-average molecular weight of described low molecular weight polyethylene imines is 500-2000Da, is preferably 600-1200Da; The molecular-weight average of described pulullan polysaccharide is 30-200kDa, is preferably 50-150kDa, is more preferably 60-120kDa; Described stearic acid grafted rate is 3-10%, and described branched chain type grafting polyethylene imine rate represents with nitrogen content: be 5-12%.
The second object of the present invention be to provide described based on pulullan polysaccharide for carrying medicament or/and the preparation method of the carrier of gene, stearic acid and low molecular weight polyethylene imines are grafted on the skeleton of pulullan polysaccharide respectively by esterification and amidate action and prepare by described carrier successively.
The present invention also provides the preferred preparation method of carrier of the present invention, comprises the steps:
(1) under nitrogen protection, stearic acid and DMAP and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine hydrochloric acid is added in pulullan polysaccharide (referred to as Pullulan), stirring reaction 30-70h at 15-50 DEG C, collect product, product, through dialysis purifying, lyophilize, obtains Propiram-stearic acid grafted polymkeric substance (referred to as Pullulan-SA); Wherein reaction solvent is dimethyl sulfoxide (DMSO);
(2) under nitrogen protection, succinyl oxide and DMAP is added in the Propiram that step (1) obtains-stearic acid grafted polymkeric substance, stirring reaction 5-30h at 30-70 DEG C, collect product, product, through dialysis purifying, lyophilize, obtains Propiram-stearic acid-succinyl oxide graftomer (referred to as Pullulan-SA-COOH); Wherein reaction solvent is dimethyl sulfoxide (DMSO);
(3) under nitrogen protection, in Propiram-stearic acid-succinyl oxide graftomer that step (2) obtains, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide, stir-activating 2-8h at 2-8 DEG C, add lower molecular weight branched chain type polymine again, stirring reaction 10-30h at 15-50 DEG C, through dialysis purifying, lyophilize, obtain Propiram-stearic acid-grafting polyethylene imine polymkeric substance (referred to as Pullulan-SA-PEI); Wherein reaction solvent is phosphate buffered saline buffer.
Further, the pulullan polysaccharide described in step (1) and stearic mol ratio are (1-5): 1-30:1, are preferably 3:1-25:1, are more preferably 4:1-20:1; The mol ratio of stearic acid, DMAP and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is 1:(0.5-2): (1-2.5), be preferably 1:(0.6-1.8): (1-2), is more preferably 1:(0.8-1.5): (1-1.5).
Further, the mol ratio of the Propiram described in step (2)-stearic acid grafted polymkeric substance and succinyl oxide is 1:(1-3), be preferably 1:(1.2-1.6), the mol ratio of DMAP and succinyl oxide is 1:(4-12), be preferably 1:(5-10), be more preferably 1:(6-9).
Further, mol ratio amino in carboxyl and low molecular weight polyethylene imines in Propiram-stearic acid-succinyl oxide graftomer described in step (3) is 1:(0.1-5), be preferably 1:(0.3-4), be more preferably 1:(0.5-2); In Propiram-stearic acid-succinyl oxide graftomer, the mol ratio of carboxyl, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1:(0.1-3): (0.1-3), be preferably 1:(0.5-2): (0.5-2), is more preferably 1:(0.8-1.8): (0.8-1.8).
In the step (1) of above-mentioned preparation method, temperature of reaction is preferably 18-45 DEG C, is more preferably 20-40 DEG C; Reaction times is preferably 35-65h, is more preferably 40-60h.
In the step (2) of above-mentioned preparation method, temperature of reaction is preferably 35-65 DEG C, is more preferably 40-60 DEG C; Reaction times is preferably 8-25h, is more preferably 10-20h.
In the step (3) of above-mentioned preparation method, temperature of reaction is preferably 18-45 DEG C, is more preferably 20-40 DEG C; Reaction times is preferably 12-28h, is more preferably 15-25h.
In above-mentioned preparation method, in the optimal technical scheme of step (1), pulullan polysaccharide is dissolved in dimethyl sulfoxide (DMSO) and obtains pulullan polysaccharide solution; Stearic acid, DMAP and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is dissolved in dimethyl sulfoxide (DMSO), at 25-50 DEG C of stir-activating 0.5-5h, obtain stearic acid activation solution, be then added in pulullan polysaccharide solution and react.
The preferred preparation method of above-mentioned carrier of the present invention, its preparation process is shown below:
The third object of the present invention is to provide medicine-gene-carrier system, and comprise carrier and load medicine on the carrier and genetic stew, wherein said carrier is carrier of the present invention.
Further, described medicine is the one in Zorubicin, taxol, hydroxycamptothecine.
Further, described genetic stew is the one in plasmid DNA, chromosomal DNA, Mitochondrial DNA.
Further, described plasmid DNA is the one in p β Gal, p53.
In described medicine-gene-carrier system, hydrophobic drug is coated in the hydrophobic core of carrier by hydrophobic interaction, genetic stew is then adsorbed on the hydrophilic outer shell of carrier by electrostatic interaction, define cationic amphiphilic polymkeric substance, this polymkeric substance can slowly-releasing hydrophobic drug, carry therapeutic gene, and ensure the integrity of hydrophobic drug and genetic stew structure in transportation.
The fourth object of the present invention is the preparation method providing medicine-gene-carrier system of the present invention, comprises the steps:
(1) carrier and hydrophobic drug are uniformly mixed, after dialysis, lyophilize, obtain carrier loaded drug system;
(2) incubated at room after being mixed with genetic stew by described carrier loaded drug system, obtains medicine-gene-carrier system;
Wherein, the mass ratio of medicine and carrier is 1:(5-30), be preferably 1:(6-25), be more preferably 1:(8-15); The mass ratio (0.01-80) of carrying medicament system and genetic stew: 1, be preferably (0.05-70): 1, be more preferably (0.1-50): 1, incubation time is 20-60min, is preferably 30-50min.
The carrier (Pullulan-SA-PEI) for carrying medicament and gene based on pulullan polysaccharide of the present invention, can self-assembly formation be hydrophobic core with stearic acid in aqueous environment, pulullan polysaccharide and branched chain type polymine be the nano-micelle of hydrophilic outer shell.The kernel of micella can load hydrophobic anticancer drug, and shell can adsorb plasmid DNA, and then realizes the common transmission of medicine and gene.The molecular structure of bifunctional vector Pullulan-SA-PEI uniqueness makes it have potential using value at medicament slow release and field of gene.Pullulan-SA-PEI of the present invention can carrying medicament separately, be applied to and prepare drug-carrier transfer system, also can be applied to and prepare gene-carrier delivery systems by load genetic stew separately, more importantly, because it has the performance of carrying medicament and gene concurrently, to medicine and genetic stew, there is higher utilization ratio, good result for the treatment of can be obtained.Experimental result shows, the carrier of the present invention (DOX/Pullulan-SA-PEI/p53) of load Zorubicin and p53 gene, after human breast cancer cell (MCF-7) is processed, cell survival rate is starkly lower than the vehicle treated group of independent load Zorubicin or p53 gene, illustrates that the cancer resistant effect of conjoint therapy is better than single pharmacological agent or gene therapy.
In the present invention, the preparation method utilizing carrier of the present invention to prepare with Zorubicin the drug-carrier transfer system being carrying medicament is: be dissolved in by Pullulan-SA-PEI in dimethyl sulfoxide (DMSO), the concentration of Pullulan-SA-PEI is 3-5mg/mL, add doxorubicin hydrochloride and triethylamine and be dissolved in the mixed solution that dimethyl sulfoxide (DMSO) formed, in mixed solution, the mol ratio of doxorubicin hydrochloride and triethylamine is 1:(1-3), the concentration of Zorubicin is 4-6mg/mL, Zorubicin is 1:(5-30 with the mixing quality ratio of Pullulan-SA-PEI), stir 0.5-2h, dialysis 48-72h, namely the medicament-carried nano micelle (DOX/Pullulan-SA-PEI) of load Zorubicin is obtained after freeze-drying.
The present invention, relative to prior art, has following advantage:
(1) one of the raw material that the Pullulan-SA-PEI that prepared by the present invention uses pulullan polysaccharide is a kind of natural polysaccharide being rich in hydroxyl, and wide material sources, biocompatibility is good.
(2) stearic acid and low molecular weight polyethylene imines are prepared Pullulan-SA-PEI respectively by esterification and amidate action are grafted on the skeleton of pulullan polysaccharide successively by the present invention, and this preparation method is efficient, reaction conditions is gentle.
(3) Pullulan-SA-PEI that prepared by the present invention can self-assembly formation be hydrophobic core with stearic acid in aqueous environment, and pulullan polysaccharide and branched chain type polymine are the nano-micelle of hydrophilic outer shell.This micella can not only wrap up hydrophobic anticancer drug Zorubicin, and can combined treatment gene type Ⅶ, realizes the common transmission of cancer therapy drug and therapeutic gene, has potential using value in technical field of biological material.
Accompanying drawing explanation
Fig. 1 prepares in the process based on the medicine/gene bifunctional vector Pullulan-SA-PEI of pulullan polysaccharide in embodiment 1, intermediate product and end product
1h NMR collection of illustrative plates, wherein: (a) is for being dissolved in D
2the pulullan polysaccharide of O, (b) is for being dissolved in DMSO-d
6propiram-stearic acid grafted polymkeric substance, (c) and (d) are respectively and are dissolved in D
2o and DMSO-d
6propiram-stearic acid-grafting polyethylene imine polymkeric substance.
Fig. 2 be different N/P than under the agarose gel electrophoresis result figure of Pullulan-SA-PEI/p β Gal mixture, wherein swimming lane 1 is that Free p β Gal, swimming lane 2-7 are respectively N/P than being Pullulan-SA-PEI/p β Gal mixture under 0.156,0.3125,0.625,1.25,2.5,5;
Fig. 3 is the transmission electron microscope picture of the Pullulan-SA-PEI bifunctional vector after bag carries Zorubicin and adsorbs p β Gal;
Fig. 4 is the drug release behavior outcome figure in free Zorubicin and the PBS damping fluid of medicament-carried nano micelle under condition of different pH;
Fig. 5 is the transfection efficiency result figure of the Pullulan-SA-PEI/p β Gal mixture under different N/P;
Fig. 6 is that Pullulan-SA-PEI/p53 mixture under different N/P is to the toxicity detection result figure of human breast cancer cell effect 48h;
Fig. 7 is that DOX/Pullulan-SA-PEI medicament-carried nano micelle, Pullulan-SA-PEI/p53 mixture, DOX/Pullulan-SA-PEI/p53 are to the toxicity detection result figure of human breast cancer cell effect 48h.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can make some transformations and distortion, these all belong to protection scope of the present invention.In following embodiment, if no special instructions, the experimental technique used is ordinary method, and material therefor, reagent etc. all can be bought from biological or chemical company.
The preparation of embodiment 1 Pullulan-SA-PEI
(1), under nitrogen protection, 1.64g pulullan polysaccharide (molecular-weight average is 100kDa, 10mM sugar unit) is dissolved in 30mL dimethyl sulfoxide (DMSO), obtains pulullan polysaccharide (referred to as Pullulan) solution; By 0.57g stearic acid (2mM), 0.61g 4-dimethylamino pyrrole and 0.96g 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (mol ratio is 1:1:1.2) is dissolved in 5mL dimethyl sulfoxide (DMSO), stir-activating 1h at 25 DEG C, obtains stearic acid activation solution; Stearic acid activation solution is added in pulullan polysaccharide solution, stirring reaction 48h at 25 DEG C, after reaction terminates, by dehydrated alcohol precipitation, with washed with diethylether precipitation several, collected by centrifugation product, be scattered in a small amount of single steaming water, dialysis purifying 72h, lyophilize, obtain Propiram-stearic acid grafted polymkeric substance (referred to as Pullulan-SA), show that stearic percentage of grafting is about 9.27% by hydrogen spectrum;
(2) under nitrogen protection, 0.95g Propiram-stearic acid grafted polymkeric substance (5mM) is dissolved in 20mL dimethyl sulfoxide (DMSO), stirring and dissolving, add 0.75g succinyl oxide (7.5mM) and 0.12g DMAP (1mM), stirring reaction 18h at 50 DEG C, after reaction terminates, precipitate with dehydrated alcohol, collected by centrifugation product, be scattered in a small amount of single steaming water, dialysis purifying, lyophilize, obtain Propiram-stearic acid-succinyl oxide graftomer (referred to as Pullulan-SA-COOH), use acid base titration to calculate carboxyl substituted degree;
(3) by 220mg Propiram-stearic acid-succinyl oxide graftomer (n
-COOH=6.50 × 10
-4mol) be dissolved in 20mL phosphate buffered saline buffer, add 187mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (9.75 × 10
-4and 112mg N-hydroxy-succinamide (9.75 × 10 mol)
-4mol), stir-activating 4h at 4 DEG C, then (weight-average molecular weight is 1000Da to add 171mg branched chain type polymine; n
-NH2=6.50 × 10
-4mol), stirring reaction 24h at 25 DEG C, dialysis purifying, lyophilize, obtains Propiram-stearic acid-grafting polyethylene imine polymer P ullulan-SA-PEI, show that the nitrogen content of polymkeric substance is about 11.09% by ultimate analysis.
The product P ullulan-SA-PEI obtained take stearic acid as hydrophobic core, and with pulullan polysaccharide and branched chain type polymine for hydrophilic outer shell, the structural formula of Pullulan-SA-PEI is:
In formula I, m is the integer between 5-24, and n is the integer between 11 to 47.
Embodiment 2 hydrogen stave is levied
Pullulan-SA-PEI and Pullulan-SA, the Pullulan that embodiment 1 are prepared are dissolved in deuterated dimethyl sulfoxide or deuterated water, by nucleus magnetic resonance (
1h NMR) carry out structure verification.
Result as shown in Figure 1, relative to Pullulan (a, D
2o) collection of illustrative plates, Pullulan-SA (b, DMSO-d
6) collection of illustrative plates in there is new nuclear-magnetism peak ,-CH on the corresponding stearic acid of δ 1.23ppm
2-characteristic peak ,-CH on the corresponding stearic acid of δ 0.85ppm
3characteristic peak.At Pullulan-SA-PEI (c, D
2o), in collection of illustrative plates, there is new nuclear-magnetism peak in δ 2.43-3.05ppm place, correspond to-NHCH in polymine
2cH
2-characteristic peak, but the characteristic peak of stearic acid δ 0.85 and δ 1.23ppm but disappears, may be because in heavy water, amphipathic pulullan polysaccharide derivative self-assembled forms nucleocapsid structure, stearic acid is owing to being wrapped the existence entering and cannot detect characteristic peak in hydrophobic core, pulullan polysaccharide and polymine are owing to having the shell of wetting ability as micella, and therefore characteristic of correspondence peak can be detected.At Pullulan-SA-PEI (d, DMSO-d
6) collection of illustrative plates in, the characteristic peak of stearic acid δ 0.85 and δ 1.23ppm occurs again, and this may be because in DMSO environment, and Pullulan-SA-PEI cannot form micellar structure, and stearic acid can not be wrapped and enter hydrophobic core.
The preparation of embodiment 3 DOX/Pullulan-SA-PEI medicament-carried nano micelle
20mg Pullulan-SA-PEI embodiment 1 prepared is dissolved in 4mL DMSO, add and be dissolved in mixed solution that 400 μ L DMSO are formed (wherein by 2.1mg doxorubicin hydrochloride and 1.03 μ L triethylamines, the mol ratio of doxorubicin hydrochloride and triethylamine is 1:2, the concentration of Zorubicin is 5mg/mL, Zorubicin is 1:10 with the mixing quality ratio of Pullulan-SA-PEI), and stirring 1h, dialysis 72h, namely obtains DOX/Pullulan-SA-PEI medicament-carried nano micelle after freeze-drying.
The preparation of embodiment 4 Pullulan-SA-PEI/p β Gal mixture and agarose gel electrophoresis experiment
The Pullulan-SA-PEI that embodiment 1 prepares is mixed with the aqueous solution of a series of different concns, constant every hole 1 μ g p β Gal, according to the different N/P value (mass ratioes of N/P:Pullulan-SA-PEI and p β Gal, 0.156, 0.3125, 0.625, 1.25, 2.5, 5) Pullulan-SA-PEI and the p of different concns β Gal equal-volume is mixed, after vortex 30s, room temperature leaves standstill 40 minutes, form stable Pullulan-SA-PEI/p β Gal mixture, the ability of bifunctional vector Pullulan-SA-PEI in conjunction with DNA is detected by agarose gel electrophoresis experiment, as Fig. 2.In Fig. 2, swimming lane 1 is Free DNA, swimming lane 2-7 be respectively different N/P than under Pullulan-SA-PEI/p β Gal mixture, the N/P that 2-7 represents respectively is 0.156,0.3125,0.625,1.25,2.5,5.As can be seen from Figure 2: obvious migration has appearred in swimming lane 1 in the electric field; For Pullulan-SA-PEI/p β Gal mixture, when N/P is less than 2.5, part p β Gal to fail by combining completely anode migration (swimming lane 2-5), and when N/P is more than or equal to 2.5, p β Gal is arrested in (swimming lane 6-7) in well by combining completely, illustrate that bifunctional vector Pullulan-SA-PEI has stronger DNA binding ability to form stable mixture.
The transmission electron microscope picture of embodiment 5 DOX/Pullulan-SA-PEI/pDNA bifunctional vector
DOX/Pullulan-SA-PEI medicament-carried nano micelle is prepared according to embodiment 3, be mixed with the certain density aqueous solution, adopt this aqueous solution, prepare according to embodiment 4 the dual-functional nanometer carrier DOX/Pullulan-SA-PEI/pDNA that N/P is 10, use determination of transmission electron microscopy Pullulan-SA-PEI to wrap up cancer therapy drug and the particle diameter adsorbed after gene and form at the same time.Result is as shown in Figure 3: after carrier carries medicine and gene at the same time, particle diameter is at about 30nm, and structure is for tightening ball-type, and size distribution is more homogeneous.
Embodiment 6 DOX/Pullulan-SA-PEI medicament-carried nano micelle vitro drug release is tested
DOX/Pullulan-SA-PEI medicament-carried nano micelle bag prepared by embodiment 3 being loaded with 200 μ g Zorubicins is dissolved in the different pH of 2mL respectively and (is respectively 3.0, 5.0, 6.5, 7.4) in PBS damping fluid, be transferred in dialysis tubing, be positioned in the PBS damping fluid of corresponding pH, 37 DEG C of constant temperature lucifuges stir dialysis on magnetic stirring apparatus, respectively 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120h takes out 3mL dialyzate, add the fresh PBS damping fluid of the corresponding pH of 3mL simultaneously, at λ ex=484nm, fluorescence intensity is recorded in spectrophotofluorometer under λ em=590nm condition, obtain drug release patterns.Result is as shown in Figure 4: free DOX (Free DOX) discharges completely when 7h, and the Zorubicin of nano-micelle discharges, and can be divided into dashes forward releases stage and slowly-releasing stage, release the stage (front 24h) prominent, nano-micelle discharges the DOX medicine of package-contained fast, thus reaches the dosage killing cancer cells; At slowly-releasing stage (rear 96h), the Zorubicin medicine of nano-micelle slow releasing package-contained, maintains drug dose, strengthens cancer resistant effect.In addition, the pH of release medium also can affect the release conditions of medicine, as can be seen from Figure 4: along with the reduction of release medium pH, the release rate of DOX is accelerated gradually, when pH is reduced to 3.0 from 7.4, when 120h, in medicament-carried nano micelle, the cumulative release amount of Zorubicin brings up to 73.47% by 51.86%.May be because: hydrophilic outer shell polymine is protonated in acid condition, and electrostatic repulsion causes hydrophilic outer shell to expand gradually, the Zorubicin in hydrophobic core is easily diffused out, causes drug releasing rate to be accelerated.Above result shows that DOX/Pullulan-SA-PEI medicament-carried nano micelle is a kind of good drug sustained release system, and drug releasing rate is accelerated along with the reduction of environmental pH.
Embodiment 7 in-vitro transfection is tested
The African green monkey kidney inoblast (COS 7) of logarithmic phase is according to 1 × 10
5/ hole is inoculated in 6 orifice plates, and every hole 2mL substratum, at 37 DEG C, 5%CO
2carefully suck original substratum after lower cultivation 24h, PBS cleaning twice, adds serum-free antibiotic-free DMEM substratum at 37 DEG C, 5%CO
2lower cultivation 1h, the Pullulan-SA-PEI/p β Gal mixture (containing 4 μ g p β Gal) of different N/P (2.5,5,10,20) is prepared according to embodiment 4, suck serum-free antibiotic-free DMEM substratum, the Pullulan-SA-PEI/p β Gal mixture of different N/P is added in corresponding hole, supplement serum-free antibiotic-free DMEM substratum to 2mL, at 37 DEG C, 5%CO
2after lower cultivation 4h, serum-free antibiotic-free DMEM substratum is replaced with normal incubation medium, at 37 DEG C, 5%CO
2clean twice with PBS after 48h is cultivated in lower continuation, add 500 μ L lysis buffers, incubated at room 5min, get appropriate amount lysate and detect beta galactosidase enzyme activity according to beta galactosidase enzyme detection system operation instructions, separately get appropriate amount lysate using modified type BCA quantification of protein detection kit and measure protein content, the expression amount of beta galactosidase enzyme represents with relative luminous intensity/milligram albumen.In transfection experiment, lipofectamine 2000 is set and is respectively positive control and negative control with free p β Gal.As can be seen from Figure 5: along with the increase of N/P, the transfection efficiency of Pullulan-SA-PEI/p β Gal mixture first increases rear reduction, and when N/P is 10, transfection efficiency is the highest, and effect is slightly worse than lipofectamine 2000, but is obviously better than free p β Gal.Above result shows: pDNA can effectively be transported in cell by Pullulan-SA-PEI bifunctional vector, and is expressed preferably.
Embodiment 8 Pullulan/p53 mixture cytotoxicity experiment
The human breast cancer cell (MCF-7) of logarithmic phase is according to 1 × 10
4/ hole is inoculated in 96 orifice plates, and every hole 100 μ L, at 37 DEG C, 5%CO
2lower cultivation 24h.The Pullulan-SA-PEI/p53 mixture of different N/P (1.25,2.5,5,10,20) is prepared, at 37 DEG C, 5%CO according to embodiment 4
2after lower cultivation 48h, every hole adds 10 μ L MTT (final concentration is 0.5mg/mL), 37 DEG C hatch 4h after, suck MTT, every hole adds 100 μ L DMSO and dissolves first a ceremonial jade-ladle, used in libations, uses microplate reader to detect the absorbancy at 570 and 630nm place immediately.Meanwhile, also detect the cytotoxicity of carrier itself, carrier concn is respectively 2,4,8,16,32 μ g/mL (correspond to carrier concn when N/P is 1.25,2.5,5,10,20), without the cell of any process as positive control.As can be seen from Figure 6: along with the rising of N/P, the toxicity of Pullulan-SA-PEI/p53 mixture to cancer cells strengthens gradually, all be better than Free p53, within the scope of studied N/P, the cell survival rate of vehicle treated group is all more than 80%, highlight the good biological safety of carrier, therapeutic gene effectively can be carried into cancer cells simultaneously, and be expressed.
Embodiment 9 DOX/Pullulan-SA-PEI/p53 cytotoxicity experiment
The human breast cancer cell (MCF-7) of logarithmic phase is according to 1 × 10
4/ hole is inoculated in 96 orifice plates, and every hole 100 μ L, at 37 DEG C, 5%CO
2lower cultivation 24h.DOX/Pullulan-SA-PEI medicament-carried nano micelle (DOX concentration is 0.36 μ g/mL) is prepared according to embodiment 3, the Pullulan-SA-PEI/p53 mixture that N/P is 2.5 is prepared according to embodiment 4, (DOX concentration is 0.36 μ g/mL to prepare DOX/Pullulan-SA-PEI/p53 according to embodiment 5, N/P is 2.5), other treatment group also comprises Pullulan-SA-PEI, Free p53, Free DOX, after different experiments group function cells 48h, every hole adds 10 μ L MTT (final concentration is 0.5mg/mL), at 37 DEG C, 5%CO
2under hatch 4h after, suck MTT, every hole adds 100 μ L DMSO and dissolves first a ceremonial jade-ladle, used in libations, uses microplate reader to detect the absorbancy at 570 and 630nm place immediately, without the cell of any process as positive control.As can be seen from Figure 7, vehicle treated group cell survival rate, more than 80%, illustrates that the biocompatibility of carrier is good; The anticancer effect of Pullulan-SA-PEI/p53 mixture is obviously better than the therapeutic gene (Free p53) dissociated, the anticancer effect of DOX/Pullulan-SA-PEI medicament-carried nano micelle is slightly better than the cancer therapy drug (Free DOX) dissociated, and shows that cancer therapy drug and therapeutic gene can effectively be transported in cancer cells to produce result for the treatment of by bifunctional vector Pullulan-SA-PEI; More importantly, the cancer cell survival rate of DOX/Pullulan-SA-PEI and Pullulan-SA-PEI/p53 treatment group is respectively 41.45 ± 1.17% and 47.85 ± 1.82%, and the cancer cell survival rate of DOX/Pullulan-SA-PEI/p53 treatment group is 21.99 ± 0.45%, illustrate that the cancer resistant effect of conjoint therapy is better than single pharmacological agent or gene therapy.
Claims (10)
1. one kind based on pulullan polysaccharide for carrying medicament or/and the carrier of gene, it is characterized in that: described carrier take stearic acid as hydrophobic core, with pulullan polysaccharide and low molecular weight polyethylene imines for hydrophilic outer shell, this carrier has the structure of formula I or formula II:
In formula I, m is the integer between 5 to 24, and n is the integer between 11 to 47; In formula II, n is the integer between 9 to 45.
2. carrier according to claim 1, is characterized in that, described low molecular weight polyethylene imines is branched chain type polymine.
3. carrier according to claim 1, it is characterized in that: the weight-average molecular weight of described low molecular weight polyethylene imines is 500-2000Da, the molecular-weight average of described pulullan polysaccharide is 30-200kDa, described stearic acid grafted rate is 3-10%, and described low molecular weight polyethylene imines percentage of grafting represents with nitrogen content: be 5-12%.
4. the preparation method of the carrier as described in any one of claim 1-3, it is characterized in that, stearic acid and low molecular weight polyethylene imines are grafted on the skeleton of pulullan polysaccharide respectively by esterification and amidate action and prepare by described carrier successively.
5. preparation method according to claim 4, is characterized in that, the method comprises the steps:
(1) under nitrogen protection, stearic acid and DMAP and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine hydrochloric acid is added in pulullan polysaccharide, stirring reaction 30-70h at 15-50 DEG C, collect product, product, through dialysis purifying, lyophilize, obtains Propiram-stearic acid grafted polymkeric substance; Reaction solvent is dimethyl sulfoxide (DMSO);
(2) under nitrogen protection, succinyl oxide and DMAP is added in the Propiram that step (1) obtains-stearic acid grafted polymkeric substance, stirring reaction 5-30h at 30-70 DEG C, collect product, product, through dialysis purifying, lyophilize, obtains Propiram-stearic acid-succinyl oxide graftomer; Reaction solvent is dimethyl sulfoxide (DMSO);
(3) under nitrogen protection, in Propiram-stearic acid-succinyl oxide graftomer that step (2) obtains, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide, stir-activating 2-8h at 2-8 DEG C, add low molecular weight polyethylene imines again, stirring reaction 10-30h at 15-50 DEG C, through dialysis purifying, lyophilize, obtains Propiram-stearic acid-grafting polyethylene imine polymkeric substance; Reaction solvent is phosphate buffered saline buffer;
Wherein:
Pulullan polysaccharide described in step (1) and stearic mol ratio are (1-5): 1-30:1, and the mol ratio of stearic acid, DMAP and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate is 1:(0.5-2): (1-2.5);
The mol ratio of the Propiram described in step (2)-stearic acid grafted polymkeric substance and succinyl oxide is 1:(1-3), the mol ratio of DMAP and succinyl oxide is 1:(4-12).
Mol ratio amino in carboxyl and branched chain type polymine in Propiram-stearic acid-succinyl oxide graftomer described in step (3) is 1:(0.1-5), in Propiram-stearic acid-succinyl oxide graftomer, the mol ratio of carboxyl, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide is 1:(0.1-3): (0.1-3).
6. medicine-gene-carrier system, comprise carrier and load hydrophobic anticancer drug on the carrier and genetic stew, wherein said carrier is the carrier described in any one of claim 1-3.
7. medicine-gene-carrier system according to claim 6, is characterized in that, described hydrophobic anticancer drug is the one in Zorubicin, taxol, camptothecine.
8. medicine-gene-carrier system according to claim 6, is characterized in that, described genetic stew is the one in plasmid DNA, chromosomal DNA, Mitochondrial DNA.
9. medicine-gene-carrier system according to claim 6, is characterized in that, described plasmid DNA is the one in p β Gal, p53.
10. the preparation method of the medicine-gene-carrier system described in any one of claim 6-9, comprises the steps:
(1) carrier and hydrophobic drug are uniformly mixed, after dialysis, lyophilize, obtain carrier loaded drug system;
(2) incubated at room after being mixed with genetic stew by described carrier loaded drug system, obtains medicine-gene-carrier system;
Wherein, the mass ratio of hydrophobic drug and carrier is 1:(5-30); The mass ratio of carrying medicament system and genetic stew is (0.01-80): 1; Described incubation time is 20-60min.
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| CN107778481A (en) * | 2016-08-25 | 2018-03-09 | 徐州医科大学 | A kind of preparation method and applications of ligand modified inositol polyethyleneimine genophore |
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| CN108498458A (en) * | 2018-05-03 | 2018-09-07 | 大连理工大学 | A kind of nano-medicament carrier, medicament carrier system and preparation method for ultrasound control release based on pulullan polysaccharide |
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| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| CN115737887A (en) * | 2022-11-07 | 2023-03-07 | 河北大学 | Skin wound dressing and preparation method thereof |
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| CN105566511A (en) * | 2016-01-27 | 2016-05-11 | 天津医科大学 | Charge reversal Pulullan derivative and synthesis method and application thereof |
| CN105566511B (en) * | 2016-01-27 | 2017-11-03 | 天津医科大学 | Electric charge overturns pulullan polysaccharide derivative and its preparation method and use |
| CN107778481A (en) * | 2016-08-25 | 2018-03-09 | 徐州医科大学 | A kind of preparation method and applications of ligand modified inositol polyethyleneimine genophore |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
| CN108078939A (en) * | 2018-01-25 | 2018-05-29 | 庄明莲 | A kind of anti-cancer medicament carrier preparation and its application |
| CN108607098A (en) * | 2018-04-28 | 2018-10-02 | 大连理工大学 | The load drug carrier of Liver targeting Intelligence Ultrasound response drug release, preparation method and applications |
| CN108498458A (en) * | 2018-05-03 | 2018-09-07 | 大连理工大学 | A kind of nano-medicament carrier, medicament carrier system and preparation method for ultrasound control release based on pulullan polysaccharide |
| CN108498458B (en) * | 2018-05-03 | 2019-10-11 | 大连理工大学 | A kind of nano-medicament carrier, medicament carrier system and preparation method for ultrasound control release based on pulullan polysaccharide |
| CN115737887A (en) * | 2022-11-07 | 2023-03-07 | 河北大学 | Skin wound dressing and preparation method thereof |
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