Targeted to the building and application of endocytoplasmic reticulum nano medicament carrying systemTechnical field
The invention belongs to field of medicaments, it is related to a kind of novel to carry medicine system targeted to the novel nano of endocytoplasmic reticulumSystem and the drug-loading system are being prepared using endoplasmic reticulum as the application in the drugs such as the antiviral and antitumor of target.
Background technique
Drug needs to reach action target spot competence exertion drug effect, the target spot of drug be generally located at intracellular protein,The functional biomolecules such as nucleic acid, enzyme and receptor.Modern drug delivery system requires drug that can be transported to target tissue, target cell, veryTo being specific organelle.Nano-carrier has many advantages, such as to protect drug, reaches sustained release and toxic side effect is small.It organizes at present and thinThe research that born of the same parents' target administration passs release system have been relatively mature, and three-level targeting, i.e. organelle targeting is ground in current drug delivery systemThe hot and difficult issue studied carefully.Three-level targeting includes that cytoplasm targets, and organelle targeting is such as Mitochondrially targeted, endoplasmic reticulum targeting, lyaseBody targeting etc., cell nucleus targeting etc..
Endoplasmic reticulum (endoplasmic reticulum, ER) is the closed mesh duct system being made of inner membrance, isIntracellular protein synthesis, lipid synthesis, matter transportation, carbohydate metabolism, its major function of the main place of removing toxic substances are synthesis eggsWhite matter and lipid.Endoplasmic reticulum is included there are many enzyme, and marker enzyme is G-6-Pase, participates in the different metabolism of glycogen, endoplasmic reticulumIntracavitary hydrolase is used for various drug metabolisms and removing toxic substances.Cytochrome P450 on liver, intestinal cell endoplasmic reticulum is human bodyThe Major Enzymes of intracellular metabolite drug can be catalyzed (including most of clinical medicines) metabolism of a variety of inside and outside source substances.Body secretesAlbumen and memebrane protein are in translation process or are transported in endoplasmic and are modified after translation, fold, correct to assemble.When interiorWhen unfolded or false folding albumen increases in matter net, stress signal can be transmitted in nucleus by endoplasmic reticulum, thenCause serial specific target gene transcriptional upregulation and protein translation horizontal down-regulation, so that cell can continue to survive, this reactionIt is exactly Non-adhesion inhibition index (UPR), the effect of the existing protection cell of UPR, and there is cytotoxic effect, cause cell to witherIt dies, so as to cause a series of diseases, such as Alzheimer's disease, the generation of cystic fibrosis, diabetes etc..Accordingly, endoplasmic reticulum canAs the novel targets of oncotherapy, some cytotoxic drugs can be used according to the access that it is induced cell apoptosis and induce endoplasmic reticulumStress, accelerate the apoptosis of cancer cell to treat cancer.Disease can also be blocked to induce using some drugs or cell factorAbnormal endoplasmic reticulum stress response reduces the apoptosis of even reverse both.
The target administration of ER is not given sufficient attention at present.The pertinent literature studied it is relatively fewer.Costin etc. contains endoplasmic reticulum N- glycosylation inhibitor n-butyl deoxynojirimycin in pH sensitive liposome (by DOPE and gallbladderSterol hemisuccinic acid ester composition) in, mouse melanin tumor cell tyrosine activity is successfully inhibited, and substantially reduce to medicamentMeasure (Gertrude-E.Costin, Mihaela Trif, et.al pH-sensitive liposomes are efficientcarriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells[J],BBRC293(2002):918–923)。
Pardaxin (PA) is to be separated to one kind from leopard sole to contain 33 amino acid residue peptide (sequences are as follows: H-GFFIt ALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH), is amphipathic cation α spiral knot isolated from fish earliestStructure has the polypeptide of membrane penetration effect, is a kind of stronger antibacterial active peptide of effect.Furthermore Pardaxin also has Inhibit proliferaton simultaneouslyInduce the effect of human carcinoma cell line's apoptosis.The structure of its 33 amino acid includes many cationics and amphiphilic acidic amino acid,It makes it easier to interact with anionic membrane, due to its special configuration, is lower than 10 in concentration-7Pardaxin can be thin when MSingle pass aperture is formed on born of the same parents' bilayer lipid membrane, is 10 in concentration-7-10-4M Shi Zehui causes cell dissolution (DoronRapaport,Yechiel Shai.Interaction of Fluorescently Labled Pardaxin and ItsAnalogues with Lipid Bilayer.[J]The Journal of Biological Chemistry 266(1991): 23769-23775；Peter I.Lelkes and Philip Lazarovici.Pardaxin inducesaggregation but not fusion of phosphatidylserine vesicles[J].FEBS LETTERS,230(1988):131-136； Bhunia A,Domadia PN,Torres J,et.al.NMR structure of pardaxin,a pore-forming antimicrobial peptide,in lipopolysaccharide micelles:mechanismOf outer membrane permeabilization [J] .Biol Chem, 285 (2010): 3883-3895), low dosageWhen, Pardaxin can avoid mitochondria, golgiosome and lysosome after entering born of the same parents, be directed to endoplasmic reticulum.Chen-Hung Ting couplePardaxin enters the positioning after born of the same parents and has carried out fluorescent tracing research, and discovery Pardaxin accumulates in endoplasmic reticulum (Chen- after entering born of the same parentsHung Ting, Han-Ning Huang,et al The mechanisms by which pardaxin,a naturalcationic antimicrobial peptide,targets the endoplasmic reticulum and inducesc-FOS[J]. Biomaterials,35(2014):3627-3640)。
Summary of the invention
The object of the present invention is to provide a kind of building targeted to endocytoplasmic reticulum nano medicament carrying system, the present invention utilizes waterDissolubility polypeptide Pardaxin has endoplasmic reticulum specificity taxis characteristic, is modified on nano-carrier surface, so promotes to receiveAfter rice carrier cell internalization, there is the selectively targeted ability of endoplasmic reticulum, realize the endoplasm of drug in nano-carrier and carrierThe accumulation of net site specific.It is realized by following scheme: by water-soluble polypeptide Pardaxin to be chemically bonded or physical absorptionMode is modified on nano-carrier surface, and the Pardaxin is containing 33 amino acid residue peptides, and amino acid sequence is:H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH。。
Particular content is as follows:
1.Pardaxin modifies nano-carrier and is realized by following chemistry and/or physical method:
(1) using the amino or carboxyl in Pardaxin molecule, chemical bonding directly occurs with nano-carrier and completes to repairDecorations.
(2) or amino or carboxyl in Pardaxin molecule is utilized first to change with a kind of compound (its connection function)Bonding is learned, then the compound for playing connection function is incorporated in and completes to modify on nano-carrier.This connection function compound is such asPolyethylene glycol, polyethyleneimine with active reactive group etc..
(3) or by Physical interactions such as charge adsorption, hydrogen bond actions realize Pardaxin polypeptide in nanoparticle sublistThe modification in face.
2. nano liposomes carrier constructing Pardaxin modification, being targeted with endoplasmic reticulum:
Using film dispersion method, injection method etc. will prepare liposome materials (phosphatide, cholesterol, DSPE-PEG-PardaxinDeng；For example fat-soluble medicine is added at this time), according to molar ratio, (phosphatide: cholesterol molar ratio is 10~0.5:1, DSPE-PEG-Pardaxin be total lipid weight 0.01%-10%) mixing, be added organic solvent dissolution (such as chloroform, methylene chloride,Ethyl alcohol etc.), film, adding water property medium (aqueous solution of saliferous and/or/person's water soluble drug) water are formed by 1) rotary evaporationChange forms liposome；Or it 2) is directly injected into aqueous medium and forms liposome.Liposomal particle size range is 1-1000 nanometers.
It is a further object to provide described targeted to endocytoplasmic reticulum nano medicament carrying system in preparation with endoplasmNet is the application in the drug of target.Contained drug can or water-soluble antitumor medicine for example fat-soluble for different physicochemical property drugsThe pro-drug that object, different therapy field drugs are for example antitumor, anti-inflammatory drug or needs are further activated in the online enzyme of endoplasmAnd the therapeutic agent of endoplasmic reticulum related disease, and the drug or the factor of er stress can be directed to.Pardaxin is exposed toOutside nano-carrier, serve as target head, can specificity will enter the medicament-carried nano liposome vectors after born of the same parents targeted to endocytoplasmic reticulum, andEndoplasmic reticulum release drug or specificity cause to cause endoplasmic reticulum behavior stress reaction (such as exciting or inhibit er stress).Involved disease includes tumour, such as liver cancer；Infective inflammation, and disease related with er stress, such as A ErziThe silent disease in sea, diabetes etc..
In this application process, mainly according to the physicochemical property of drug, (such as lipophilic is hydrophilic for the preparation method of drug-carrying nanometer particleProperty, electrical property etc.) from the difference of nano-carrier and it is different, similar phase dissolution method can be taken to complete package fat-soluble medicine, canIt will complete to load after drug and lipid mixed dissolution film forming.Water soluble drug can be completed using its acid-base property or charging propertyThe loading of Nano medication.Water-soluble pesticide such as cyclophosphamide, adriamycin, fat-soluble medicine such as dexamethasone acetate etc..
The present invention is that water-soluble polypeptide Pardaxin is integrated to nano-carrier surface.The present invention typically willPardaxin is modified in surface of liposome.Utilize DSPE-PEG-NH2Amino and Pardaxin carboxyl carry out dehydrating condensation it is anti-More stable amido bond should be formed.DSPE-PEG-Pardaxin is formd after modifying, the modifier be it is amphipathic, DSPE isHydrophobic side, PEG-Pardaxin are water-wet side.Hydrophobic side can be used as " anchor ", and rivet divides in the double of lipid carrier such as liposomeOn sublayer film, water-wet side is exposed to " target head " outer, as targeting endoplasmic reticulum.
Endoplasmic reticulum target liposomes constructed by the present invention can load water-soluble and fat-soluble medicine, and by drug deliveryTo endoplasmic reticulum position, enhance pharmacotherapeutic efficacy, reduces toxic side effect.Nano-carrier of the present invention is not limited solely to lipidBody is also possible to solid lipid nano granule, nano-emulsion, polymer micelle and inorganic nano material etc..It is new constructed by the present inventionType endoplasmic reticulum targeted nano carrier can significantly improve the concentration for acting on target area at it as the drug of target using endoplasmic reticulum, beThe curative effect performance of these drugs provides a new approach.
Detailed description of the invention
Fig. 1 is DSPE-PEG, the nuclear magnetic resonance figures hydrogen spectrum of Pardaxins, DSPE-PEG-Pardaxin.
The influence of the comparison liposome encapsulation of phosphatide and drug when Fig. 2 is film dispersion method preparation.
Fig. 3 is the influence of the comparison liposome encapsulation of phosphatide and drug when being prepared with Calcium acetate gradient gradient method.
Fig. 4 is endoplasmic reticulum targeting cyclophosphamide liposome endoplasmic reticulum common location figure.
Fig. 5 is endoplasmic reticulum targeting cyclophosphamide liposome lysosome escape figure.
Fig. 6 is endoplasmic reticulum targeting cyclophosphamide liposome to SKOV-3, the cell toxicity test of HepG-2.
Specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1
The synthesis of DSPE-PEG-Pardaxin: precision weighs Pardaxin, is dissolved in 3mL-10mL anhydrous DMF solvent,(BOC) is added under ice bath being protected from light2O reagent protects 4 free amine groups on Pardaxin polypeptide, (BOC)2O:PardaxinMolar ratio is 5.2:1, is protected from light nitrogen-sealed and carries out reaction 12h.Through (BOC)2After the protection of O reagent, EDC and NHS activation is addedCarboxyl on Pardaxin polypeptide, EDC:Pardaxin molar ratio are 10:1, and NHS:Pardaxin molar ratio is living under 5:1 room temperatureChange 2 hours of reaction.After activation, DSPE-PEG-NH is added2, react 24 hours under magnetic agitation, DSPE-PEG-NH2:Pardaxin molar ratio is 1:1.After reaction, 2 hours are stirred to react with 1ml 12M HCI slough BOC protection, it is rear to use3M NaOH (1.2g is dissolved in 10mL water) recalls to neutrality, dialysis, is lyophilized up to DSPE-PEG-Pardaxin, structural formula is as follows:
DSPE-PEG-Pardaxin structural identification: using nuclear magnetic resonance (Nuclear Magnetic Resonance,NMR), DSPE-PEG-Pardaxin is carried out1H NMR and IR spectrum scanning structural identification (Fig. 1).DSPE-PEG-In Pardaxin hydrogen nuclear magnetic resonance spectrogram, at chemical shift 6.81-8.23ppm, there are multiple sharp signal peaks, it shouldThe characteristic peak that peak belongs to as amido bond；At chemical shift 4.46-4.65ppm, there is an apparent bimodal signal, it shouldPeak belongs to methine signals peak adjacent with amido bond in Pardaxin polypeptide；In addition, Pardaxin polypeptide is in chemical shiftTwo unimodal signals of carboxyl of 12.15-12.76 ppm have disappeared, and illustrate to have successfully synthesized DSPE-PEG-Pardaxin。
Fig. 1 is DSPE-PEG, the nuclear magnetic resonance figures hydrogen spectrum of Pardaxins, DSPE-PEG-Pardaxin.It is as shown in the figure:In DSPE-PEG-Pardaxin hydrogen nuclear magnetic resonance spectrogram, at chemical shift 6.81-8.23ppm, there are multiple sharp lettersNumber peak, the characteristic peak which belongs to as amido bond；At chemical shift 4.46-4.65ppm, occur one it is apparent bimodalSignal, the peak belong to, the methine signals peak adjacent with amido bond in Pardaxin polypeptide；In addition, Pardaxin polypeptide existsTwo unimodal signals of carboxyl of chemical shift 12.15-12.76ppm have disappeared, and illustrate to have successfully synthesized DSPE-PEG-Pardaxin。
Embodiment 2
The synthesis of Thioctic Acid (TA)-PEG-Pardaxin: pass through NH2-PEG-NH2With lipoic acid (ThiocticAcid, TA) between dehydration synthesize NH2-PEG-TA.Firstly, DCC, NHS (molar ratio: 1:5:10) are dissolved in by TA2 hours are stirred in DMF and at 60 DEG C to activate the carboxyl on TA.Then, a certain amount of NH is added2-PEG-NH2 (NH2-PEG-NH2: TA=1:2, mol/mol) and continue stirring 24 hours.Crude product distilled water is dialysed 48 hours, is then lyophilized,Obtain NH2-PEG-TA。
Before synthesizing Pardaxin-PEG-TA, using the above method, the amino on pardaxin peptide is also by (BOC)2OProtection.Later, the carboxyl on FAL is activated using EDC and NHS (Pardaxin:EDC:NHS=1:5:10, mol/mol).SoAfterwards, NH is added2-PEG-TA(Pardaxin:NH2- PEG-TA=1:1, mol/mol) and continue stirring 24 hours.Terminate in reactionWhen, blocking group is removed using HCl, and pH value is adjusted by NaOH.After further dialysis and freeze-drying, Pardaxin- is collectedPEG-TA is simultaneously stored using preceding in 4 DEG C.
Embodiment 3
The synthesis of PCL-PEG-Pardaxin: (BOC) is added under ice bath being protected from light2O reagent protects the upstream PardaxinFrom amino, (BOC)2O:Pardaxin molar ratio is 5.2:1, is protected from light nitrogen-sealed and carries out reaction 12h.Through (BOC)2O reagent is protectedAfter shield, carboxyl on EDC and NHS activation Pardaxin polypeptide is added, EDC:Pardaxin molar ratio is 10:1, NHS:PardaxinMolar ratio is 2 hours of priming reaction under 5:1 room temperature.After activation, the polyethylene glycol-polycaprolactone with aminoterminal is added(PCL-PEG-NH2), it reacts 24 hours under magnetic agitation, PCL-PEG-NH2: Pardaxin molar ratio is 1:1.After reaction,2 hours are stirred to react with 1ml 12M HCI and slough BOC protection, with 3M NaOH (1.2g is dissolved in 10mL water) recall to neutrality afterwards,Dialysis is lyophilized up to PCL-PEG-Pardaxin.
Embodiment 4
The composition of endoplasmic reticulum targeting cyclophosphamide liposome:
The endoplasmic reticulum Targeting Effect and pharmacodynamic results of endoplasmic reticulum target liposomes prepared by the present invention are investigated, the present invention is with realityFor the cyclophosphamide endoplasmic reticulum target liposomes of example 4, studied.
Cyclophosphamide is current clinically most common alkylating agents antineoplastic, into after in vivo, in hepatomicrosome enzymeLower decompose of catalysis releases the very strong chloroethyl phosphamide of alkanisation or phosphamide mustargen, and generates cell toxicant to tumour cellEffect, furthermore this product also has significant immunosuppressive action.It is clinically used for malignant lymphoma, Huppert's disease, leukaemia, creamGland cancer, oophoroma, cervical carcinoma, prostate cancer, colon cancer, bronchiolar carcinoma, lung cancer etc., there is certain curative effect.It can also be used for rheumatoidThe treatment of arthritis, primary nephrotic syndrome of children and autoimmune disease.Pro-drug cyclophosphamide is in vitro without antitumor workProperty, into first drug effect can be played by being converted to aldophosphamide by the particle body function oxidizing ferment on hepatocyte endoplasmic reticulum afterwards in vivo.
The cyclophosphamide of building endoplasmic reticulum targeting can effectively improve the activation efficiency of cyclophosphamide, reduce dosage, subtractFew toxic side effect.Cyclophosphamide is water soluble drug, and pH value, between 5.6-6.5, aqueous solution shows faintly acid, therefore this hairIt is bright that endoplasmic reticulum targeting cyclophosphamide liposome drug delivery system is constructed using Calcium acetate gradient for the first time, and carried out placeSide investigates, cellular uptake, the research such as cell toxicant.
1, the physicochemical property characterization of endoplasmic reticulum target liposomes
The prescription of cyclophosphamide liposome is studied first, it is found that best preparation method is Calcium acetate gradient,Phosphatide is hydrogenated soya phosphatide, using the encapsulating of high-efficient liquid phase technique (HPLC) measurement endoplasmic reticulum target liposomes cyclophosphamideRate, result are 40% or more (Fig. 2 and 3).Using dynamic light scattering method (dynamic light scattering, DLS) to itPartial size and current potential are measured.It the results are shown in Table 1.Table 1 is that endoplasmic reticulum targets cyclophosphamide liposome physicochemical property, using transmission electricitySub- microscope (transmission electron microscope, TEM), to the configurations of surface of endoplasmic reticulum target liposomes intoRow observation.It can be seen that, the particle diameter distribution of endoplasmic reticulum target liposomes is more uniform, liposome encapsulation and drugloading rate from table 1Within tolerance interval.The partial size of liposome is less than 200nm and more uniform.
1 endoplasmic reticulum of table targets cyclophosphamide liposome physicochemical property
| Drugloading rate (%) | Encapsulation rate (%) | Partial size | PDI |
| 1.89±0.07 | 48.99±2.405 | 184±10.75 | 0.206±0.045 |
Fig. 3 is the influence of the comparison liposome encapsulation of phosphatide and drug when being prepared with Calcium acetate gradient gradient method.?HSPC effect is better than S100 under identical prescription.And film dispersion method is compared using the liposome encapsulation that pH gradient method is preparedIt is high.The present invention preferred Calcium acetate gradient prepares cyclophosphamide liposome.
2, endoplasmic target experimental verification (laser co-focusing)
By humanized oophoroma SKOV-3, humanized liver cancer HepG-2, mouse colon cancer CT-26 cell presses 5 × 104A/hole is inoculated in the 10mm in the every hole of 24 orifice plates2Slide on.After adherent for 24 hours, every hole is separately added into containing 500 μ g/mL of liposomeThe non-targeted liposome of blank endoplasmic reticulum target liposomes and blank by FITC fluorescent marker.It is sopped up in hole after being incubated for 12hCulture medium washs every hole 3 times with PBS.Core dye is carried out to cell with hochest33342 dyestuff, it is 5ug/mL that concentration, which is added, in every holeCore dyestuff 100uL sop up dye liquor after 37 DEG C are protected from light and are incubated for 30min, washed three times with PBS；It is 5ug/mL's that concentration, which is added,Endoplasmic reticulum dyestuff ER-tracker 100uL, which is protected from light, is incubated for 30min, is marked to endocytoplasmic reticulum, sucks dye liquor and use PBSWashing 2-3 times, every hole are added the 4% fixed cell of paraformaldehyde solution, take out slide after twenty minutes and carry out mounting, be placed in laserIt is observed under Laser Scanning Confocal Microscope.As a result see Fig. 4, targeting group endoplasmic reticulum fluorescence signal and liposome fluorescence signal repeatability are preferable,Non-targeted group lower with endoplasmic reticulum registration.Show the endoplasmic reticulum targeting of Pardaxins work well (red fluorescence representEndoplasmic reticulum, green fluorescence represent liposome.)
3, lysosome escape experiment (laser co-focusing)
With method by humanized oophoroma SKOV-3, humanized liver cancer HepG-2, mouse colon cancer CT-26 cell by 5 ×104A/hole is inoculated in culture vessel with glass bottom.After adherent for 24 hours, every ware be separately added into the 500 μ g/mL containing liposome by FITCThe non-targeted liposome of blank endoplasmic reticulum target liposomes and blank of fluorescent marker.The culture medium in ware is sopped up after incubation 12h, is usedPBS washs every hole 3 times.Core dye is carried out to cell with hochest33342 dyestuff, the core dyestuff that concentration is 5ug/mL is added in every hole100uL sops up dye liquor, is washed three times with PBS after 37 DEG C are protected from light incubation 30min；The lysosome Lys- that concentration is 5ug/mL is addedTracker dyestuff 100uL, which is protected from light, is incubated for 30min, is marked to Cytolysosome, sucks dye liquor and washed 2-3 times with PBS,The 4% fixed cell of paraformaldehyde solution is added in every hole, takes out slide after twenty minutes and carries out mounting, it is micro- to be placed in laser co-focusingUnder the microscope.As a result see Fig. 5, targeting group lysosome fluorescence signal and liposome fluorescence signal registration are lower, non-targeted group withEndoplasmic reticulum registration is higher.Show that Pardaxin has the function of helping to escape from lysosome.
4, cytotoxicity experiment
By humanized oophoroma SKOV-3, humanized liver cancer HepG-2, mouse colon cancer CT-26 cell presses 5 × 104A, the amount in 200 holes μ L/ is grafted on 96 orifice plates.After cell for 24 hours is adherent, the non-targeted liposome of blank, drug-loaded liposome,Blank target liposomes and carry medicine endoplasmic reticulum target liposomes, according to carrier series of concentrations (25ug/mL 50ug/mL,100ug/mL, 250ug/mL) it is added in tumour cell.It is incubated for 72h.The MTT aqueous solution (5mg/mL) of 20 μ L is added in every hole again,Continue after being incubated for 4h, discard culture solution, every hole is added the DMSO of 100 μ L, vibrates after twenty minutes on shaking table, microplate reader is in 570nmPlace's measurement light absorption value.As a control group, free cyclophosphamide group is used as sun for blank liposome group and blank target liposomes groupProperty control.As a result see Fig. 6, free cyclophosphamide is common because it is water-soluble and is prodrug so vitro cytotoxicity is weakerThe born of the same parents that enter that cyclophosphamide liposome increases cyclophosphamide lead, and improve the amount of active cyclophosphamide metabolites in system, cell in vitroToxicity specific ionization significantly increases that drug is big, illustrates that Liposomal formulation can improve intake of the cell to drug, enhances curative effect of medication.With cyclophosphamide can be further increased after DSPE-PEG-Pardaxin modified liposome to the toxicity of cell, and with DSPE-The increase of PEG-Pardaxin ratio does not increase significantly to cytotoxic effect, and showing may DSPE-PEG-PardaxinHave in the different cell lines of saturability two that drug is big to the cytotoxicity ratio SKOV-3 of HepG-2 from the identification of endoplasmic reticulum, this canIt can further illustrate and be targeted in endoplasmic reticulum because there is the cytochromes enzyme of more activation cyclophosphamide in liver cancer cells endoplasmic reticulumIt acts on lower cyclophosphamide activation degree to increase, cytotoxic effect enhancing.
Embodiment 5
The composition of endoplasmic reticulum targeting peptide-doxorubicin liposome:
HSPC, Cholesterol, DSPE-PEG of recipe quantity2000And DSPE-PEG-PAR, it is placed in eggplant-shape bottle, is addedIt is dissolved in organic solvent (chloroform), vacuum decompression rotary evaporation is stayed overnight in 55 DEG C of water-baths, and bottle wall forms film.Add into eggplant-shape bottleEnter the ammonium sulfate of pH=5.4,60 DEG C of water-baths are hydrated 1h, and film separation dissolves and forms multilamellar liposome solution.Use spyHead is ultrasonic by above-mentioned solution ultrasound to required partial size.Resulting liposome solutions are crossed into Sephadex G50 column, with PBS (pH=7.4) it elutes, removes the ammonium sulfate of liposome foreign minister.The adriamycin that 2mg/mL is added in endoplasmic reticulum target liposomes is moltenLiquid, makes medicine rouge mass ratio 1:20, and 50 DEG C of stirred in water bath are incubated for 30min.Using Sephadex G50 column, remove freeDoxorubicin hydrochloride is finally collected as carrying the endoplasmic reticulum target liposomes of adriamycin.
Adriamycin is a kind of antitumor antibiotics, can inhibit the synthesis of RNA and DNA, most strong to the inhibiting effect of RNA, is resistedTumor spectrum is wider, has effect to kinds of tumors, belongs to cell cycle nonspecific agent (CCNSA), kill to the tumour cell of various growth cyclesThe effect of going out.It is primarily adapted for use in acute leukemia, it is effective to acute lymphoblastic leukemia and granulocytic leukemia.The present invention existsPardaxin is added in the classical prescription of Evacet, and Pardaxin can help Evacet to enter born of the same parents and be targeted to endoplasmOn nethike embrane, Evacet can bypass lysosome to protect drug.The present invention can reduce the dosage of adriamycin, dropLow poisonous side effect of medicine.
Embodiment 6
The composition of endoplasmic reticulum targeting dexamethasone acetate liposome:
The Dex-Ac of recipe quantity, HSPC, Cholesterol, DSPE-PEG2000And DSPE-PEG-PAR, it is placed in eggplant-shape bottleIn, dissolution in organic solvent (chloroform) is added, vacuum decompression rotary evaporation is stayed overnight in 55 DEG C of water-baths, and bottle wall forms film.To eggplantThe PBS of pH=7.4 is added in shape bottle, 60 DEG C of water-baths are hydrated 1h, and film separation dissolves and forms multilamellar liposome solution.Use spyHead is ultrasonic by above-mentioned solution ultrasound to required partial size.Resulting liposome solutions are crossed into Sephadex G50 column, with PBS (pH=7.4) it elutes, removes the dexamethasone acetate drug to dissociate in liposome.The liposome with opalescence eluted is collected, i.e.,For the endoplasmic reticulum target liposomes for being loaded with dexamethasone acetate.
Dexamethasone acetate is a kind of cortex hormone of aadrenaline medicine.With anti-inflammatory, antiendotoxin, inhibit immune, Hemorrhagic shockAnd the pharmacological actions such as enhancing stress reaction, it is widely used in each section and treats a variety of diseases, such as autoimmune disease, allergy, inflammationDisease, asthma and dermatology, ophthalmology disease.Dexamethasone acetate adverse reaction is more, mostly occurs in application pharmacological dose, and withThe course for the treatment of, dosage, types of medicines, usage and administration route etc. have substantial connection.Common adverse reactions have following a few classes: iatrogenic libraryGlad syndrome face and posture, digestive tract ulcer, spiritual abnormal, glucocorticoid withdrawal syndrome, glycosuria and class Ke Xing are comprehensiveClose disease etc..
It is metabolized to dexamethasone under the action of liver drug enzyme after dexamethasone acetate administration, is liver microsomalinducer, for a long timeUsing liver drug enzyme increased activity can be caused, lead to the increase of Cytochrome P450 content, promotes liver endoplasmic reticulum hyperplasia and particleThe synthesis of body embrane-associated protein.It can dissolve in ethyl alcohol or chloroform, therefore film dispersion method can be used and fill in rice with preparing acetic acidRosin plastid.Dexamethasone acetate is prepared into the liposome with endoplasmic reticulum targeting using Pardaxin target head.The inventionThree advantages: first is that drug can be improved in the concentration of target organ, drug is reduced in the distribution of other organs, second is that directly by medicineObject can improve the activation efficiency of body Dichlorodiphenyl Acetate dexamethasone targeted to endoplasmic reticulum using the enzyme in endoplasmic reticulum, and three are to confer to drugAfter three-level targeting, drug administration dosage can be preferably controlled, the effect that liver microsomalinducer is played it is controlled.Acetic acidThe adverse reaction of dexamethasone is more, and the Liposomal formulation for being prepared into that there is endoplasmic reticulum to target can be effectively reduced administrationDosage reduces adverse reaction, controls its inducing action to liver drug enzyme.
Embodiment 7
The composition of endoplasmic reticulum target drug-carrying hemoglobin liposome:
E80, Cholesterol, DSPE-PEG of recipe quantity2000And DSPE-PEG-PAR, it is placed in eggplant-shape bottle, addition hasIt is dissolved in solvent (chloroform), vacuum decompression rotary evaporation is stayed overnight in 55 DEG C of water-baths, and bottle wall forms film.It is added into eggplant-shape bottlePBS containing Hb recipe quantity, 60 DEG C of water-baths are hydrated 1h, and film separation dissolves and forms multilamellar liposome solution.It is super using probeSound is by above-mentioned solution ultrasound to required partial size.Resulting liposome solutions are crossed into Sephadex G50 column, with PBS (pH=7.4)Elution removes the Hb to dissociate in liposome.The liposome with opalescence eluted is collected, the endoplasmic reticulum targeting of Hb is as loaded withLiposome.Oxygen molecule can be directly delivered to endocytoplasmic reticulum position in combination with oxygen molecule by the liposome, improve the position oxygenContent, and then be conducive to the treatment of the subsequent oxygen dependence for endoplasmic reticulum position.
Embodiment 8
Endoplasmic reticulum targeting carries the composition of paclitaxel solid lipid nano particle:
Taxol 5mg
Glycerin monostearate 100mg
DSPE-PEG-Pardaxin 2mg
Taxol, glycerin monostearate and the DSPE-PEG-Pardaxin of recipe quantity, are added to absolute ethanol dissolution,It is injected into the buffer solution of PBS, then required partial size is obtained by ultrasound or homogenizer.By resulting nanoparticle solution mistakeSephadex G50 column is purified by flash with PBS (pH=7.4), is as loaded with the endoplasmic reticulum targeting solid lipid nano of taxolGrain.
Embodiment 9
Endoplasmic reticulum targeting carries the composition of Docetaxel nano-micelle:
Docetaxel 5mg
Polyethylene glycol-polycaprolactone (PEG-PCL) 100mg
PCL-PEG-Pardaxin 10mg
Docetaxel, polyethylene glycol-polycaprolactone and the PCL-PEG-Pardaxin of recipe quantity, are added to absolute ethanolDissolution, is injected into the buffer solution of PBS, then obtains required partial size by ultrasound or homogenizer.By resulting nanoparticle solutionSephadex G50 column is crossed, is purified by flash with PBS (pH=7.4), the endoplasmic reticulum targeted nano glue of Docetaxel is as loaded withBeam.
Embodiment 10
Target the synthesis of Jenner's grain of rice of endoplasmic reticulum: the 2mg Pardaxin-PEG-TA synthesized in Example 2, room temperatureUnder in aqueous medium with the hollow gold nano-material of 10mg (HAuNS) (size 40nm has Idiotype absorption peak at 800nm),It is incubated for 24 hours altogether, centrifugal purification must have endoplasmic reticulum target function to obtain hollow Jenner's grain of rice.The nanoparticle enter after cellEndoplasmic reticulum accumulation, further by extraneous stimulation (near infrared light), it can be achieved that being directed to special sexual behaviour (the photo-thermal thorn of endoplasmic reticulumSwash, enhancing er stress is horizontal).
Sequence table
<110>Zhejiang University
<120>targeted to the building and application of endocytoplasmic reticulum nano medicament carrying system
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> PRT
<213> Pardaxin
<400> 1
Gly Phe Phe Ala Leu Ile Pro Lys Ile Ile Ser Ser Pro Leu Phe Lys
1 5 10 15
Thr Leu Leu Ser Ala Val Gly Ser Ala Leu Ser Ser Ser Gly Gly Gln
20 25 30
Glu