CN113350512A - PEG2,nLipid derivative modified nano-carrier, preparation method and application - Google Patents

PEG2,nLipid derivative modified nano-carrier, preparation method and application Download PDF

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CN113350512A
CN113350512A CN202110629173.XA CN202110629173A CN113350512A CN 113350512 A CN113350512 A CN 113350512A CN 202110629173 A CN202110629173 A CN 202110629173A CN 113350512 A CN113350512 A CN 113350512A
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peg
dspe
modified
lipid derivative
molecular weight
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CN113350512B (en
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邓意辉
刘敏
李�杰
闫娜
赵丹
刘梦阳
张红霞
汤雪莹
宋艳志
刘欣荣
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Shenyang Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

PEG2,nThe invention relates to a lipid derivative modified nano-carrier, a preparation method and application, belonging to the technical field of medicine, and the method for eliminating ABC phenomenon caused by PEG nano-carrier provided by the invention is characterized in that the modification material used by the nano-carrier is PEG2,nPEG of (2)2,nLipid derivatives, which form a dense hydrated layer on the surface of the carrier, improve the physical and biological stability of the carrier. In addition, the present invention selects PEG2,nPEG of (2)2,nThe lipid derivative is used for modifying the nano-carrier, so that not only can the ABC phenomenon caused by the PEG nano-carrier be eliminated, but also the severe problem of the current PEG nano-carrier is greatly solved; yet ensureThe in vivo circulation time of the nano-carrier is ensured, the defect of insufficient circulation time of a plurality of PEG substitute materials is overcome, and a firmer foundation is laid for the clinical transformation of the PEG nano-preparation.

Description

PEG2,nLipid derivative modified nano-carrier, preparation method and application
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a novel method capable of eliminating the phenomenon that PEG nano-carrier accelerates blood clearance.
Background
In order to overcome the defects that the traditional nano-carrier is easily identified and taken by a mononuclear-macrophage system (MPS), has short blood circulation half-life and poor targeting property and the like, researchers modify the surface of the nano-carrier by utilizing PEG lipid derivatives, so that the physical, chemical and biological stability of the carrier and the medicine is greatly improved, and the process is called PEGylation. Since the 90 s of the 20 th century, the technology is widely applied to the field of nano-carrier surface modification. Despite the increasing research that other synthetic hydrophilic polymers for surface modification may exhibit long-circulating properties similar to pegylation, the unique advantages of PEG, such as low toxicity, low immunogenicity, and amphiphilicity, continue to hold a difficult alternative in the field of pharmaceutical formulations. At present, the most widely used PEG-lipid derivative is linear mPEG2000DSPE, which has become the "gold standard" for extending the cycling time of nanocarriers.
However, studies in recent years have shown that the PEG-based nano-carrier has a problem that cannot be ignored, namely, the generation of an Accelerated Blood Clearance (ABC) phenomenon. The ABC phenomenon refers to that PEG liposome is injected into the same animal body repeatedly (every few days), which causes the abnormal changes of pharmacokinetics and tissue distribution of the liposome injected twice, generally showing that the blood clearance is accelerated, and the aggregation quantity of the liver and the spleen is obviously increased. This phenomenon is present in the repeated administration of various PEGylated formulations, such as PEGylated micelles (Koide H, Asai T, Hatanaka K, et al, particulate size-dependent mutagenesis of acidic membrane clear peptides [ J ]. Int J Pharm,2008,362(1-2):197-200.), solid lipid nanoparticles (ZHAO Y, Wang L, Yan M, et al, reproduced information of PEGylated hydrophobic nanoparticles and copolymers [ J ]. Int J. Nano-molecular peptides, 2012, 2891-2900., emulsion (Wang C, chemical X, Sui Y, chemical a. A. peptide: chemical polypeptide clear peptides: polysaccharide J. (Biochemical J.), "PEG J. adsorbed peptide J.," adsorbed protein, polysaccharide J., "adsorbed protein", PEG 3. adsorbed protein J., "adsorbed protein, polysaccharide J.," adsorbed protein, adsorbed by adsorbed protein, adsorbed, 2012,14(3):261 and 266), etc. Furthermore, it has been found that the ABC phenomenon is induced by the first injection of non-PEGylated Common Liposomes (CL) (Ishida T, Harada M, Wang XY, et al. Accerated lipid clearance of PEGylated lipids following the introduction of a prediction lipid injection: effects of lipid and PEG surface-sensitivity and chain length of the first-domain lipids [ J ]. Journal of controlled release,2005,105(3): 305. 317.). This suggests that the ABC phenomenon may be a problem commonly encountered with nano-formulations. In addition, it has been reported that ABC phenomenon is found in animal models such as mice, rats, guinea pigs, rabbits, rhesus monkeys, Beagle dogs, and the like. More seriously, several studies have shown that anti-PEG antibodies can be detected in the blood of 40% to 72% of healthy subjects (Kinam, park, impact of anti-PEG antibodies on PEG-based nanoparticles in vivo. Journal of Controlled Release office Journal of the Controlled Release society.2018.), which means that humans cannot avoid the ABC phenomenon.
The generation of the ABC phenomenon not only greatly weakens the long-circulating advantage of the PEG nano-carrier, but also causes serious toxic and side effects of the encapsulated drug in vivo due to the change of pharmacokinetics and tissue distribution behaviors, and seriously hinders the clinical transformation of the nano-preparation.
Researchers have attempted to replace PEG with PVP, PDMA, HPMA, and like materials (Hoang Thi, Thai Thanh, et al. the immunity of Poly (ethylene glycol) Alternatives for over-communicating PEG immunity in Drug Delivery and bioconjugation. polymers12.2(2020):298.) although these material-modified nanopreparations do not induce the ABC phenomenon, these materials suffer from other drawbacks, such as short blood circulation half-life. Moreover, in view of the great amount of time, energy and financial resources in the field of PEG modification, it is a natural but inexorable method to find PEG substitutes, and therefore, it is urgent to directly solve the immunogenicity of PEG.
Compared to linear PEG, studies have shown that branched PEG (PEG) is used2) The protein medicine is modified, so that the stability of the protein medicine can be improved, and the circulation time is prolonged. Current PEG2Has been widely used in the modification of enzymes, monoclonal antibodies, oligonucleotides and small molecule drugs, such as the preparation on the market
Figure BDA0003102926480000021
And the like. When PEG is used2When modified, PEG chains are distributed in an "umbrella" structure on the surface of the protein drug modified by PEG chains, and the coverage is wider, which prevents the drug from being recognized and taken up by MPS, thereby increasing stability and prolonging cycle time (Monfardini, Cristina, et al. "a branched monomethoxypoly (ethylene glycol) for protein modification." Bioconjugate chemistry 6.1(1995):62-69. h.nishimura.t.takahashi.k.sakurai.a.matrishima and y.inada, Gann,73(I982) 470.). It has been reported that "brush-like" branched PEG does not bind to anti-PEG IgM and does not bind non-specifically to proteins and cells. Therefore, we speculate that branched PEG may have great potential in reducing the antigenicity of the formulation, thereby reducing or even eliminating the ABC phenomenonForce.
In summary, we believe that the use of PEG2Modifying the nanocarriers may break the dilemma of pegylation of the nanocarriers. At present, there is little concern about the use of PEG2Related reports of modified nano-carriers, no more than research personnel for investigating PEG2Transforming the ABC phenomenon of the nano-carrier. Our research just fills the gap in this field. We used PEG of different molecular weights2Lipid derivatives, DSPE-mPEG2,nModifying emulsion, liposome and micelle as nano carrier, and researching repeated injection of PEG with different molecular weights2The induction condition of the nanometer carrier to the ABC phenomenon provides a new method for eliminating the ABC phenomenon of the PEG nanometer carrier.
The classical immune mechanism of the ABC phenomenon is currently generally recognized as: the PEG nano-carrier injected for the first time stimulates B cells in the marginal zone of the spleen to proliferate and differentiate in a TI-2 type antigen mode and generates anti-PEG IgM, after the PEG nano-carrier is injected for the second time, the anti-PEG IgM is rapidly identified and combined with the PEG IgM to activate a complement system, so that the complement C3 fragment has a conditioning effect on particles, the ingestion of the PEG nano-carrier by MPS is accelerated, the PEG nano-carrier is rapidly eliminated from blood, and the ABC phenomenon occurs.
It follows that anti-PEG IgM is a major cause of ABC phenomenon, and it is now known that pegylation induces anti-PEG antibodies in both animals and humans. Clinical experience with pegylation therapy has shown that anti-PEG antibodies not only increase clearance and lose efficacy, but also cause severe allergic or anaphylactic reactions. POVSIC, Thomas J., et al, Pre-existant-PEG antibodies associated with a segment immunological interactions to pegivacogin, a PEGylated aptamer, journal of Allergy and Clinical Immunology,2016,138.6: 1712. Oc.1715; (ii) RAU, Rachel E., et al, Outcome of particulate substrates with access to particulate having a particulate diameter&cancer,2018,65.3:e26873.；LONGO,Nicola,et al.Single-dose,subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria:an open-label,multicentre,phase 1dose-escalation trial.The Lancet,2014,384.9937:37-44.]Notably, the FDA currently requires that patients be tested for anti-PEG antibodies prior to treatment with experimental PEGylated compounds. Laboratory studies have shown that this is in contrast to PEG2k(straight-chain PEG), first injecting PEG into rat body2,nThe modified nanocarriers produced significantly less anti-PEG IgM, suggesting that we used PEG2,nThe modified nano-carrier can reduce the generation of anti-PEG antibody; in addition, studies have shown that the first injection of PEG2,nThe emulsion was modified and repeated injections seven days later were made with different levels of formulation to antibody binding. Following PEG2,nThe molecular weight increases and the slower the binding of the preparation to the antibody, wherein PE2,40kBinding to antibodies was the slowest. This also suggests that we are high molecular weight PEG2,nThe nano-carrier is modified, so that the circulation time of the nano-carrier is prolonged, and a foundation is provided for the research and development of nano-preparations.
Disclosure of Invention
In view of the above problems, the main object of the present invention is to provide a PEG2,nThe lipid derivative modified nano carrier and the preparation method thereof are used for eliminating the ABC phenomenon caused by the PEG nano carrier and solving the problems of high blood clearing speed, obvious increase of liver and spleen aggregation amount and the like caused by the ABC phenomenon of the PEG nano carrier.
The invention is realized by adopting the following technical scheme:
the method for eliminating ABC phenomenon caused by PEG nano carrier provided by the invention uses branched chain PEG (PEG) with different molecular weights2,n) PEG of (2)2,nLipid derivatives to modify the nanocarriers.
The nano-carrier comprises: emulsions, liposomes, micelles.
The PEG2,nTwo linear PEG chains with methoxy end, and a compound which is respectively connected with two amino groups of lysine in a covalent bond, wherein n represents the total molecular weight of the two linear PEG chains. Theoretically, such PEGs2,nThere can be countless, two linear PEG chains (PEG)2) Of (a) a moleculeThe amount was 200-400000 Da. Preferably, PEG2,nThe molecular weight of (B) is 20000-40000 Da.
The branched PEG of the invention also comprises branched PEG comprising three or more than three PEG chains.
The PEG of the present invention2,nPEG of (2)2,n-lipids in the lipid derivative are natural phospholipids, semi-synthetic phospholipids or synthetic phospholipids. The method comprises the following steps: soybean lecithin, egg yolk lecithin (E80), phosphatidylglycerol, EPG (egg yolk phosphatidylglycerol), phosphatidic acid, cardiolipin, sphingomyelin, phosphatidic acid serine, phosphatidylinositol, phosphatidylethanolamine, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, didecanoylphosphatidylcholine, dioctanoylphosphatidylcholine, dihexanoylphosphatidylcholine, distearoylphosphatidylglycerol and salts thereof, dipalmitoylphosphatidylglycerol and salts thereof, L- α -dimyristoylphosphatidylglycerol and salts thereof, dilauroylphosphatidylglycerol, didecanoylphosphatidylglycerol, dioctanoylphosphatidylglycerol, dihexanoylphosphatidylglycerol, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, phosphatidylethanolamine, Dioleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, distearoylphosphatidylglycerol and salts thereof, dipalmitoylphosphatidylglycerol and salts thereof, dimyristoylphosphatidylglycerol and salts thereof, dilauroylphosphatidylglycerol, distearoylphosphatidylinositol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilauroylphosphatidylinositol, palmitoleoylphosphatidylcholine, stearoylileoylphosphatidylcholine, stearoyleicosanoylphosphatidylcholine, Distearoylphosphatidylethanolamine (DSPE).
In the present invention, PE represents a linear or branched PEG-modified emulsion, PE2kRepresents a linear PEG with a molecular weight of 2000; PE (polyethylene)2,2k，PE2,10k，PE2,20kAnd PE2,40kDenoting end as a2 linear PEG chains of oxygen groups, and lysine through covalent bond, wherein the total molecular weight of the 2 PEG chains is 2000, 10000, 20000 and 40000 respectively.
In the present invention, PL denotes a linear or branched PEG-modified liposome, and PL2kRepresents a linear PEG with a molecular weight of 2000; PL2,2k，PL2,10kAnd PL2,20kRepresents a compound obtained by connecting 2 linear PEG chains with methoxyl at the tail end and lysine through covalent bond, wherein the total molecular weight of the 2 PEG chains is 2000, 10000 and 20000.
The PEG of the present invention2,n-lipid derivative modified emulsions, the composition and preparation method of which comprises:
the components: oil phase, emulsifier, PEG2,n-lipid derivatives, sterile water for injection; wherein the mass ratio of the oil phase to the emulsifier is (3-5) to 1, and PEG2,nThe molar ratio of the lipid derivative to the E-80 is 1 (10-100).
The preparation method comprises the following steps:
weighing the membrane material (oil phase, emulsifier, PEG) according to the prescription2,n-lipid derivative) is completely dissolved in a water bath at 55-65 ℃, and sterilized water for injection heated to the same temperature is injected under stirring;
continuously stirring and incubating in 55-65 deg.C water bath for 10-30min to obtain primary emulsion;
subjecting the obtained primary emulsion to ultrasonic dispersion treatment (for reducing particle size), and sequentially filtering with 0.80, 0.45 and 0.22 μm microporous filter membrane to obtain PEG2,n-lipid derivative modified emulsions.
Further, DSPE-mPEG with different molecular weights2,nModified emulsion, prescription and preparation method:
TABLE 1 different molecular weights of DSPE-mPEG2,nModified emulsion formula
Figure BDA0003102926480000051
The preparation method comprises the following steps: weighing the membrane material with the prescription amount, completely dissolving in a water bath at 55 ℃, and injecting sterilized water for injection which is heated to the same temperature under the stirring condition. And further stirring and incubating in 55 deg.C water bath for 20min to obtain primary emulsion. Subjecting the obtained primary emulsion to ultrasonic dispersion treatment (power and time: 200 Wx 4min +400 Wx 6min, 1s interval 1s), sequentially filtering with 0.80, 0.45 and 0.22 μm microporous filter membrane to obtain emulsion.
The PEG of the present invention2,nLipid derivative-modified liposomes, the composition and the preparation method of which comprise:
the components: phospholipid, cholesterol, PEG2,n-lipid derivatives and sterile water for injection; wherein the mass ratio of phospholipid to cholesterol (3-5) is 1, PEG2,nThe molar ratio of the lipid derivative to the (phospholipid + cholesterol) is 1 (10-100).
The preparation method comprises the following steps:
weighing the membrane material (phospholipid, cholesterol, PEG) according to the prescription2,n-a lipid derivative), adding absolute ethyl alcohol, stirring and dissolving in a water bath at 55-75 ℃, volatilizing 50-80% of absolute ethyl alcohol to obtain a concentrate;
dripping the sterilized water for injection preheated to the same temperature into the concentrate;
continuously stirring in water bath at 55-75 deg.C for 15-30min to obtain liposome primary product;
subjecting the obtained liposome primary product to ultrasonic dispersion treatment, sequentially passing through 0.80, 0.45, 0.22 μm microporous filter membrane to obtain PEG2,n-lipid derivative modified liposomes.
Further, DSPE-mPEG with different molecular weights2,nThe modified liposome prescription and the preparation method are as follows:
TABLE 2 different molecular weights of DSPE-mPEG2,nModified liposome formulations
Figure BDA0003102926480000052
The preparation method comprises the following steps: weighing the membrane material (hydrogenated soybean phospholipid, cholesterol and DSPE-mPEG)2,n) Placing the mixture into a 10mL penicillin bottle, adding 300-500 mu L absolute ethyl alcohol, stirring and dissolving the mixture in a water bath at 65 ℃, and volatilizing most of the absolute ethyl alcohol. At a rate of 4 mL/min-1Will be preheated to the same speedSterile water for injection (5mL) at temperature was injected into the membrane. Stirring in 65 deg.C water bath for 20min to obtain liposome primary product. Subjecting the obtained primary product to ultrasonic dispersion treatment (power and time: 200 Wx 4min +400 Wx 6min, 1s operation interval 1s), and sequentially passing through 0.80, 0.45, and 0.22 μm microporous filter membrane to obtain liposome.
The invention has the advantages that:
the invention can eliminate ABC phenomenon caused by PEG nano carrier. The modification material used by the nano-carrier is PEG2,nPEG of (2)2,nLipid derivatives, which form a dense hydrated layer on the surface of the carrier, improve the physical and biological stability of the carrier. In addition, the present invention selects PEG2,nPEG of (2)2,nThe lipid derivative is used for modifying the nano-carrier, so that not only can the ABC phenomenon caused by the PEG nano-carrier be eliminated, but also the severe problem of the current PEG nano-carrier is greatly solved; the in vivo circulation time of the nano-carrier is ensured, the defect of insufficient circulation time of a plurality of PEG substitute materials is overcome, and a firmer foundation is laid for the clinical transformation of the PEG nano-preparation.
Drawings
FIG. 1 shows DSPE-PEG with a total molecular weight of 2000Da after the first tail vein injection of PEG in example 1 of the present invention2000DSPE-PEG with molecular weight of 2000Da for secondary tail vein injection of PEG by modified emulsion2000The effect of modifying the time profile of the emulsion;
in the figure, PE2kDSPE-PEG representing PEG with total molecular weight of 2000Da2000A modified emulsion; 5. mu. mol/kg PE2k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5. mu. mol/kg PEG and total molecular weight of 2000Da2000A modified emulsion; 5. mu. mol/kg PE2k-2 represents the first and second injections of a DSPE-PEG with a phospholipid concentration of 5. mu. mol/kg PEG and a total molecular weight of 2000Da2000A modified emulsion.
FIG. 2 shows DSPE-PEG with a total molecular weight of 2000Da after the first tail vein injection of PEG in example 2 of the present invention2,2kDSPE-PEG with molecular weight of 2000Da for secondary tail vein injection of PEG by modified emulsion2,2kThe effect of modifying the time profile of the emulsion;
in the figure, PE2,2kDSPE-PEG representing PEG with total molecular weight of 2000Da2,2kA modified emulsion; 5. mu. mol/kg PE2,2k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5. mu. mol/kg PEG and total molecular weight of 2000Da2,2kA modified emulsion; 5. mu. mol/kg PE2,2k-2 represents the first and second injections of a DSPE-PEG with a phospholipid concentration of 5. mu. mol/kg PEG and a total molecular weight of 2000Da2,2kA modified emulsion.
FIG. 3 shows DSPE-PEG with a total molecular weight of 10000Da for the first tail vein injection of PEG in example 3 of the present invention2,10kThe modified emulsion is used for secondary tail vein injection of DSPE-PEG with PEG total molecular weight of 10000Da2,10kThe effect of modifying the time profile of the emulsion;
in the figure, PE2,10kDSPE-PEG representing PEG with total molecular weight of 10000Da2,10kA modified emulsion; 5. mu. mol/kg PE2,10k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5 mu mol/kg PEG and total molecular weight of 10000Da2,10kA modified emulsion; 5 μmol/kg PEG2,10k-2 represents DSPE-PEG with total molecular weight of 10000Da of PEG with phospholipid concentration of 5 mu mol/kg for the first and the second injection2,10kA modified emulsion.
FIG. 4 shows DSPE-PEG with total molecular weight of 20000Da injected into tail vein for the first time in example 4 of the present invention2,20kThe modified emulsion is used for the DSPE-PEG with the total molecular weight of 20000Da for the secondary tail vein injection of PEG2,20kThe effect of modifying the time profile of the emulsion;
in the figure, PE2,20kDSPE-PEG with PEG total molecular weight of 20000Da2,20kA modified emulsion; 5. mu. mol/kg PE2,20k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5 mu mol/kg and PEG total molecular weight of 20000Da2,20kA modified emulsion; 5 μmol/kg PEG2,20k-2 represents the first and second injections of DSPE-PEG with phospholipid concentration of 5. mu. mol/kg, total PEG molecular weight of 20000Da2,20kA modified emulsion.
FIG. 5 shows DSPE with total molecular weight of 40000Da after first tail vein injection of PEG in example 5 of the present invention-PEG2,40kThe modified emulsion is used for the DSPE-PEG with the total molecular weight of 40000Da of PEG injected into the tail vein twice2The effect of modifying the time profile of the emulsion;
in the figure, PE2,40kDSPE-PEG with total PEG molecular weight of 40000Da2,40kA modified emulsion; 5. mu. mol/kg PE2,40k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total PEG molecular weight of 40000Da2,40kA modified emulsion; 5 μmol/kg PEG2,40k-2 represents the first and second injections of a DSPE-PEG with a phospholipid concentration of 5. mu. mol/kg, a total molecular weight of the PEG of 40000Da2,40kA modified emulsion.
FIG. 6 shows DSPE-PEG with a total molecular weight of 2000Da after the first tail vein injection of PEG in example 6 of the present invention2000Modified liposome DSPE-PEG with molecular weight of 2000Da for secondary tail vein injection of PEG2000The effect of the curve when modifying the liposome drug;
PL in the figure2kDSPE-PEG representing PEG with total molecular weight of 2000Da2000A modified liposome; 5. mu. mol/kg PL2k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5. mu. mol/kg PEG and total molecular weight of 2000Da2000A modified liposome; 5. mu. mol/kg PE2k-2 represents the first and second injections of a DSPE-PEG with a phospholipid concentration of 5. mu. mol/kg PEG and a total molecular weight of 2000Da2000Modified liposomes.
FIG. 7 shows DSPE-PEG with total molecular weight of 2000Da after first end intravenous injection of PEG in example 7 of the present inventors2,2kModified liposome DSPE-PEG with molecular weight of 2000Da for secondary tail vein injection of PEG2,2kThe effect of the curve when modifying the liposome drug;
PL in the figure2,2kDSPE-PEG representing PEG with total molecular weight of 2000Da2,2kA modified liposome; 5. mu. mol/kg PL2,2k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5. mu. mol/kg PEG and total molecular weight of 2000Da2,2kA modified liposome; 5. mu. mol/kg PL2,2k-2 represents DSPE-PE with a total molecular weight of 2000Da of PEG with 5 mu mol/kg phospholipid concentration for the first and second injectionsG2,2kModified liposomes.
FIG. 8 shows DSPE-PEG with a total molecular weight of 10000Da for the first tail vein injection of PEG in example 8 of the present invention2,10kModified liposome DSPE-PEG with PEG molecular weight of 10000Da for secondary tail vein injection2,10kThe effect of the curve when modifying the liposome drug;
PL in the figure2,10kDSPE-PEG representing PEG with total molecular weight of 10000Da2,10kA modified liposome; 5. mu. mol/kg PL2,10k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5 mu mol/kg PEG and total molecular weight of 10000Da2,10kA modified liposome; 5. mu. mol/kg PL2,10k-2 represents DSPE-PEG with total molecular weight of 10000Da of PEG with phospholipid concentration of 5 mu mol/kg for the first and the second injection2,10kModified liposomes.
FIG. 9 shows DSPE-PEG with total molecular weight of 20000Da injected into tail vein for the first time in example 9 of the present invention2,20kModified liposome DSPE-PEG with PEG molecular weight of 20000Da for secondary tail vein injection2,20kThe effect of the curve when modifying the liposome drug;
PL in the figure2,20kDSPE-PEG with PEG total molecular weight of 20000Da2,20kA modified liposome; 5. mu. mol/kg PL2,20k-1 represents the first injection of 5% glucose injection, the second injection of DSPE-PEG with phospholipid concentration of 5 mu mol/kg and PEG total molecular weight of 20000Da2,20kA modified liposome; 5. mu. mol/kg PL2,20k-2 represents the first and second injections of DSPE-PEG with phospholipid concentration of 5. mu. mol/kg, total PEG molecular weight of 20000Da2,20kModified liposomes.
FIG. 10 shows the effect of first tail vein injection of different branched PEG-modified PEs on anti-PEG IgM levels in examples 1-5 of the present invention.
FIG. 11 shows the effect of first tail vein injection of different branched PEG modified PEs on anti-PEG IgM level in examples 6-9 and 11 of the present invention.
FIG. 12 shows the first PE injection in example 12 of the present invention2,nRepeated injections of PE 7 days later2,nMeasurement of anti-PEG IgM levels in plasma at different later time points.
FIG. 13 shows the first injection of PL in example 13 of the present invention2,nRepeated injections of PL 7 days later2,nMeasurement of anti-PEG IgM levels in plasma at different later time points.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
Example 1:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 2000Da2000The modified emulsion is used for secondary tail vein injection of DSPE-PEG with the total molecular weight of PEG of 2000Da2000Effect of modified emulsion timing Curve
Experimental animals:
wistar rats (180-210 g,
Figure BDA0003102926480000081
lei yun Shang pharmaceutical laboratory animal center provides)
The administration scheme is as follows:
taking male Wistar rats with the weight of 180-210 g, randomly dividing the rats into 2 groups, wherein one group is a control group, the other group is an experimental group, and 6 rats are subjected to tail vein injection administration. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with PEG total molecular weight of 2000Da at phospholipid dosage of 5 mu mol/kg for the first time2000Modifying the emulsion. After the first injection, every 7 days later, all groups were intravenously injected with 5. mu. mol phospholipid/kg of DSPE-PEG with a PEG total molecular weight of 2000Da2000Modifying the emulsion, taking blood through orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the concentration of the drug in the plasma by using an enzyme-labeling instrument after treatment.
The processing is specifically operative to: 0.5mL of plasma was placed in a 1.5mL EP tube, 0.9mL of absolute ethanol was added, vortexed for 5min, and mixed well. The resulting suspension was centrifuged at 10000rpm for 10min, the resulting supernatant (0.6mL) was transferred to another 1.5mL EP tube and centrifuged at 10000rpm for 10min twice, 200. mu.L of the supernatant was applied to a 96-well plate, and the fluorescence intensity F was measured at a wavelength of. lambda. ex 750nm and. lambda. em 790 nm. The following experiments were conducted in the same manner as in example 1.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total molecular weight of PEG of 2000Da2000Modifying the emulsion, injecting DSPE-PEG with phospholipid concentration of 5 μmol/kg and total molecular weight of 2000Da at an interval of 7 days2000The time curve of the modified emulsion is shown as 5 mu mol/kg PE in figure 12kCurve denoted by-2, ABC index(0-0.5h)The results are shown in Table 1, and the results of the anti-PEG IgM assay are shown in FIG. 10, and the anti-PEG IgM content is high, and the OD value is 3.27. + -. 0.07.
The results show that the first injection of DSPE-PEG with PEG total molecular weight of 2000Da2000Modifying the emulsion, inducing two repeated injections of DSPE-PEG with a total PEG molecular weight of 2000Da2000Rapid clearance of modified emulsion in blood, its ABC index(0-0.5h)0.17. + -. 0.01, inducing a strong ABC phenomenon.
Example 2:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 2000Da2,2kThe modified emulsion is used for secondary tail vein injection of DSPE-PEG with the total molecular weight of PEG of 2000Da2,2kEffect of modified emulsion timing Curve
Experimental animals:
wistar rat (180 ~ 210g, Lishui Shang pharmaceutical laboratory animal center)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with PEG total molecular weight of 2000Da at phospholipid dosage of 5 mu mol/kg for the first time2,2kModifying the emulsion. After the first injection, every 7 days later, all groups were intravenously injected with 5. mu. mol phospholipid/kg of DSPE-PEG with a PEG total molecular weight of 2000Da2,2kModifying emulsion, collecting blood via orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h, and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting with enzyme-labeling instrumentThe concentration of the drug in (a).
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total molecular weight of PEG of 2000Da2,2kModifying the emulsion, injecting DSPE-PEG with phospholipid concentration of 5 μmol/kg and total molecular weight of 2000Da at an interval of 7 days2,2kThe time curve of the modified emulsion is shown in figure 2, wherein the PE is 5 mu mol/kg2,2kCurve denoted by-2, ABC index(0-0.5h)The results are shown in Table 1, and the results of the anti-PEG IgM assay are shown in FIG. 10, with a higher content of anti-PEG IgM and an OD of 1.97. + -. 0.10.
The results show that the first injection of DSPE-PEG with PEG total molecular weight of 2000Da2,2kModifying the emulsion by injecting DSPE-PEG with total molecular weight of 2000Da2,2kThe clearance rate of the modified emulsion in blood has almost no influence, and the ABC index thereof(0-0.5h)Is 1.00 +/-0.03, and obviously eliminates the ABC phenomenon.
Example 3:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 10000Da2,10kThe modified emulsion is used for secondary tail vein injection of DSPE-PEG with PEG total molecular weight of 10000Da2,10kEffect of modified emulsion timing Curve
Experimental animals:
wistar rat (180 ~ 210g, Lishui Shang pharmaceutical laboratory animal center)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with PEG total molecular weight of 10000Da at phospholipid dosage of 5 mu mol/kg for the first time2,10kModifying the emulsion. 7 days after the first injection, all groups were intravenously injected with DSPE-PEG of 5. mu. mol phospholipid/kg PEG with a total molecular weight of 10000Da2,10kModifying the emulsion, taking blood through orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the concentration of the drug in the plasma by using an enzyme-labeling instrument after treatment.
As a result:
first tail vein injectionDSPE-PEG with phospholipid concentration of 5 mu mol/kg and PEG total molecular weight of 10000Da2,10kModifying the emulsion, injecting DSPE-PEG with phospholipid concentration of 5 μmol/kg and total molecular weight of 10000Da for two times at an interval of 7 days2,10kThe time curve of the modified emulsion is shown in figure 3, 5 μmol/kg PEG2,10kCurve denoted by-2, ABC index(0-0.5h)See table 1, the results of the anti-PEG IgM assay are shown in fig. 10, the anti-PEG IgM content is high, and the OD value is 1.06 ± 0.09.
The result shows that the first injection of the DSPE-PEG with the total molecular weight of the PEG being 10000Da2,10kModifying the emulsion by injecting DSPE-PEG with total molecular weight of 10000Da in the process of two times2,10kThe clearance rate of the modified emulsion in blood has almost no influence, and the ABC index thereof(0-0.5h)Is 1.01 +/-0.02, and obviously eliminates the ABC phenomenon.
Example 4:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 20000Da2,20kThe modified emulsion is used for the DSPE-PEG with the total molecular weight of 20000Da for the secondary tail vein injection of PEG2,20kEffect of modified emulsion timing Curve
Experimental animals:
wistar rat (180 ~ 210g, Lishui Shang pharmaceutical laboratory animal center)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with total molecular weight of 20000Da at phospholipid dosage of 5 μmol/kg2,20kModifying the emulsion. 7 days after the first injection, all groups were intravenously injected with DSPE-PEG of 5. mu. mol phospholipid/kg PEG with a total molecular weight of 20000Da2,20kModifying the emulsion, taking blood through orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the concentration of the drug in the plasma by using an enzyme-labeling instrument after treatment.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total PEG molecular weight of 20000Da2,20kModifying the emulsion, injecting phosphorus twice at an interval of 7 daysDSPE-PEG with lipid concentration of 5 mu mol/kg and PEG total molecular weight of 20000Da2,20kThe time curve of the modified emulsion is shown in figure 4, 5 μmol/kg PEG2,20kCurve denoted by-2, ABC index(0-0.5h)See table 1, the results of the anti-PEG IgM assay are shown in fig. 10, the anti-PEG IgM content is high, and the OD value is 0.68 ± 0.06.
The result shows that the first injection of the DSPE-PEG with the total molecular weight of the PEG of 20000Da2,20kModifying emulsion, and repeatedly injecting DSPE-PEG with total molecular weight of 20000Da2,20kThe clearance rate of the modified emulsion in blood has almost no influence, and the ABC index thereof(0-0.5h)Is 1.03 +/-0.04, and obviously eliminates the ABC phenomenon.
Example 5:
the first tail vein injection of DSPE-PEG with PEG total molecular weight of 40000Da2,40kThe modified emulsion is used for the DSPE-PEG with the total molecular weight of 40000Da of PEG injected into the tail vein twice2,40kEffect of modified emulsion timing Curve
Experimental animals:
wistar rats (180-210 g,
Figure BDA0003102926480000111
lei yun Shang pharmaceutical laboratory animal center provides)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with total molecular weight of 40000Da of PEG according to phospholipid dosage of 5 mu mol/kg for the first time2,40kModifying the emulsion. 7 days after the first injection, all groups were intravenously injected with 5. mu. mol phospholipid/kg of DSPE-PEG with a PEG total molecular weight of 40000Da2,40kModifying the emulsion, taking blood through orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the concentration of the drug in the plasma by using an enzyme-labeling instrument after treatment.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total molecular weight of PEG of 40000Da2,40kModifying the emulsion, injecting twice at an interval of 7 daysDSPE-PEG with total PEG molecular weight of 40000Da and with belamcanda concentration of 5 mu mol/kg2,40kThe time curve of the modified emulsion is shown in figure 5, 5 μmol/kg PEG2,40kCurve denoted by-2, ABC index(0-0.5h)See table 1, the results of the anti-PEG IgM assay are shown in fig. 10, the anti-PEG IgM content is high, and the OD value is 0.38 ± 0.08.
The result shows that the first injection of the DSPE-PEG with the total molecular weight of the PEG being 40000Da2,40kModifying emulsion, and repeatedly injecting DSPE-PEG with total molecular weight of 40000Da2,40kThe clearance rate of the modified emulsion in blood has almost no influence, and the ABC index thereof(0-0.5h)Is 1.01 +/-0.01, and obviously eliminates the ABC phenomenon.
Example 6:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 2000Da2000Modified liposome DSPE-PEG with total molecular weight of 2000Da for secondary tail vein injection of PEG2000Effect of modified Liposomal drug timing Curve
Experimental animals:
wistar rat (180 ~ 210g, Lishui Shang pharmaceutical laboratory animal center)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with PEG total molecular weight of 2000Da at phospholipid dosage of 5 mu mol/kg for the first time2000The liposomes are modified. 7 days after the first injection, all groups were intravenously injected with DSPE-PEG of 5. mu. mol phospholipid/kg PEG with a total molecular weight of 2000Da2000Modifying liposome, collecting blood via orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration with enzyme labeling instrument.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total molecular weight of PEG of 2000Da2000Modifying liposome, injecting DSPE-PEG with phospholipid concentration of 5 μmol/kg and total molecular weight of 2000Da at an interval of 7 days2000The time curve of the modified liposome is shown in5. mu. mol/kg PE in FIG. 62kCurve denoted by-2, ABC index(0-0.5h)See table 2, the results of the anti-PEG IgM assay are shown in fig. 11, the anti-PEG IgM content is high, and the OD value is 3.28 ± 0.06.
The results show that the first injection of DSPE-PEG with PEG total molecular weight of 2000Da2000Modifying liposome, inducing twice repeated injections of DSPE-PEG with PEG total molecular weight of 2000Da2000Modified liposomes rapidly cleared in blood, their ABC index(0-0.5h)0.21. + -. 0.02, a strong ABC phenomenon is induced.
Example 7:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 2000Da2,2kModified liposome DSPE-PEG with total molecular weight of 2000Da for secondary tail vein injection of PEG2,2kEffect of modified Liposomal drug timing Curve
Experimental animals:
wistar rats (180-210 g,
Figure BDA0003102926480000121
lei yun Shang pharmaceutical laboratory animal center provides)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with PEG total molecular weight of 2000Da at phospholipid dosage of 5 mu mol/kg for the first time2,2kThe liposomes are modified. 7 days after the first injection, all groups were intravenously injected with DSPE-PEG of 5. mu. mol phospholipid/kg PEG with a total molecular weight of 2000Da2,2kModifying liposome, collecting blood via orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration with enzyme labeling instrument.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total molecular weight of PEG of 2000Da2,2kModifying liposome, injecting DSPE-PEG with phospholipid concentration of 5 μmol/kg and total molecular weight of 2000Da at an interval of 7 days2,2kThe time curve of the modified liposome is shown in5 μmol/kg PL in FIG. 72,2kCurve denoted by-2, ABC index(0-0.5h)See table 2, the results of the anti-PEG IgM assay are shown in fig. 11, the anti-PEG IgM content is high, and the OD value is 1.77 ± 0.07.
The results show that the first injection of DSPE-PEG with PEG total molecular weight of 2000Da2,2kModifying liposome, and repeatedly injecting DSPE-PEG with total molecular weight of 2000Da2,2kThe clearance rate of the modified liposome in blood has little influence, and the ABC index thereof(0-0.5h)Is 1.02 +/-0.03, and obviously eliminates the ABC phenomenon.
Example 8:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 10000Da2,10kModified liposome DSPE-PEG with total molecular weight of 10000Da for secondary tail vein injection of PEG2,10kEffect of modified Liposomal drug timing Curve
Experimental animals:
wistar rats (180-210 g,
Figure BDA0003102926480000131
lei yun Shang pharmaceutical laboratory animal center provides)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with PEG total molecular weight of 10000Da at phospholipid dosage of 5 mu mol/kg for the first time2,10kThe liposomes are modified. 7 days after the first injection, all groups were intravenously injected with DSPE-PEG of 5. mu. mol phospholipid/kg PEG with a total molecular weight of 10000Da2,10kModifying liposome, collecting blood via orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration with enzyme labeling instrument.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total PEG molecular weight of 10000Da2,10kModifying liposome, injecting DSPE-PEG with phospholipid concentration of 5 μmol/kg and total molecular weight of 10000Da for two times at an interval of 7 days2,10kModified lipidsThe time curve of the body is shown in figure 8, 5 μmol/kg PL2,10kCurve denoted by-2, ABC index(0-0.5h)See table 2, the results of the anti-PEG IgM assay are shown in fig. 11, the anti-PEG IgM content is high, and the OD value is 0.95 ± 0.04.
The result shows that the first injection of the DSPE-PEG with the total molecular weight of the PEG being 10000Da2,10kModifying liposome, and repeatedly injecting DSPE-PEG with total PEG molecular weight of 10000Da for two times2,10kThe clearance rate of the modified liposome in blood has little influence, and the ABC index thereof(0-0.5h)1.10 +/-0.05, and obviously eliminates the ABC phenomenon.
Example 9:
first tail vein injection of DSPE-PEG with PEG total molecular weight of 20000Da2,20kThe modified liposome is used for secondary tail vein injection of DSPE-PEG with PEG total molecular weight of 20000Da2,20kEffect of modified Liposomal drug timing Curve
Experimental animals:
wistar rats (180-210 g,
Figure BDA0003102926480000141
lei yun Shang pharmaceutical laboratory animal center provides)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 2 groups and administered by tail vein injection. The control group is injected with 5% glucose solution for the first time, and the experimental group is injected with DSPE-PEG with total molecular weight of 20000Da at phospholipid dosage of 5 μmol/kg2,20kThe liposomes are modified. 7 days after the first injection, all groups were intravenously injected with DSPE-PEG of 5. mu. mol phospholipid/kg PEG with a total molecular weight of 20000Da2,20kModifying liposome, collecting blood via orbital venous plexus for 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration with enzyme labeling instrument.
As a result:
the first tail vein is injected with DSPE-PEG with phospholipid concentration of 5 mu mol/kg and total PEG molecular weight of 20000Da2,20kModifying liposome, injecting two times DSPE-containing agent with phospholipid concentration of 5 μmol/kg and total molecular weight of PEG of 20000Da at intervals of 7 daysPEG2,20kThe time curve of the modified liposome is shown in figure 9, which shows 5 μmol/kg PL2,20kCurve denoted by-2, ABC index(0-0.5h)See table 2, the results of the anti-PEG IgM assay are shown in fig. 11, the anti-PEG IgM content is high, and the OD value is 0.46 ± 0.06.
The result shows that the first injection of the DSPE-PEG with the total molecular weight of the PEG of 20000Da2,20kModifying liposome, and repeatedly injecting DSPE-PEG with total molecular weight of 20000Da2,20kThe clearance rate of the modified liposome in blood has little influence, and the ABC index thereof(0-0.5h)1.08 +/-0.03, and obviously eliminates the ABC phenomenon.
In order to quantitatively compare the occurrence intensity of the ABC phenomenon, the concept of ABC index is introduced, namely the ratio of AUC of a two-injection preparation to AUC of a first-injection preparation is taken as a standard for measuring the intensity. The ABC index calculation method comprises the following steps: ABC index is AUC of the second injection(0～t)AUC of first injection(0～t). The larger the ABC index value, the smaller the change in pharmacokinetic behavior of the second injection caused by the first formulation, i.e., the weaker the ABC phenomenon. The early work in the laboratory shows that ABC index(0-0.5h)The ABC phenomenon is considered to be absent in the range of 0.9-1.0, and the ABC phenomenon is considered to be weak in the range of 0.7-0.9. As can be seen from Table 3, the ABC index of each group of PE was modified by branched PEG(0-0.5h)The molecular weight is more than 0.95, and ABC phenomenon is not induced by repeatedly injecting each group of branched PE. As can be seen from Table 4, the ABC index of each PL group was modified with branched PEG(0-0.5h)All of them were 0.95 or more, and it is considered that repeated injections of each group of branched chains PL did not induce ABC phenomenon.
The ABC phenomenon is an important reason for hindering the clinical transformation of pegylated nanoformers, and therefore, exploring the mechanism of generating the ABC phenomenon, and finding an effective way to attenuate or eliminate this phenomenon is an unavoidable problem for drug delivery system researchers. A branched chain PEG modified nano carrier is adopted, so that a new method is provided for eliminating the ABC phenomenon of a PEG nano preparation. It is worth noting that the modification of the branched chain PEG can not only eliminate the ABC phenomenon of the PEGylated nano preparation, but also has good circulation time. In conclusion, the branched-chain PEG modified nano-carrier can improve the curative effect of the preparation, reduce the toxic and side effects, greatly increase the clinical transformation probability of the PEG nano-preparation, and has good application prospect in the field of nano-preparation modification.
TABLE 3 Primary and Secondary injections of groups of branched PEABC indexes(0-0.5h)Value of
Figure BDA0003102926480000151
TABLE 4 Primary and Secondary injections of groups of branched chains PLABC index(0-0.5h)Value of
Figure BDA0003102926480000152
Example 10:
determination of anti-PEG IgM
After the first tail vein injection of different branched chain PE, blood is respectively taken through orbital venous plexus before the second administration (after the first injection for 7 d), and serum is separated for standby.
Coating: 50 μ L of the coating solution was added to a 96-well plate and allowed to air dry overnight at room temperature.
The coating liquid comprises: 2.80mg of DSPE-mPEG2000 was dissolved in a 5mL volumetric flask with absolute ethanol and diluted to the mark, and shaken to give a 0.56mg/mL DSPE-mPEG2000 solution.
And (3) sealing: take 150. mu.L of blocking solution, add to 96-well plate, incubate for 1h at room temperature.
The sealing liquid is prepared by the following steps: 1g BSA was diluted to the mark with PBS buffered saline in a 100mL volumetric flask and shaken up to obtain blocking solution (PBS buffered saline containing 1% BSA).
Cleaning: firstly, after a 96-well plate is patted dry, filling washing liquid into each well, standing for 30-60 s, pouring out the washing liquid, then patting the plate dry gently, and repeatedly washing for 5 times.
Washing liquid: 0.5g BSA was diluted to the mark with PBS buffer solution in a 500mL volumetric flask and shaken up to obtain the washing solution (PBS buffer solution containing 0.1% BSA).
Sample adding: mu.L of diluted (1:1000) serum samples were added to a 96-well plate (except for blank wells), and 3 wells of each serum sample were run in parallel and incubated for 1h at room temperature.
Cleaning: the same as the above washing step.
Adding an enzyme labeling conjugate: 100. mu.L of diluted enzyme-labeled conjugate was placed in a 96-well plate (except for blank wells), and incubated at room temperature for 1 h.
Cleaning: the same as the above washing step.
Color development: and precisely adding 100 mu L of newly prepared color developing agent into each hole, and incubating for 15min at room temperature.
Color developing agent: 0.184g of disodium hydrogen phosphate and 0.047g of citric acid are put into a 10mL volumetric flask, diluted to the scale by redistilled water and shaken up to obtain the phosphate-citric acid buffer solution with the pH value of 5.0. And accurately weighing 10.00mg of o-phenylenediamine in a 10mL volumetric flask, diluting the o-phenylenediamine to a scale by using a phosphoric acid-citric acid buffer solution with the pH value of 5.0, adding 10 mu L of 30% hydrogen peroxide, and shaking up to obtain the color developing agent. It should be noted that the preparation is carried out before use and is stored in the dark.
And (3) terminating the reaction: 100. mu.L of stop solution was added precisely to each well.
And (3) measuring the absorbance: the absorbance was measured within 5min after termination of the reaction by a microplate reader, dual wavelength method, wavelengths 490nm and 630 nm.
The results show that: the first injection of 5. mu. mol phospholipid/kg of different branched PEG-modified PE stimulated the production of anti-PEG IgM in rats, but at different levels. As can be seen from FIG. 10, as the molecular weight of PEG in the first injection preparation increases, the secretion of anti-PEG IgM decreases, and the amounts of production become PE in order from large to small2,2k>PE2,10k>PE2,20k>PE2,40kWherein PE is injected for the first time2,40kProduction of anti-PEG IgM in minimal amounts (. about.P)<0.05,**P<0.01,***P<0.001.). This is probably due to the fact that high molecular weight branched PEG modification leads to too high epitope density and difficulty in effectively stimulating splenic B cells, resulting in reduced antibody secretion by B cells.
Example 11:
determination of anti-PEG IgM
After the first tail vein injection of different branched chains PL, blood was collected via orbital venous plexus before the second administration (after the first injection for 7 days), and serum was separated for use.
The coating solution, sealing solution, developer, washing solution and washing process described in the following steps are the same as those of example 10.
Coating: 50 μ L of the coating solution was added to a 96-well plate and allowed to air dry overnight at room temperature.
And (3) sealing: take 150. mu.L of blocking solution, add to 96-well plate, incubate for 1h at room temperature.
Cleaning: firstly, after a 96-well plate is patted dry, filling washing liquid into each well, standing for 30-60 s, pouring out the washing liquid, then patting the plate dry gently, and repeatedly washing for 5 times.
Sample adding: mu.L of diluted (1:1000) serum samples were added to a 96-well plate (except for blank wells), and 3 wells of each serum sample were run in parallel and incubated for 1h at room temperature.
Cleaning: the same as in example 10.
Adding an enzyme labeling conjugate: 100. mu.L of diluted enzyme-labeled conjugate was placed in a 96-well plate (except for blank wells), and incubated at room temperature for 1 h.
Cleaning: the same as in example 10.
Color development: and precisely adding 100 mu L of newly prepared color developing agent into each hole, and incubating for 15min at room temperature.
And (3) terminating the reaction: 100. mu.L of stop solution was added precisely to each well.
And (3) measuring the absorbance: the absorbance was measured within 5min after termination of the reaction by a microplate reader, dual wavelength method, wavelengths 490nm and 630 nm.
The results show that: the first injection of 5. mu. mol phospholipid/kg of different branched PEG modified PL stimulated the production of anti-PEG IgM in rats, but at different levels. As can be seen from FIG. 11, as the molecular weight of PEG in the first injection preparation increases, the secretion of anti-PEG IgM decreases, and the amount of production is PL in order from large to small2,2k>PL2,10k>PL2,20kWherein PL is first injected2,20kProduction of anti-PEG IgM in minimal amounts (. about.P)<0.05,**P<0.01,***P<0.001.). This is probably due to the fact that high molecular weight branched PEG modification leads to too high epitope density and difficulty in effectively stimulating splenic B cells, resulting in reduced antibody secretion by B cells. The above data indicate that branched PEG was selected20kPerhaps with good prospects for modifying nano-preparations。
Example 12:
first tail vein injection DSPE-PEG2,nEmulsion modification, repeated tail vein injection DSPE-PEG 7 days later2,nEmulsion modification, determination of antibody levels over different time periods.
Experimental animals:
wistar rats (180-210 g,
Figure BDA0003102926480000171
lei yun Shang pharmaceutical laboratory animal center provides)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 4 groups and administered by tail vein injection. The DSPE-PEG is injected for the first time according to the phospholipid dosage of 5 mu mol/kg2,nModified emulsion (the PEG)2,nIs PE2k，PE2,2k，PE2,10k，PE2,20k，PE2,40k). After 7 days, all groups were injected repeatedly with the corresponding DSPE-PEG2,nModifying the emulsion, taking blood through orbital venous plexus at 0.016, 0.5, 1, 4 and 8h after the second administration, standing for 2h, centrifuging at 5000rpm for 20min, separating serum, treating the sample according to the method of example 5, and determining the antibody content.
As a result:
the results of the anti-PEG IgM assay are shown in FIG. 12.
As can be seen from FIG. 12, PE2,2k，PE2,10k，PE2,20kAfter repeated injections for 1min, the amount of anti-PEG IgM decreased rapidly, indicating that the formulation rapidly bound to the antibody after repeated injections; and PE2,40kWithin 0.5h of repeated injection, the amount of the anti-PEG IgM has no significant change, and the content of the anti-PEG IgM begins to be reduced after 0.5h, indicating that PE2,40kThe binding rate with the antibody was slow after repeated injections.
In the first injection of PE2,nRepeated PE injections 7 days later2,nThe results of the determination of the rate of decrease of anti-PEG IgM levels in plasma at different time points are shown in table 5.
TABLE 5 PE in first injection2,nRepeated PE injections 7 days later2,nanti-P in plasma at different later time pointsResults of measurement of the Rate of decrease of EG IgM level
Figure BDA0003102926480000181
Example 13:
first tail vein injection DSPE-PEG2,nModifying liposome, and repeating tail vein injection DSPE-PEG 7 days later2,nLiposomes were modified and antibody levels were measured over different time periods.
Experimental animals:
wistar rat (180 ~ 210g, Lishui Shang pharmaceutical laboratory animal center)
The administration scheme is as follows:
male Wistar rats weighing 180-210 g are randomly divided into 4 groups and administered by tail vein injection. The DSPE-PEG is injected for the first time according to the phospholipid dosage of 5 mu mol/kg2,nThe liposomes are modified. After 7 days, all groups were injected repeatedly with the corresponding DSPE-PEG2,nModifying liposome, collecting blood via orbital venous plexus at 0.016, 0.5, 1, 4, and 8 hr after secondary administration, standing for 2 hr, centrifuging at 5000rpm for 20min, separating serum, treating sample according to example 5, and determining antibody content.
As a result:
the results of the anti-PEG IgM assay are shown in FIG. 13.
As can be seen from FIG. 13, PL2,2k，PL2,10k，PL2,20kThe amount of anti-PEG IgM decreased rapidly after 1min of repeated injections, indicating that the formulation bound rapidly to the antibody after repeated injections.
To accurately assess the binding of the preparation to anti-PEG IgM, the antibody reduction rate was calculated and the results are shown in table 4.
In the first injection of PL2,nRepeated injections of PL 7 days later2,nThe results of the determination of the rate of decrease of anti-PEG IgM levels in plasma at different time points are shown in table 6.
TABLE 6 PL at first injection,nRepeated injections of PL 7 days later2,nResults of determination of the Rate of decrease of anti-PEG IgM levels in plasma at different later time points
Figure BDA0003102926480000182
Figure BDA0003102926480000191
Example 14:
application of branched PEG in liposome of irinotecan and the like
Irinotecan liposomes were prepared according to the prescription composition of irinotecan liposomes (ONIVYDE), a product marketed in the United states. The composition of the different formulations is shown in table 7.
TABLE 7 different molecular weights of DSPE-mPEG2,nModified liposome formulations
Figure BDA0003102926480000192
Remarking: replacing with branched chain PEG (DSPE-mPEG) according to the equal proportion of the molar charge of the DSPE-PEG2,n；PL2,2k、PL2,10k、PL2,20k)
Weighing the liposome membrane material with the prescription amount, adding absolute ethyl alcohol with the final volume of 20% (v/v) of the blank preparation, and stirring and dissolving in a water bath at 65 ℃. Injecting citric acid-sodium citrate solution (200mM, pH 4.0) preheated to the same temperature into the membrane material at a speed of 3mL/min, and stirring in water bath for 0.5h to obtain blank liposome primary product. And (3) sequentially extruding the blank liposome primary product by polycarbonate membranes with the diameters of 5 times 400nm, 10 times 200nm, 10 times 100nm and 5 times 80nm to obtain blank liposome suspensions with different formulas. Using 500mM sodium phosphate solution as pH regulator, regulating pH of external water phase to 7.0, establishing gradient liposome with delta pH of 3.0, adding medicine at a medicine-to-lipid ratio of 1:10, incubating at 70 deg.C for 30min, loading medicine, placing in ice-water bath for 5min to terminate medicine loading, determining encapsulation efficiency, placing in refrigerator (4 + -2 deg.C), and examining encapsulation efficiency stability, the results are shown in Table 8.
TABLE 8 Liposomal encapsulation efficiency stability of irinotecan
Figure BDA0003102926480000193
Remarking: replacing with branched chain PEG according to the equal proportion of the DSPE-PEG (straight chain PEG) molar charge
It is clear that branched PEG is superior to linear PEG, especially in terms of shelf stability, PL2,10kAnd (4) optimizing.
Similarly, doxorubicin liposomes were prepared and formulated in accordance with irinotecan liposomes, and the results are shown in table 9.
TABLE 9 Adriamycin (Doxorubicin) Liposome encapsulation Rate stability
Figure BDA0003102926480000201
It is clear that branched PEG is superior to linear PEG, especially in terms of shelf stability, PL2,2k、PL2,10kAnd PL2,20kAre all superior to linear PEG (DSPE-PEG). The adriamycin liposome of the prescription is superior to that of the commercial one
Figure BDA0003102926480000202
PEG has low density, does not cause hand-foot syndrome, and does not have ABC phenomenon even if the PEG is administrated in small dose.
In addition, other antitumor drugs (epirubicin, mitoxantrone, pixantrone and vincristine) and antibiotic drug liposome are prepared, and the liposome shows better stability.
Example 15:
coenzyme Q10 injection
The existing coenzyme Q10 injection can be deposited after being placed for 6 months, and the problem is not solved so far. Feeding coenzyme Q10 and Tween 80 according to the mass ratio of 1:100 to prepare the coenzyme Q10 injection. Branched PEG (DSPE-PEG2000) and branched PEG were added to the injections and the values of the branched PEG were compared and the results are shown in Table 10.
TABLE 10 formulation composition and shelf stability of different coenzyme Q10 injections
Figure BDA0003102926480000203
It is clear that the linear PEG did not solve the problem of drug precipitation, and that branched PEG in which PEG is present2,10kPreferably, the problem of drug precipitation is completely solved.
The preparation can be used for preparing medicines such as vitamin A, D, E, K.
In addition, propofol injection emulsion is prepared, common emulsion is slightly yellow after being placed for one year, and PEG2,10kAnd the chemical stability of the preparation is improved.
The branched PEG is used for preparing paclitaxel/taxane and derivatives thereof (including oleate, DHA; disulfide bond S-S, and monosulfide-S-derivatives), also shows good value and has great development prospect.

Claims (10)

1. PEG (polyethylene glycol)2,n-lipid derivative-modified nanocarriers, characterized in that PEGs of different molecular weights are used2,nPEG of (2)2,n-lipid derivatives to modify the nanocarriers; the nano-carrier includes: emulsions, liposomes, micelles; the lipid is natural phospholipid, semisynthetic phospholipid or synthetic phospholipid; the PEG2,nTwo or more linear PEG chains with methoxy end groups are respectively connected with two amino groups of lysine by covalent bonds, wherein n represents the total molecular weight of the two linear PEG chains, and n is 200-400000 Da.
2. A PEG according to claim 12,n-lipid derivative modified nanocarriers, characterized in that said PEG is2,nTwo linear PEG chains with methoxy end, a compound which is respectively connected with two amino groups of lysine by covalent bonds, wherein n is 20000-40000 Da;
the lipid is soybean lecithin, yolk lecithin, phosphatidyl glycerol, yolk phosphatidyl glycerol, phosphatidic acid, cardiolipin, sphingomyelin, phosphatidic acid serine, phosphatidylinositol, phosphatidylethanolamine, hydrogenated soybean lecithin, hydrogenated yolk lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dilauroyl phosphatidylcholine, didecanoyl phosphatidylcholine, dioctanoyl phosphatidylcholine, dihexanoyl phosphatidylcholine, distearoyl phosphatidylglycerol and salts thereof, dipalmitoyl phosphatidylglycerol and salts thereof, L-alpha-dimyristoyl phosphatidylglycerol and salts thereof, dilauroyl phosphatidylglycerol, didecanoyl phosphatidylglycerol, dioctanoyl phosphatidylglycerol, dihexanoyl glycerol, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, etc, Dioleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, distearoylphosphatidylglycerol and salts thereof, dipalmitoylphosphatidylglycerol and salts thereof, dimyristoylphosphatidylglycerol and salts thereof, dilauroylphosphatidylglycerol, distearoylphosphatidylinositol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dilauroylphosphatidylinositol, palmitoyloleoylphosphatidylcholine, palmitoleoylphosphatidylcholine, stearoylileoylphosphatidylcholine, stearoyloleoylphosphatidylcholine, stearoylarachidonic acid phosphatidylcholine, and DSPE.
3. A PEG according to claim 12,n-lipid derivative modified nanocarriers, wherein said PEG is a lipid derivative modified nanocarrier2,nThe application of the lipid derivative in the preparation of the PEG nano carrier for eliminating the phenomenon of accelerating blood clearance.
4. The PEG of claim 12,n-use of lipid derivative modified nanocarriers for eliminating the phenomenon of accelerated blood clearance caused by pegylated nanocarriers.
5. A PEG according to claim 12,n-lipid derivative-modified nanocarriers, wherein the nanocarriers comprise an oil phase and an emulsion when the nanocarriers are emulsionsAgent, PEG2,n-lipid derivatives, sterile water for injection; wherein the mass ratio of the oil phase to the emulsifier is (3-5) to 1, and PEG2,nThe molar ratio of the lipid derivative to the emulsifier is 1 (10-100).
6. A PEG according to claim 52,n-lipid derivative-modified nanocarriers, wherein when the nanocarriers are emulsions, the oil phase is MCT, the emulsifier is egg yolk lecithin, the PEG is conjugated to a lipid derivative, and the oil phase is MCT, and the emulsifier is egg yolk lecithin2,nThe lipid derivative is DSPE-mPEG2,2k，DSPE-mPEG2,10k，DSPE-mPEG2,20kOr DSPE-mPEG2,40kThe PEG2,n-the molar ratio of lipid derivative to emulsifier is 1: 10.
7. The PEG of claim 52,n-a method for preparing lipid derivative-modified nanocarriers, wherein the method for preparing the nanocarriers is an emulsion, the method comprising the steps of:
weighing oil phase, emulsifier and PEG2,n-a lipid derivative which is completely dissolved at a temperature of 55-65 ℃, and sterilized water for injection heated to the same temperature is injected under stirring;
continuously stirring and incubating at 55-65 deg.C for 10-30min to obtain primary emulsion;
subjecting the obtained primary emulsion to ultrasonic dispersion treatment, and sequentially filtering with 0.80, 0.45 and 0.22 μm microporous filter membrane to obtain PEG2,n-lipid derivative modified emulsions.
8. A PEG according to claim 12,n-lipid derivative-modified nanocarriers, wherein the nanocarriers comprise phospholipids, cholesterol, PEG when the nanocarriers are liposomes2,n-lipid derivatives and sterile water for injection; wherein the mass ratio of phospholipid to cholesterol is (3-5): 1, PEG2,nThe molar ratio of the lipid derivative to the phospholipid plus cholesterol is 1 (10-100).
9. The method of claim 8PEG (polyethylene glycol)2,n-lipid derivative-modified nanocarriers, wherein the phospholipids are hydrogenated soy phospholipids and the PEG is2,n-the molar ratio of lipid derivative to phospholipid + cholesterol is 1: 10.
10. The PEG of claim 82,nA method for preparing a lipid derivative-modified nanocarrier, wherein when the nanocarrier is a liposome, the method comprises the steps of:
mixing phospholipid, cholesterol, and PEG2,n-mixing the lipid derivatives, adding absolute ethanol, stirring and dissolving at 55-75 ℃, volatilizing 50-80% of absolute ethanol to obtain a concentrate;
dripping the sterilized water for injection preheated to the same temperature into the concentrate;
continuously stirring at 55-75 deg.C for 15-30min to obtain liposome primary product;
subjecting the obtained liposome primary product to ultrasonic dispersion treatment, sequentially passing through 0.80, 0.45, 0.22 μm microporous filter membrane to obtain PEG2,n-lipid derivative modified liposomes.
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