CN105012956A - Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof - Google Patents
Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof Download PDFInfo
- Publication number
- CN105012956A CN105012956A CN201410153928.3A CN201410153928A CN105012956A CN 105012956 A CN105012956 A CN 105012956A CN 201410153928 A CN201410153928 A CN 201410153928A CN 105012956 A CN105012956 A CN 105012956A
- Authority
- CN
- China
- Prior art keywords
- acid
- drug
- liposome
- phospholipid
- organic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention belongs to the field of pharmaceutic preparations in medical technology and discloses a difunctional tumor targeted liposome drug-delivery system and a preparation method and an application thereof. By directly coupling a cell-penetrating peptide rich in arginine residues and liposome surface long-chain organic acid and with the combination of a polyethylene glycol-hydrazone bond-long-chain organic acid conjugate, a tumor targeted liposome loading a fat soluble drug and a water-soluble anthraquinone slightly alkaline drug is prepared by a thin-film rehydration method, wherein the cell-penetrating peptide is polyarginine short peptide, preferably nonaarginine; molecular weight of polyethylene glycol is 500 Da-5000 Da, preferably 2000 Da; optimal cell-penetrating peptide density is 2-8% (preferably 4%) of density of phospholipid; and density of the polyethylene glycol-hydrazone bond-long-chain organic acid conjugate is 5-20% (preferably 8%) of density of phospholipid. The system provided by the invention solves the problem that existing liposome cellular internalization is difficult, and provides possibility for antitumor drug targeting subcellular organelle. The drug-delivery system basically is nontoxic, has a simple preparation technology and is easy for industrial application.
Description
Technical field
The present invention relates to liposome administration system, particularly relate to cancer target liposome administration system and preparation method thereof, belong to medical art.
Background technology
Due to biocompatibility, biodegradability, medicine carrying multiformity, and can medicine stability be improved, liposome has become a kind of nano-carrier system [1] be widely used.Nineteen ninety-five, first antitumor medicinal liposome preparation Evacet was ratified first by FDA, and enter clinical [2], it significantly improves the cardiac toxicity of amycin, but its drug effect does not significantly improve.Drug effect is improved further in the accumulation of target area in order to increase medicine, successively there is the pH sensitive liposome [3 with Physical Target tropism in liposome research field, 4], thermal sensitive liposome [5], magnetosensitive liposome [6,7], the immunoliposome [8,9] that can realize active targeting of part or antibody modification and multi-functional liposome [10].
Over nearly 20 years, the modification of target liposomes mainly concentrates on receptor-mediated [11-13], and receptor-mediated endocytosis achieves medicine concentrating target cell.But, after medicine and carrier thereof enter born of the same parents with endocytosis, the first step forms endosome, then engulfed by lysosome, and be degraded enrich enzyme effect in lysosome under, cause medicine to be difficult to escape from lysosome enter Cytoplasm thus, limit medicine and arrive real target spot and realize drug effect as the subcellular organelle such as nucleus or mitochondrion plays a role.
In recent years, research finds cell-penetrating peptides (cell penetrating peptides, CPP) because it is rich in arginine residues, can with the anionic species of cell surface, comprise glycosaminoglycans, membrane phospholipid head etc., there is strong interaction, the picked-up [14,15] of active cell.Especially the medicine carried as CPP enter cell formed endosome by lysosome fusion after, with positive charge after amino absorption H+ on CPP, can with the negative charge generation electrostatic interaction of lysosome inner surface, lysosome is inside contracted, form fenestra road, cause medicine to be released to Cytoplasm from lysosome thus, provide chance [16] for entering nucleus further, this effect of CPP is referred to as " proton sponge effect ".Not. Huffman-Laroqie Co., Ltd had once applied for PCT Chinese patent CN103096932A, it has invented a kind of tat peptide with aminoacid sequence GRKKRRQRRRPPQ, illustrate that it has stronger internalization effect (at least 80%, cell is dissolved) in the effect of preferred at least 90%, nucleic acid can be carried and egg is inverting or the one or more cell of transfection, there is certain purposes preventing and/or treating in the disease being selected from cancer, immunological diseases, cardiovascular disease, sacred disease, infection and inflammatory diseases.But regrettably, the tat peptide invented so far or all inorganizable and cell-specific [17] of cell-penetrating peptides (CPP), it is while penetrating tumor cell, also has short picked-up effect, cause the infringement of normal tissue thus for normal cell.Therefore, up to the present, the internalization that penetrates of tat peptide or CPP peptide gains public acceptance, but lacks the selectivity of targeted cells just because of it, and the application therefore in drug-supplying system is restricted.
For playing CPP further to the selectivity of tumor tissues and targeting, have bibliographical information [18] target cell specificity part such as RGD and CPP jointly to modify carrier system, what utilize RGD etc., to the specificity of tumor, CPP " band " is realized CPP to tumor penetrates internalization.In addition, Chinese patent CN102552929A has invented by shielding peptide, enzymolysis peptide substrate, has worn film peptide and be linked in sequence that formed can active cell penetrating peptide, a kind of drug-supplying system that utilized PSA sensitivity to invent, and improves the targeting of CPP.But this system design comparatively complexity not easily realizes large production, and remains in problems such as immunogenicities, because which limit border application in fact.
In addition, as will be appreciated, in liposome evolution, be the milestone [19] of a history with Polyethylene Glycol (poly (ethylene glycol), the PEG) modification of derivant to liposome rete.But the modification of PEG to liposome is one " double-edged sword ".It is while having slackened liposome and opsonification, also will weaken when long circulating liposomes reaches target site and the interaction of target cell, even the hydrated sheath of PEG chain hinders " targeting " effect of connected part, antibody, reduces the efficiency [20] of targeted and cellular uptake.In above-mentioned Chinese patent CN102552929A, CPP modifies in surface of liposome after namely connecting with Polyethylene Glycol, and this Polyethylene Glycol hydrated sheath has " obstruction " effect for the performance of CPP effect, can not give full play to the effect of CPP.
As everyone knows, the pH of tumor locus is starkly lower than normal structure and blood [21], and hydrazone key is easy hydrolytic cleavage when low pH.Therefore; PEG is connected to surface of liposome by hydrazone key and makes this acid-sensitive liposome optimumly can play its effect at tumor locus; that is under body circulation pH7.0 condition; PEG stable existence plays " protective effect " in surface of liposome; and at the acidic region of tumor low ph value, the hydrazone bond fission of link PEG makes PEG " leave " surface of liposome to promote that cell is to the picked-up of liposome.Kale, A.A.and Torchilin, V.P. [22]. the pH sensitive liposome that a kind of TATp modifies once was proposed, wherein TATp inserts surface of liposome by forming TATp-PEG1000-PE with short chain polyalkylene glycol (PEG1000)-PE coupling, and polyethylene glycol long chain mPEG2000 inserts by hydrazone key and PE coupling (mPEG2000-HZ-PE) long circulating that surface of liposome realizes acid-sensitive.Although this acid-sensitive drug-supplying system improves the selectivity for tumor of CPP to a certain extent, still do not consider and improve the inhibition of the hydrated sheath of PEG, not making the effect of CPP give full play of.In addition, PE used in long circulating acid-sensitive part mPEG2000-HZ-PE is the phospholipid molecule with two long chain organic acids, it has less critical micelle concentration, in this acid-sensitive liposomal preparation, more easily form micelle and be difficult to insert surface of liposome, in preparation, there is the problem that acid-sensitive conjugates modification rate is low.
For Kale, A.A.and Torchilin, V.P. Problems existing in this acid-sensitive liposome designed, we devise a kind of simple and effective difunctional Tumor Targeting Drug Delivery System, the i.e. acid-sensitive liposome of CPP modification, wherein CPP and stearoyl (STR) directly coupling modified liposome surface, avoid " obstruction " effect of PEG, in addition, mPEG20000-Hz-STR has been synthesized with strand organic acid (as stearic acid STR), wherein instead of double-strand PE with strand STR, comparatively (mPEG2000-HZ-PE) has higher critical micelle concentration, not easily micelle is formed in liposomal preparation, easily insert surface of liposome all the better, realize long circulating.This simple and effective targeted liposome preparation has long circulating in body, tumor physical targeting and cell-penetrating internalization three kinds of characteristics concurrently, can realize simultaneously antitumor drug concentrate in the efficient location of tumor area and rapid cellular penetrate two objects.And said preparation preparation is simple, is easy to large production, has the wide spectrum suitability to all tumors, be expected to help cancer target drug-delivery preparation breakthrough of zero in clinical practice.
List of references:
[1].Cullis，P.R.and A.Chonn，Recent advances in liposome techno1ogies and their applications for systemic genedelivery.Adv Drug Deliv Rev，1998.30(1-3)：p.73-83.
[2].Ning，Y.M.，et a1.，Liposomal doxorubicin in combination with bortezomib for relapsed or refractory multiplemyeloma.Onco1ogy(Williston Park)，2007.21(12)：p.1503-8；discussion1511，1513，1516passim.
[3].Evjen，T.J.，et a1.，Lipid membrane composition influences drug release fromdioleoylphosphatidylethanolamine-based liposomes on exposure to ultrasound.Int J Pharm，2011.406(1-2)：p.114-6.
[4].Legendre，J.Y.and F.J.Szoka，Delivery ofplasmid DNA into mammalian cell1ines using pH-sensitive liposomes：comparison with cationic liposomes.Pharm Res，1992.9(10)：p.1235-42.
[5].Kullberg，M.，K.Mann and J.L.Owens，A two-component drug delivery system using Her-2-targetingthermosensitive liposomes.J Drug Target，2009.17(2)：p.98-107.
[6].Kiwada，H.，et a1.，Feasibility of magnetic 1iposomes as a targeting device for drugs.Chem Pharm Bull(Tokyo)，1986.34(10)：p.4253-8.
[7].Petrichenko，O.，et a1.，Bilayer properties of giant magnetic liposomes fornmed by cationic pyridine amphiphile andprobed by active deformation under magnetic forces.Eur Phys J E Soft Matter，2013.36(1)：p.9.
[8].Apte，A.，et a1.，Doxorubicin in TAT peptide-modified multifunctional immunoliposomes demonstratesincreased activity against both drug-sensitive and drug-resistant ovarian cancer models.Cancer Biol Ther，2013.15(1).
[9].Yu，B.，et al.，Targeted drug delivery and cross-linking induced apoptosis with anti-CD37based dual-ligandimmunoliposomes in B chronic lymphocytic leukemia cells.Biomaterials，2013.34(26)：p.6185-93.
[10].Xiang，B.，et a1.，PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostatecancer.Biomaterials，2013.34(28)：p.6976-91.
[11].Lee，R.J.and P.S.Low，Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis.JBiol Chem，1994.269(5)：p.3198-204.
[12].Kawakami，S.，et a1.，Mannose receptor-mediated gene transfer into macrophages using novel mannosylatedcationic liposomes.Gene Ther，2000.7(4)：p.292-9.
[13].Hendriks，B.S.，et a1.，Impact of tumor HER2／ERBB2expression level on HER2-targeted liposomaldoxorubicin-mediated drug delivery：multiple low-affinity interactions lead to a threshold effect.Mol Cancer Ther，2013.12(9)：p.1816-28.
[140.Frankel， A.D.and C.O.Pabo，Cellular uptake of the tat protein from human immunodeficiency virus.Cell，1988.55(6)：p.1189-93.
[15].Mai，J.C.，et a1.，Efficiency ofprotein transduction is cell type-dependent and is enhanced by dextran sulfate.J BiolChem，2002.277(33)：p.30208-18.
[16].El-Sayed，A.，S.Futaki and H.Harashima，Delivery of macromolecules using arginine-rich cell-penetratingpeptides：ways to overcome endosomal entrapment.AAPS J，2009.11(1)：p.13-22.
[17].Vives，E.，P.Brodin and B.Lebleu，A truncated HIV-1Tat protein basic domain rapidly translocates through theplasma membrane and accumulates in the cell nucleus.J Biol Chem，1997.272(25)：p.16010-7.
[18].Koren，E.，et a1.，Multifunctional PEGylated2C5-immunoliposomes containing pH-sensitive bonds and TATpeptide for enhanced tumor cell internalization and cytotoxicity.J Contro1Release，2012.160(2)：p.264-73.
[19].Blume，G.and G.Cevc，Liposomes for the sustained drug release in vivo.Biochim Biophys Acta，1990.1029(1)：p.9l-7.
[20].Khalid，M.N.，et a1.，Long circulating poly(ethylene glyco1)-decorated lipid nanocapsules deliver docetaxel to solidtumors.Pharm Res，2006.23(4)：p.752-8.
[21].Lee，E.S.，z.Gao and Y.H.Bae，Recent progress in tumor pH targeting nanotechno1ogy.J Contto1Release，2008.132(3)：p.164-70.
[22]Kale AA，Torchilin VP.″Smart″drug carriers：PEGylated TATp-modified pH-sensitive liposomes.JLiposome Res2007 2007-01-20；17(3-4)：197-203.
Summary of the invention
An object of the present invention is to provide one and has bifunctional cancer target liposome administration system;
Two of object of the present invention is to provide the synthetic method of acid-sensitive part Polyethylene Glycol-hydrazone key-mono-long chain organic acid conjugate;
Three of object of the present invention provides the preparation method of the difunctional cancer target Liposomal formulation of acid-sensitive with cell-penetrating and proton sponge effect (lysosome escape).
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
For the shortage cellular uptake existing for existing Liposomal formulation, be difficult to lysosome escape, targeting subcellular organelle can not be realized, and PEG hydrated sheath is to defects such as " obstruction " effects of decorating molecule, first the present inventor finds by studying with keen determination, by a kind of inactive, the small peptide being rich in arginine residues of non-immunogenicity is as decorating molecule, larger with the use of critical micelle concentration, there is Polyethylene Glycol-hydrazone key-mono-long chain organic acid conjugate of acid-sensitive characteristic, adopt and be easy to the thin film aquation method of suitability for industrialized production or high pressure homogenization method and prepare there is bifunctional Liposomal formulation, solve existing Liposomal formulation and realize the technical problem in cancer target.Said preparation envelop rate, particle diameter meet liposome medication requirement, there is stronger sensitivity to acid, hemolytic is lower, belong to non-toxic carrier, there is cellular uptake, in cell in vitro drug effect and body, the distribution of tumor-bearing mice tumor locus preparation is all higher than long circulating liposomes dosage form commercially available at present, thus completes the present invention.
1. first aspect present invention provides a kind of difunctional cancer target liposome administration system, comprises liposome and active constituents of medicine; Wherein said liposome is by phospholipid, and cholesterol comprises phospholipid, cholesterol and derivant thereof, cell-penetrating peptides-long chain organic acid conjugate (as CPP-STR) and Polyethylene Glycol-hydrazone key-long chain organic acid conjugate (as mPEG-Hz-STR) and forms.The wherein said particle diameter with the cancer target liposome of internalization between 50nm ~ 500nm, between preferred 100nm ~ 300nm.
In drug-supplying system of the present invention, the Polyethylene Glycol-hydrazone key-long chain organic acid conjugate synthetic method of acid-sensitive characteristic is shown in a second aspect of the present invention.In drug-supplying system, Polyethylene Glycol mPEG molecular weight is 500Da ~ 5000Da, preferred 2000Da; The density of this conjugate is 5% ~ 20% of the amount of phospholipid material.Preferably 8%.
In drug-supplying system of the present invention, CPP is poly arginine small peptide, preferably nine poly arginines; CPP is by the direct coupling of long chain organic acid of the activation of surface of liposome, and preparation technology is shown in a third aspect of the present invention.In drug-supplying system, CPP density is 2% ~ 8% of the amount of phospholipid material.Preferably 4%.
Phospholipid described in drug-supplying system of the present invention selects natural, semi-synthetic, complete synthesis phospholipid and derivant thereof.Preferred scheme is that described phospholipid is selected from but is not limited to the soybean lecithin (SPC) of subordinate, Ovum Gallus domesticus Flavus lecithin (EPC), EPG (EPG), polyene phosphatidylcholine, phosphatidic acid, cardiolipin, sphingomyelins, phosphatidic acid serine, phosphatidylinositols, PHOSPHATIDYL ETHANOLAMINE, hydrogenated soya phosphatide (HSPC), hydrogenated yolk lecithin (HEPC), complete synthesis phospholipid, as the various synthetic phospholipids of C3 to C30, as distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidyl choline (DPPC), DOPC (DOPC), dimyristoyl phosphatidyl choline (DMPC), DLPC (DLPC), DSPG (sodium salt DSPG), DPPG (sodium salt DPPG), L-α-GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (sodium salt DMPG), PE (DLPG), DSPE (DSPE), DPPE (DPPE), DOPE (DOPE), DMPEA (DMPE), two lauroyl PHOSPHATIDYL ETHANOLAMINE (DLPE), two DSPG (sodium salt), two DPPG (sodium salt), two GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (sodium salt), two PE, distearoylphosphatidyl inositol, two palmityl phosphatidylinositols, two myristoyl phosphatidylinositols, two lauroyl phosphatidylinositols, POPC, the sub-oleoyl phosphatidylcholine of palmityl, the sub-oleoyl phosphatidylcholine of stearoyl, stearoyl has phosphatidyl choline, stearoyl palmitoylphosphatidylcholine.
Preferably soya lecithin in the specific embodiment of the invention.
Other liposomees such as the cholesterol described in drug-supplying system of the present invention and derivant (as Cholesteryl hemisuccinate), sitosterol.Prepare in liposome, add cholesterol, sitosterol as the supplementary element of liposome, be used for reducing the mobility of phospholipid, be conducive to load lipophilic drugs.
Preferred cholesterol in the specific embodiment of the invention.
Long chain organic acid described in drug-supplying system of the present invention, selects stearic acid, Palmic acid, oleic acid, myristic acid.
Preferred stearic acid in the specific embodiment of the invention.
The contained medicine of drug-supplying system of the present invention is antitumor drug, comprise Anthraquinones antibiotic doxorubicin hydrochloride, amycin, epirubicin, daunorubicin, platinum medicine, as carboplatin, JM-216, eptalatin, acetic acid platinum, cisplatin, cyclophosphamide, D actinomycin D, bleomycin, rubidomycin, mitomycin, methotrexate, 5-fluorouracil, carmustine (BCNU), Semustine, etoposide, etoposide, interferon, camptothecine and various derivant, phenesterin, taxol (Taxol) and derivant thereof, docetaxel and derivant thereof, vinblastine, vincristine, vinorelbine and derivant thereof, tamoxifen, A-20968, tetracyclic triterpenoid etc.
Preferred doxorubicin hydrochloride in the specific embodiment of the invention.
2. a second aspect of the present invention provides the synthetic method of acid-sensitive Polyethylene Glycol-hydrazone key-stearic acid conjugate, first adopt the Polyethylene Glycol of aldehyde radical and the reaction of 3-(2-pyridine two sulfur) propionyl hydrazine (PDPH) to generate PEG-hydrazone key-PDPH, then generate acid-sensitive conjugate required for the present invention with the coupling of sulfhydrylation stearic acid phase.Concrete synthetic method and compound are differentiated see embodiment.
3. third aspect present invention provides the preparation method of difunctional loading liposomes fat-soluble medicine preparation, and the method comprises: (1) adopts and reduction vaporization after the dissolving of phospholipid, cholesterol, stearic acid and fat-soluble medicine oil phase is obtained lipid membrane; (2) normal saline is added or 5% D/W aquation obtains pastille conventional liposome; (3) long chain organic acid in 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) EDCI and sulfhydrylation-N-hydroxysuccinimide (s-NHS) activated lipid body, adjustment pH is strong basicity, then adds CPP and obtain CPP modified liposome; (5) acid-sensitive Polyethylene Glycol-hydrazone key-long chain organic acid conjugate overnight incubation is added; (6) bag filter or super filter tube purification, to obtain final product.
Wherein, after phospholipid, cholesterol, long chain organic acid, fat-soluble medicine dissolve with oil phase by step (1), at 25 ~ 60 DEG C, reduction vaporization obtains lipid membrane; Strong basicity belonging to step (3) is pH is 10 ~ 12.The bag filter dialysis 4h ~ 12h of step (6) molecular cut off used 5000Da ~ 300000Da or with the super filter tube of molecular cut off 5000Da ~ 300000Da at the centrifugal 10min ~ 30min of 4000g ~ 10000g.Preferred molecular cut off is 10000Da, dialysis time 12h, and centrifugal speed is 10000g, and centrifugation time is 20min.
Preferred Coumarin-6 in the specific embodiment of the invention.
4. third aspect present invention provides difunctional loading liposomes water soluble drug, as the preparation method of Anthraquinones weakly basic drugs preparation, the method comprises: (1) adopts and phospholipid, cholesterol, long chain organic acid oil phase are dissolved rear reduction vaporization and obtain lipid membrane; (2) add and obtain blank conventional liposome containing organic acid or inorganic aqueous acid aquation; (3) regulate outer aqueous pH values to alkalescence with alkaline buffer again; (4) add Anthraquinones weakly basic drugs active component aqueous solution, stir, overnight incubation, obtains pastille conventional liposome; (5) long chain organic acid in 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCl) EDCI and sulfhydrylation-N-hydroxysuccinimide (s-NHS) activated lipid body is added, adjustment pH is strong basicity, then adds CPP and obtain CPP modified liposome; (6) add acid-sensitive Polyethylene Glycol-hydrazone key-long chain organic acid conjugate overnight incubation, (7) bag filter or super filter tube purification, to obtain final product.
Wherein, after phospholipid, cholesterol, stearic acid dissolve with oil phase by step (1), at 25 ~ 60 DEG C, reduction vaporization obtains lipid membrane; Organic acid in step (2) or mineral acid comprise citric acid, lactic acid, fumaric acid, tartaric acid, acetic acid, gluconic acid, lactobionic acid, sorbic acid, maleic acid, ascorbic acid, oxalic acid, formic acid, benzenesulfonic acid, benzoic acid, glutamic acid, aspartic acid, hydrochloric acid, arginine etc., optimization citric acid; Alkalescence pH value described in step (3) is 7.5-8.0; Step (adds active constituents of medicine aqueous solution at 50 DEG C in (4) at 3 temperature; Strong basicity belonging to step (5) is pH is 10 ~ 12; The bag filter dialysis 4h ~ 12h of step (7) molecular cut off used 5000Da ~ 300000Da or with the super filter tube of molecular cut off 5000Da ~ 300000Da at the centrifugal 10min ~ 30min of 4000g ~ 10000g.Preferred molecular cut off is 30000Da, dialysis time 12h, and centrifugal speed is 10000g, and centrifugation time is 20min.
Preferred doxorubicin hydrochloride in the specific embodiment of the invention.
Preparation method of the present invention can also comprise filtration sterilization.The suspension particle diameter that the present invention obtains is less, and very easy membrane filtration is degerming after filtration.
0.2 μm of filtering with microporous membrane is comprised degerming at the preferred embodiment of the specific embodiment of the invention.
Drug-supplying system provided by the present invention is acid-sensitive liposome turbid liquor or the freeze-dried powder with internalization.
Drug-supplying system provided by the present invention has stronger sensitivity to acid.
Drug-supplying system provided by the present invention has stronger selectivity to tumor, normal tissue not damaged.
Drug-supplying system hemolytic concentration provided by the present invention, higher than clinical practice concentration, belongs to non-toxic carrier.
Compared with prior art, novelty is in the present invention:
(1) dual function of Polyethylene Glycol PEG in liposome structure is considered, the designed this acid-sensitive liposome administration system with internalization can ensure the long circulating action in body circulation, all CPP can be exposed again at tumor locus, and be no longer subject to the inhibition of PEG, thus at utmost play CPP internalization effect, utilize the sour environment of tumor area to achieve the targeting of CPP;
(2) in preparation method, CPP is connected to surface of liposome by reaction method.Through the system of optimized choice strong basicity PH12 in reaction system, CPP reaction yield is brought up to more than 75%, belong to and innovating first.
(3) in preparation method, optimize the optimum density that in this drug-supplying system, CPP modifies, its effect is at utmost played, research method belongs to pioneering in CPP research;
(4) preparation technology of this drug-supplying system at load Anthraquinone has been invented, the envelop rate of the amycin that this technique obtains is more than 95%, and not by the impact of CPP density, avoid the interaction of CPP and the amycin (DOX) mentioned in CN201310100731.9, thus the phenomenon that the DOX envelop rate caused increases along with CPP density and declines;
(5) this drug-supplying system not only can load fat-soluble medicine, and can load Anthraquinones alkalescent water soluble drug.The preparation process thereof obtained is simple, and raw material is easy to get, and is easy to the large quality of production and controls.
Accompanying drawing explanation
The synthetic route of Fig. 1 acid-sensitive component Polyethylene Glycol-hydrazone key-stearic acid conjugate.
The HPLC spectrogram of Fig. 2 Polyethylene Glycol-hydrazone key-stearic acid conjugate.
The 1HNMR spectrogram of Fig. 3 Polyethylene Glycol-hydrazone key-stearic acid conjugate.
Fig. 4 has preparation technology's schematic diagram of internalization acid-sensitive liposome.
The sensitivity to acid evaluation (HPLC method) of Fig. 5 drug-supplying system of the present invention.
The sensitivity to acid evaluation (flow cytometry) of Fig. 6 drug-supplying system of the present invention, * * p<0.01.CL and SSL is matched group, and CPL is the experimental group of drug-supplying system
The optimization (flow cytometry) of CPP density in Fig. 7 drug-supplying system of the present invention.
The hemolytic result of Fig. 8 drug-supplying system of the present invention, SSL is matched group, and CPL is the experimental group of drug-supplying system.
Detailed description of the invention
The present invention is further illustrated below in conjunction with embodiment.Should be appreciated that the following examples for illustration of content of the present invention non-limiting content of the present invention, any pro forma variation and/or accommodation all will fall within protection scope of the present invention.
The synthesis of embodiment 1 acid-sensitive Polyethylene Glycol-hydrazone key-stearic acid conjugate and purification
Synthetic route as shown in Figure 1.MPEG
2000-CHO is dissolved in the chloroformic solution containing the PDPH little over amount, and room temperature reaction 48h, stirs in argon shield, synthesis mPEG
2000-HZ-PDP.STR-SH is dissolved in anhydrous chloroform, containing excessive mPEG in chloroform
2000-HZ-PDP, adds triethylamine, stirs and spend the night in room temperature under argon shield.Chloroform removed under pressure, conjugate Sepharose CL-4B is purified, and lyophilizing obtains white powder.It is 99% (Fig. 2) that Agilent HP1100/ELSD method measures this conjugates purity.1NNMR collection of illustrative plates (Fig. 3) demonstrates this conjugates structure.
The preparation of embodiment 2 difunctional loading liposomes fat-soluble medicine coumarin
Soybean lecithin, cholesterol, stearic acid, coumarin is dissolved in chloroform with 2: 1: 0.08: 0.02 mol ratio, and vacuum rotary steam forms thin film on bottle wall.Pastille conventional liposome (CL) is formed after PBS aquation.Under magnetic agitation, add EDCI, add S-NHS after about 1min, in reaction, the mol ratio of stearic acid and EDCI and S-NHS is 1: 40: 100.Adjusting PH after activating 15min under room temperature is 12, adds the CPP with STP equivalent, stirring at room temperature 12 hours, then adds 8%STR-HZ-PEG2000 and hatch 12 hours.10000g ultrafiltration (MW cut off=30000) 20min removes unreacted EDCI, S-NHS and CPP.Preparation process is shown in Fig. 4. the acid-sensitive liposomal particle size with internalization of the load coumarin obtained is 100 ~ 150nm, and envelop rate is greater than 95%, and within 4 hours, slip is less than 1%.
The CPP provided in the present invention and activated lipid precursor reactant condition are strong basicity pH12, make CPP reaction yield reach more than 75%, belong to and innovating first.
The preparation of the difunctional loading liposomes amycin of embodiment 3
Membrane process: soybean lecithin, cholesterol, stearic acid, is dissolved in chloroform with 2: 1: 0.08 mol ratios, and at 40 DEG C, vacuum rotary steam forms thin film on bottle wall.Blank conventional liposome is formed containing after Fructus Citri Limoniae aqueous acid (pH4 ~ 5) aquation.Outer aqueous phase pH7.5 ~ 8.0 of sodium carbonate buffer adjustment, add amycin aqueous hydrochloric acid solution, 50 DEG C of water-bath overnight incubation, obtain amycin conventional liposome.Under magnetic agitation, add EDCI, add S-NHS after about 1min, in reaction, the mol ratio of stearic acid and EDCI and S-NHS is 1: 40: 100.Adjusting PH after activating 15min under room temperature is 12, adds the CPP with STR equivalent, stirring at room temperature 12 hours, then adds 8%STR-HZ-PEG2000 and hatch 12 hours.10000g ultrafiltration (MW cut off=30000) 20min removes unreacted EDCI, S-NHS and CPP.The acid-sensitive liposomal particle size with internalization of the load amycin obtained is 100 ~ 150nm, and envelop rate is greater than 95%, and within 4 hours, slip is less than 3%.
First prepare blank liposome in this technique, then utilize pH gradient to load amycin, amycin is loaded as after intraliposomal aqueous phase, then pass through the reaction of pastille liposome and CPP, CPP is coupled to surface of liposome (Fig. 4).This technique avoids the interaction of DOX and CPP in the membrane formation process China and foreign countries aqueous phase mentioned in Chinese patent 201310100731.9.The preparation process of this load amycin provided by the present invention is effective and feasible, can not affect the envelop rate of amycin, is an important improvement.
The evaluation of embodiment 4 difunctional liposome pH sensitivity
Adopt HPLC/ELSD method, utilize gel chromatographic columns Shodex KW-804 molecular-exclusion chromatography post to be effectively separated liposome and PEG.Result shows (Fig. 5), acid-sensitive liposome prepared by the present invention stable existence when pH7.0, and at pH6.5, under 6.0 and 4.5 conditions, the hydrazone key of acid-sensitive liposome ruptures completely, makes PEG leave liposome.Under three kinds of acid conditions, the PEG relative quantity that ruptures is consistent with recipe quantity, as long as illustrate under faintly acid pH6.5, this drug-supplying system just can rupture PEG completely, has stronger sensitivity to acid.
Cellular uptake experiment shows (Fig. 6), the picked-up of acid-sensitive liposome prepared by the present invention under pH7.0 condition is consistent with the long circulating liposomes that PEG exists (SSL), and the picked-up under pH6.0 condition is consistent with the conventional liposome (CL) not containing PEG.Further illustrate the sensitivity to acid of drug-supplying system of the present invention, achieve the purpose of design of drug-supplying system of the present invention.
The optimization of CPP density in the difunctional liposome of embodiment 5
Pass through flow cytometry, determine the acid-sensitive liposome of prepared different CPP density under tumor area acidity (pH6.0) condition, the relation of cellular uptake and CPP density, and compared with normal structure environment (pH7.0) (Fig. 7).Result shows, under acidic cancer environment, and cellular uptake and the positive correlation of CPP density, and without saturability; But under normal structure environment, when CPP density is less than 4%, cellular uptake is consistent with the SSL modified without CPP, and when CPP density is increased to 8%, cellular uptake suddenly increases.Result illustrates under normal structure environment, and drug-supplying system PEG of the present invention does not rupture, can most multipotency effectively cover 4% CPP.Thus, the modification density of CPP in the present invention is selected to be decided to be 4%.Its implication represents in body circulation, this system normal tissue not damaged, but for tumor cell, can effectively impel it to play internalization.
Further, when acid-sensitive PEG density is 15%, it is 4% that the best of CPP modifies density.
The difunctional liposome of embodiment 6 has obvious cell-penetrating and lysosome escape effect
By confocal laser scanning microscope, drug-supplying system of the present invention can effectively be escaped from lysosome, being distributed widely in Cytoplasm, providing condition for entering cell nucleus further.And matched group, SSL, invisible lysosome is escaped, and contained coumarin and lysosome are located altogether.Result describes drug-supplying system of the present invention and has stronger subcellular organelle targeting.
The difunctional liposome of embodiment 7 has comparatively low hemolytic, substantially nontoxic
Measured by simple hemolytic experiment and find, the hemolytic of drug-supplying system of the present invention (CPL) and matched group SSL do not have difference (Fig. 8), in drug-supplying system, phospholipid concentration hemolytic when below 400nmole/mL is less than 10%, regards as nontoxic. clinically
phospholipid Clinical practice dosage be 1.2-2.4mg/kg, administering mode is for instiling, and in prescription, the ratio of general DOX and phospholipid is 1: 10 (mass ratio), and adult's blood volume is 86mL/kg, calculate thus, phospholipid concentration is 186 ~ 372nmole/mL.As can be seen here, drug-supplying system of the present invention has certain safety in clinical practice.
The difunctional liposome of embodiment 8 has stronger tumor-targeting
Drug-supplying system of the present invention (CPL), after mice with tumor tail intravenously administrable, in the distribution of tumor much larger than matched group (SSL), along with the prolongation of time, weakens in the picked-up of tumor locus, but administration 8 hours, experimental group is still greater than SSL.On the contrary, SSL has certain distribution at spleen, but this drug-supplying system distributes less in spleen.Distribute in liver relatively many, at brain, the heart, lung, the distribution of kidney is all less.As can be seen here, this drug-supplying system is lower in the intake of normal organ, is morely distributed in tumor, further illustrates the targeting that drug-supplying system of the present invention improves CPP.
Claims (12)
1. a difunctional cancer target liposome administration system, is characterized in that cell-penetrating peptides is directly coupled to surface of liposome by long chain organic acid, has the Polyethylene Glycol-hydrazone key-long chain organic acid conjugate modified liposome surface of sensitivity to acid simultaneously.Its preparation technology comprises the steps:
(1) preparation of medicine carrying conventional liposome: the method for load fat-soluble medicine: appropriate phospholipid, cholesterol and derivant thereof, long chain organic acid, fat-soluble medicine is dissolved in organic solvent, rotates and flash to film at 25 ~ 60 DEG C, isosmotic solution aquation and get final product; Method for water-soluble anthraquinone class weakly basic drugs: appropriate phospholipid, cholesterol and derivant thereof, long chain organic acid, be dissolved in organic solvent, rotary evaporation film forming, add acidity (comprising organic acid or mineral acid) aqueous solution aquation, and then regulate outer aqueous phase to be alkalescence pH7.5 ~ 8.0, add water-soluble anthraquinone class weakly basic drugs overnight incubation, to obtain final product.
(2) CPP modified liposome preparation: in the medicine carrying conventional liposome solution that step (1) obtains, add the long chain organic acid in appropriate 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) EDCI and sulfhydrylation-N-hydroxysuccinimide (s-NHS) activated lipid body, pH is regulated to be 10 ~ 12, add the CPP modified liposome that CPP obtains having internalization again, wherein CPP modifies density is 4% ~ 8% of the amount of phospholipid material, preferably 4%.
(3) preparation of difunctional liposome: add Polyethylene Glycol-hydrazone key-long chain organic acid, overnight incubation.Its density is 8% ~ 20% of the amount of phospholipid material, preferably 8%.
(4) with the bag filter dialysis 4h ~ 48h of molecular cut off 5000Da ~ 300000Da or with the super filter tube of molecular cut off 5000Da ~ 300000Da at the centrifugal 10min ~ 30min of 4000g ~ 15000g, to obtain final product.Preferred molecular cut off is 10000Da, dialysis time 12h, and centrifugal speed is 10000g, and centrifugation time is 20min.
2. the composition of drug-supplying system according to claim 1, is characterized in that: described cell-penetrating peptides is poly arginine small peptide, and preferred sequence is nine poly arginines.
3. according to the drug-supplying system described in claim 1; it is characterized in that: the synthetic method of Polyethylene Glycol-hydrazone key-long chain organic acid is: aldehyde radical polyethylene glycol (mPEG-CHO) is dissolved in the chloroformic solution containing 3-(2-pyridine two sulfur) the propionyl hydrazine (PDPH) little over amount; room temperature reaction 48h; stir in argon shield, synthesis mPEG-HZ-PDP.Sulfhydrylation long chain organic acid is dissolved in anhydrous chloroform, containing excessive mPEG-HZ-PDP in chloroform, adds triethylamine, stirs and spend the night in room temperature under argon shield.Chloroform removed under pressure, conjugate Sepharose CL-4B is purified, and lyophilization obtains white powder.
4. the composition of drug-supplying system according to claim 1, is characterized in that described molecular weight polyethylene glycol is 500Da ~ 5000Da, preferred 2000Da.
5. drug-supplying system according to claim 1, is characterized in that long chain organic acid is selected from stearic acid, lauric acid, oleic acid, Palmic acid, myristic acid.Preferred stearic acid.
6. drug-supplying system according to claim 1, is characterized in that: phospholipid is selected from natural, semi-synthetic, complete synthesis phospholipid and derivant thereof.Preferred scheme is that described phospholipid is selected from but is not limited to following soybean lecithin (SPC), Ovum Gallus domesticus Flavus lecithin (EPC), EPG (EPG), polyene phosphatidylcholine, phosphatidic acid, cardiolipin, sphingomyelins, phosphatidic acid serine, phosphatidylinositols, PHOSPHATIDYL ETHANOLAMINE, hydrogenated soya phosphatide (HSPC), hydrogenated yolk lecithin (HEPC), complete synthesis phospholipid, as the various synthetic phospholipids of C3 to C30, as distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidyl choline (DPPC), DOPC (DOPC), dimyristoyl phosphatidyl choline (DMPC), DLPC (DLPC), DSPG (sodium salt DSPG), DPPG (sodium salt DPPG), L-α-GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (sodium salt DMPG), PE (DLPG), DSPE (DSPE), DPPE (DPPE), DOPE (DOPE), DMPEA (DMPE), two lauroyl PHOSPHATIDYL ETHANOLAMINE (DLPE), two DSPG (sodium salt), two DPPG (sodium salt), two GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (sodium salt), two PE, distearoylphosphatidyl inositol, two palmityl phosphatidylinositols, two myristoyl phosphatidylinositols, two lauroyl phosphatidylinositols, POPC, the sub-oleoyl phosphatidylcholine of palmityl, the sub-oleoyl phosphatidylcholine of stearoyl, stearoyl has phosphatidyl choline, stearoyl palmitoylphosphatidylcholine.
7. according to the drug-supplying system described in claim 1, it is characterized in that, comprise cholesterol and derivant (as Cholesteryl hemisuccinate) thereof and sitosterol.
8. according to the drug-supplying system described in claim 1, it is characterized in that, described medicine is antitumor drug, comprise Anthraquinones antibiotic doxorubicin hydrochloride, amycin, epirubicin, daunorubicin, platinum medicine, as carboplatin, JM-216, eptalatin, acetic acid platinum, cisplatin, cyclophosphamide, D actinomycin D, bleomycin, rubidomycin, mitomycin, methotrexate, 5-fluorouracil, carmustine (BCNU), Semustine, etoposide, etoposide, interferon, camptothecine and various derivant, phenesterin, taxol (Taxol) and derivant thereof, docetaxel and derivant thereof, vinblastine, vincristine, vinorelbine and derivant thereof, tamoxifen, A-20968, tetracyclic triterpenoid etc.
9. according to the drug-supplying system described in claim 1, it is characterized in that, the organic acid that described in step of preparation process (1), load water soluble medicine is used or mineral acid comprise citric acid, lactic acid, fumaric acid, tartaric acid, acetic acid, gluconic acid, lactobionic acid, sorbic acid, maleic acid, ascorbic acid, oxalic acid, formic acid, benzenesulfonic acid, benzoic acid, glutamic acid, aspartic acid, hydrochloric acid, arginine etc., optimization citric acid.
10. drug-supplying system according to claim 1, is characterized in that: be suspension or the freeze-dried powder of liposome.
11. drug-supplying systems according to claim 1, it is characterized in that, it is degerming that preparation method comprises filter membrane.
12. drug-supplying systems according to claim 1, is characterized in that, without hemolytic, stronger cellular uptake, stronger tumor-selective is a kind of promising Tumor Targeting Drug Delivery System.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410153928.3A CN105012956A (en) | 2014-04-17 | 2014-04-17 | Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410153928.3A CN105012956A (en) | 2014-04-17 | 2014-04-17 | Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105012956A true CN105012956A (en) | 2015-11-04 |
Family
ID=54403490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410153928.3A Pending CN105012956A (en) | 2014-04-17 | 2014-04-17 | Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105012956A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105315345A (en) * | 2015-10-29 | 2016-02-10 | 南京大学 | Targeting arginine nonamer and application thereof to RNA interference |
| CN106750254A (en) * | 2017-01-25 | 2017-05-31 | 郑州大学 | The 10 HCPT water-soluble macromolecule conjugates and preparation method and application of the sensitive releases of pH |
| CN107226840A (en) * | 2017-05-04 | 2017-10-03 | 西安交通大学 | A kind of cell-penetrating peptide skin penetration enhancer and its preparation method and application |
| CN112618728A (en) * | 2020-12-18 | 2021-04-09 | 绍兴文理学院 | Dual-response prodrug containing polysialic acid group, synthetic method thereof and application thereof in pharmaceutical preparation |
| CN114209853A (en) * | 2022-01-14 | 2022-03-22 | 广西大学 | Preparation and application of dual-ligand modified liver cancer targeted liposome |
| CN114276404A (en) * | 2021-12-24 | 2022-04-05 | 五邑大学 | Glycyrrhetinic acid glycerophospholipid compound, secretory phospholipase A2 sensitive liposome thereof and application thereof |
| CN114652680A (en) * | 2020-12-24 | 2022-06-24 | 沈阳药科大学 | Method for preparing liposome |
| US11826468B2 (en) | 2021-02-18 | 2023-11-28 | Amorepacific Corporation | Insoluble active substance carrier comprising transfersome |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150024033A1 (en) * | 2013-07-21 | 2015-01-22 | Kimia Zist Parsian Co | Method and system for synthesizing nanocarrier based long acting drug delivery system for buprenorphine |
| CN104784118A (en) * | 2014-01-21 | 2015-07-22 | 谢辉 | Acid-sensitive liposome drug delivery system with internalization effect, and preparation method and applications thereof |
-
2014
- 2014-04-17 CN CN201410153928.3A patent/CN105012956A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150024033A1 (en) * | 2013-07-21 | 2015-01-22 | Kimia Zist Parsian Co | Method and system for synthesizing nanocarrier based long acting drug delivery system for buprenorphine |
| CN104784118A (en) * | 2014-01-21 | 2015-07-22 | 谢辉 | Acid-sensitive liposome drug delivery system with internalization effect, and preparation method and applications thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105315345A (en) * | 2015-10-29 | 2016-02-10 | 南京大学 | Targeting arginine nonamer and application thereof to RNA interference |
| CN105315345B (en) * | 2015-10-29 | 2019-05-10 | 南京大学 | A kind of nine poly arginine of targeting type and its application in the RNA interference |
| CN106750254A (en) * | 2017-01-25 | 2017-05-31 | 郑州大学 | The 10 HCPT water-soluble macromolecule conjugates and preparation method and application of the sensitive releases of pH |
| CN107226840A (en) * | 2017-05-04 | 2017-10-03 | 西安交通大学 | A kind of cell-penetrating peptide skin penetration enhancer and its preparation method and application |
| CN112618728A (en) * | 2020-12-18 | 2021-04-09 | 绍兴文理学院 | Dual-response prodrug containing polysialic acid group, synthetic method thereof and application thereof in pharmaceutical preparation |
| CN114652680A (en) * | 2020-12-24 | 2022-06-24 | 沈阳药科大学 | Method for preparing liposome |
| US11826468B2 (en) | 2021-02-18 | 2023-11-28 | Amorepacific Corporation | Insoluble active substance carrier comprising transfersome |
| CN114276404A (en) * | 2021-12-24 | 2022-04-05 | 五邑大学 | Glycyrrhetinic acid glycerophospholipid compound, secretory phospholipase A2 sensitive liposome thereof and application thereof |
| CN114209853A (en) * | 2022-01-14 | 2022-03-22 | 广西大学 | Preparation and application of dual-ligand modified liver cancer targeted liposome |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kuerban et al. | Doxorubicin-loaded bacterial outer-membrane vesicles exert enhanced anti-tumor efficacy in non-small-cell lung cancer | |
| Shan et al. | Current approaches of nanomedicines in the market and various stage of clinical translation | |
| Qin et al. | Liposome formulated with TAT-modified cholesterol for improving brain delivery and therapeutic efficacy on brain glioma in animals | |
| Kiaie et al. | Axial pharmaceutical properties of liposome in cancer therapy: Recent advances and perspectives | |
| Wang et al. | Biomembrane-based nanostructures for cancer targeting and therapy: from synthetic liposomes to natural biomembranes and membrane-vesicles | |
| CN105012956A (en) | Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof | |
| Gu et al. | Nanotechnology-mediated immunochemotherapy combined with docetaxel and PD-L1 antibody increase therapeutic effects and decrease systemic toxicity | |
| Wang et al. | Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates | |
| Ju et al. | Liposomes, modified with PTDHIV-1 peptide, containing epirubicin and celecoxib, to target vasculogenic mimicry channels in invasive breast cancer | |
| Yu et al. | Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma | |
| Allahou et al. | Investigating the application of liposomes as drug delivery systems for the diagnosis and treatment of cancer | |
| Meng et al. | Integrin-targeted paclitaxel nanoliposomes for tumor therapy | |
| CN106137967B (en) | Target the preparation and application of the dual modified liposome drug delivery system of glioma | |
| Chen et al. | Novel glycyrrhetinic acid conjugated pH-sensitive liposomes for the delivery of doxorubicin and its antitumor activities | |
| CN104784118A (en) | Acid-sensitive liposome drug delivery system with internalization effect, and preparation method and applications thereof | |
| Desai et al. | Pancreatic cancer: Recent advances in nanoformulation-based therapies | |
| WO2019158060A1 (en) | Polyionic complex formed by polysialic acid and cationic compound, preparation method therefor and application thereof | |
| He et al. | Liposomes and liposome-like nanoparticles: From anti-fungal infection to the COVID-19 pandemic treatment | |
| Jia et al. | Increasing the antitumor efficacy of doxorubicin liposomes with coupling an anti-EGFR affibody in EGFR-expressing tumor models | |
| Li et al. | Polysialic acid-functionalized liposomes for efficient honokiol delivery to inhibit breast cancer growth and metastasis | |
| Song et al. | Dual-targeting nanovesicles enhance specificity to dynamic tumor cells in vitro and in vivo via manipulation of αvβ3-ligand binding | |
| WO2018172942A1 (en) | Quercetin nanoparticles | |
| Liu et al. | Transferrin-conjugated liposomes loaded with carnosic acid inhibit liver cancer growth by inducing mitochondria-mediated apoptosis | |
| EP2384743A1 (en) | Siosomal formulation for intracellular delivery and targeting of therapeutic agents | |
| Abbasi et al. | What We Need to Know about Liposomes as Drug Nanocarriers: An Updated Review |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151104 |