CN103239405A - Preparation method of propranolol stealth liposome modified by vascular endothelial growth factor receptor-2 (VEGFR-2) monoclonal antibody and casein phosphopeptides (CPP) jointly, and products thereof - Google Patents
Preparation method of propranolol stealth liposome modified by vascular endothelial growth factor receptor-2 (VEGFR-2) monoclonal antibody and casein phosphopeptides (CPP) jointly, and products thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a propranolol stealth liposome modified by a vascular endothelial growth factor receptor-2 (VEGFR-2) monoclonal antibody and casein phosphopeptides (CPP) jointly; the method comprises the following steps of: (1) confirming the composition and the proportion of liposome compound phosphatide; (2) preparing a propranolol stealth liposome modified by the CPP; and (3) connecting the VEGFR-2 monoclonal antibody and the surface of the propranolol stealth liposome modified by the CPP to obtain the propranolol stealth liposome modified by the VEGFR-2 monoclonal antibody and the CPP jointly. The preparation method has the characteristics of high stability, long circulation, active targeting, high-efficiency endocytosis and the like, and can guarantee the toxicity reducing and efficacy enhancing of anti-hemangioma medicines. The research result of the method provides a preparation technology platform for the clinical targeted therapy of the anti-hemangioma medicines.
Description
Technical field
The present invention relates to molecular cloning and biomedical sector, relate in particular to VEGFR-2 monoclonal antibody and CPP combined modification the Propranolol hidden liposome preparation method and products thereof and use.
Background technology
Hemangioma (Hemangiomas) is common benign tumor, and its morbidity exists tangible race and sex difference, and its sickness rate in low birth weight infant and premature infant may be higher.Hemangioma to the patient particularly infant physiology and psychology potential hazard can not be ignored, especially the hemangioma of privileged sites, specific type must be taked positive therapeutic intervention in its propagation phase.The main mechanism that the scholar proposes the hemangioma generation is that the unbalance of short angiogenesis factor and inhibitive factor causes.Blood vessel is the neoplastic key factor of blood vessel, and its main feature is the endotheli ocytosis of blood capillary.Topmost VEGF (vascular endothelial growth factor in the short angiogenesis factor, VEGF), (basic fibroblast growth factor BFGF) is considered to promote the angiogenesis factor that vascular endothelial cell proliferation is active to basic fibroblast growth factor.In the hemangioma endotheliocyte of propagation phase, the molecular marked compound of angiogenesis such as VEGF etc. present overexpression.
Often side effect is bigger for clinical treatment hemangioma medicine at present, and in order to reach the purpose of attenuation synergistic, targeted therapy becomes the focus of present hemangioma treatment.The characteristics of targeted therapy just are to adopt carrier that medicine is wrapped up, and under the effect of decorative material, carrier can conduct drugs to site of action specifically after the administration, thereby realize the maximum of operational efficiency.
A large amount of research reports that surperficial Propranolol is the angiomatous active drug for the treatment of.Up to now, have about using Propranolol and successfully treat facial area, eye, respiratory tract, liver and other angiomatous a large amount of reports of serious symptom with complication, many diseased regions even serious threat infant life, and confirmed that Propranolol can change angiomatous natural medical history, block the early apoptosis that it is bred, mediates and promote IH.Further studies show that: Propranolol is to being in the propagation phase and paracmastic hemangioma all has significant curative effect.In addition, using Propranolol treatment hemangioma also to be proved definite curative effect separately, is the doubt of being dominated by the other treatment means thereby eliminated its therapeutic effect.Many scholars recommend it to replace corticosteroid hormone becomes angiomatous first-line treatment means, China's stomatology can Oral and Maxillofacial Surgery Professional Committee's vascular disease group determined that also Propranolol is the angiomatous line medicine for the treatment of, and include " oromaxillo-facial region hemangioma treatment guide " in.
Though Propranolol treatment hemangioma is effective, serious adverse reactions such as the bradycardia that may exist the Propranolol medication, hypotension, hypoglycemia also can not be adopted a casual attitude.Generally speaking, the hemangioma infant should basically form normal food intake when taking the Propranolol treatment, and at frequent feeding for the treatment of initial stage, and strengthen monitoring dynamics to important vital sign and blood sugar level, to avoid the generation of above serious adverse reaction as far as possible.Especially untoward reaction that it should be noted that the Propranolol medication may have higher incidence rate for Chinese, because the Chinese is relatively poor to the tolerance of Propranolol, and the sensitivity height.Existing document proof Chinese is high at least 2 times than white people to the sensitivity of beta-receptor blockader.Propranolol has the serious adverse reaction of causing possibility as a kind of nonselective receptor blocking agent, and blindly strengthening dosage tends to follow bigger risk.In addition, the untoward reaction of Propranolol is relevant with route of administration to a great extent, and oral administration has been proved to be bioavailability low (being about 30%), individual variation is big, has significant first pass effect, so really arrive the affected part and bring into play the dose of curative effect limited.
Liposome (liposomes) is one of carrier of greatest concern among the present TDDS, mainly formed by matrix materials such as phospholipid and cholesterol, has bilayer, can coated water-soluble or fat-soluble medicine, have cellular affinity and histocompatibility in vivo, and can modify as required.As pharmaceutical carrier, liposome is demonstrating good prospect aspect the hemangioma treatment.
Hidden liposome (stealth liposomes is called long circulating liposomes again) is the main flow of present antitumor liposome.This liposome generally adopts DSPE-Macrogol 2000, by improving the hydrophilic of surface of liposome, reduce the combination of opsonin (opsonin) and liposome, thereby avoid being identified the time that prolongation is detained by vivo immuning system in blood circulation, increase by the probability of target cell identification and specificity combination.
It must be noted that, present active target liposomes is modified at surface of liposome PEG chain end mostly, though can realize long circulation and active targeted double effect, the sterically hindered and strong polarity of PEG chain also is unfavorable for that liposome fast, fully enters target cell.
Therefore, modify hidden liposome if adopt the VEGFR monoclonal antibody separately, be attached to the blood vessel surface of hemangioma pathological changes though can realize long circulation (being trapped in the blood circulation for a long time) and targeting, but because the obstruction of surface of liposome PEG chain and hemangioma position vascular endothelial cell abnormality proliferation often, vessel wall thickening causes and the contained drug delivery intravasation of liposome interior tumor inside can't be played a role.
Adopt corresponding antibody to carry out the active targeting modification on the lipid surface and can specifically the medicine orientation be transported to the hemangioma position.Cultivate if can select a lymphocyte of making a kind of single-minded antibody, just can obtain the cell mass that formed through division growth by unicellular, i.e. monoclonal.Monoclonal will synthesize the antibody at a kind of antigenic determinant, and such antibody becomes monoclonal antibody.
Because vascularization is a quite complicated process, be subjected to the positive negative regulation of the multiple factor, wherein VEGF acts on one of the strongest positivity regulatory factor, it is by (vascular endothelial growth factor receptor is VEGFR) in conjunction with the formation that stimulates new vessels with corresponding vascular endothelial growth factor receptor.VEGFR-2 is that the III type of the about 210KD of molecular weight is striden the film tyrosine protein kinase, is one of three kinds of vascular endothelial growth factor receptors.VEGFR-2 is the extracellular region that contains 7 immunoglobulin like domain by, strides film district and an intracellular region composition that has tyrosine kinase domain for one.The 1st Ig domain of VEGFR-2 extracellular region is that the VEGF part is in conjunction with necessary, the binding site of part is on the 2nd and the 3rd Ig domain, the 4th Ig domain is that the formation homodimer is necessary behind the receptor activation, and the combination of all the other several Ig domains and part there is no much relations.In the hemangioma endotheliocyte of propagation phase, molecular marked compound such as VEGF and the VEGFR etc. of angiogenesis present overexpression.
Carbamide treatment hemangioma is one of clinical the most frequently used method.But because carbamide treatment hemangioma lacks the targeted therapy effect, be difficult in the tumor body, form high concentration and assemble, need the multiple injection treatment, treatment cycle is prolonged, increase the patient suffering; And because urea concentration is too high, unit interval innerlich anwenden amount is excessive, and local injection can cause the skin histology necrosis, also can cause the developmental disorder of normal adjacent tissue organ.With the monoclonal antibody of VEGFR as coupling antibody modification carbamide liposome, make its active targeting in angiomatous vascular endothelial cell, experiment in vitro is found, blank liposome itself does not have any injury effect to people's hemangioma endotheliocyte, the cell killing rate of 2.6% carbamide group is 27.94%, the cell killing rate of 2.6% carbamide liposome group is 84.01%, adopting the cell killing rate of the carbamide immunoliposome group after monoclonal antibody is initiatively modified is 99.91%, as seen after adopting initiatively targeting modification carrier coated urea, the lethal effect of target cell is significantly improved.
Cell-penetrating peptides (cell-penetrating peptide, CPP) be that a class can quick penetration cytolipin plasma membrane less than 30 amino acid whose small peptides.CPP can carry the macromole bioactive substance even the nano-carrier that are difficult to permeates cell membranes and enter cell, and toxicity is low, can the damaging cells film, thereby be widely used in neoplasm targeted therapy.CPP and macromolecular conjugate are that the endocytosis by energy dependence enters cell.
The application of CPP has greatly improved from the drug molecule to the nano-carrier efficient by cell membrane.Studies show that in a large number CPP has very powerful delivery potential, wear film mechanism about its internalization and have dispute all the time, but in CPP and the membrane interaction, CPP positively charged make its be easy to cell membrane in mutually combine by electrostatic interaction such as phospholipid.Think that at present the main three aspect mechanism of CPP membrane penetration effect are: A. directly is penetrated into cell membrane by electrostatic interaction; B. a temporary structure that forms by transposition promotes infiltration; C. mediate into film by endocytosis.The main CPP of report is as shown in the table at present:
Common cell-penetrating peptide aminoacid sequence
Studies show that in a large number CPP can carry materials such as protein, polypeptide, DNA, chemical small-molecule substance, oligonucleotide, liposome by non-covalent bond and covalent bond ways of connecting.Wang Wei etc. are with eight poly arginines (Arg8) and DPPE-PEG(DPPE-Polyethylene Glycol) synthetic Arg8-PEG-DPPE, be used for modified liposome, found that: even the conventional liposome of modifying without Arg8 can not enter cell with lung carcinoma cell NCI-H446 co-cultivation 24h, and behind liposome that Arg8 modifies and cancerous cell co-cultivation 1h, tangible cellular uptake is arranged namely.
CPP lacks cell-specific, if adopt initiatively targeting modification and CPP combined modification, can address this problem, after the special receptors bind of the part of active targeting modification and cell surface, be hidden in the CPP in the PEG chain owing to approach with the distance of cell membrane, also can bring into play membrane penetration effect, thereby solve the cell-specific problem equally, and also solve the initiatively problem of targeting modification liposome permeates cell membranes inefficiency simultaneously.
Summary of the invention
Goal of the invention: the preparation method that the purpose of this invention is to provide the Propranolol hidden liposome of VEGFR-2 monoclonal antibody and CPP combined modification, the product that another object of the present invention provides preceding method to obtain, a further object of the invention provide preceding method for the preparation of the application on the treatment hemangioma medicine.
Technical scheme: for achieving the above object, the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody of the present invention and CPP combined modification is characterized in that may further comprise the steps:
1) the liposome composite phospholipid is formed and determination of ratio;
2) the Propranolol hidden liposome preparation of CPP modification:
A) pNP-PEG-DPPE's is synthetic;
B) CPP-PEG-DPPE's is synthetic;
C) the Propranolol hidden liposome preparation of CPP modification;
3) the VEGFR-2 monoclonal antibody is connected the Propranolol hidden liposome that namely gets VEGFR-2 monoclonal antibody and CPP combined modification with the Propranolol stealthy liposome surface that CPP modifies.
Described liposome composite phospholipid adopts distearoyl phosphatidylcholine, hydrogenated soy phosphatidyl choline as high phase transition temperature phospholipid; Adopting dipalmitoyl phosphatidyl choline and dimyristoyl phosphatidyl choline is low transformation temperature phospholipid, and the combined preparation composite phospholipid liposome has 4 kinds of combinations in twos.
Described liposome composite phospholipid consists of dipalmitoyl phosphatidyl choline and hydrogenated soy phosphatidyl choline.
Described dipalmitoyl phosphatidyl choline and hydrogenated soy phosphatidyl choline ratio are 6:4.
The mol ratio of a kind of composite phospholipid of combined preparation and DSPE-PEG2000 is 95:5 in above-mentioned 4, obtains the DSPE-PEG2000 composite phospholipid.
Selected DSPE-PEG2000 composite phospholipid and the Propranolol that contains 5% mol ratio made the Propranolol hidden liposome, temperature is bathed 2h in 37 ℃ of water-baths, with step 2) in the synthetic CPP-PEG-DPPE that obtains directionally be inserted into according to 5% mol ratio on the outer membrane of Propranolol hidden liposome and obtain the Propranolol hidden liposome that CPP modifies.
The method of attachment of the Propranolol stealthy liposome surface that described step 3) VEGFR-2 monoclonal antibody and CPP modify is as follows: earlier the VEGFR-2 monoclonal antibody is dissolved in the HEPESpH8.0 buffer, mix with the SPDP solution of 6.25mg/ml, reaction generates PDP-IgG, fully after the reaction, the centrifugal removal of ultrafiltration is SPDP and the by-product of coupling not, collects PDP-IgG, it is 50mmol/L that adding DTT makes its final concentration, stir 25 ℃ of reaction 25min, make the PDP mercaptanization on the antibody, form IgG-SH; IgG antibody-SH the solution of mercaptanization is mixed with the Propranolol hidden liposome that CPP modifies, molar ratio range is 1:1000-1:50, the dark place stirred overnight at room temperature, ultrafiltration is centrifugal to remove unconjugated IgG-SH, namely obtains the Propranolol hidden liposome of VEGFR-2 monoclonal antibody and CPP combined modification.
The envelop rate of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody and CPP combined modification is 80%, and the cumulative release degree of release: 10h is about 15%, setting-out stability: setting-out 70d is stable, targeting efficient: 60%.
The VEGFR-2 monoclonal antibody that described preparation method obtains and the Propranolol hidden liposome of CPP combined modification are for the preparation of the application on the treatment hemangioma medicine.
Beneficial effect: the present invention adopts the stability of composite phospholipid liposome technical guarantee liposome in blood circulation, and the stealthy liposome body technique is realized the long circulating effect of drug-loaded liposome in blood circulation after the intravenous injection, thereby provides possible for targeted delivery; Adopt the VEGFR-2 monoclonal antibody to modify drug-loaded liposome and realize that initiatively the targeted medicine arrives the hemangioma position; Adopt the CPP modified liposome to realize efficiently going into born of the same parents after the targeted, thus the present invention have high stability, long circulation, initiatively targeting, efficiently go into characteristic such as born of the same parents, can guarantee the attenuation synergistic of anti-angiogenic tumor medicine.Achievement in research of the present invention will provide the preparation technique platform for the clinical targeted therapy of anti-angiogenic tumor medicine.
Description of drawings
Fig. 1 is the mechanism of action sketch map of VEGFR-2 monoclonal antibody of the present invention and CPP combined modification drug-loaded liposome and target cell;
Fig. 2 is the mathematical law figure of composite phospholipid ratio of the present invention and phase change temperature of liposome.
The specific embodiment
Embodiment 1
Set up the HPLC-FID analytical method of measuring body fluid Propranolol concentration:
Employing HPLC-FID(fluorescence) detection method is measured the concentration of Propranolol in blood, tissue and the cell culture fluid.
Concrete grammar: be interior mark with carvedilol.Detect wavelength: exciting light 290nm, emission light 340nm.Chromatographic column: C18 post (250 * 4.6mm, 2.5 μ m).Biological sample adds inner mark solution 10 microlitres, (the vortex mixed 3min of normal hexane-dichloromethane-isopropyl alcohol=65:30:5), the centrifugal 10min of 4000rpm divide and get supernatant to add the mixed solvent of 50 μ L saturated sodium carbonates and 2.5mL, nitrogen dries up, and 100 μ L methanol redissolution sample introduction, 50 μ L measure.
Embodiment 2
Composite phospholipid is formed and determination of ratio: adopt distearoyl phosphatidylcholine (DSPC), hydrogenated soya phosphatide (HSPC) as high phase transition temperature phospholipid; Adopting dipalmitoyl phosphatidyl choline (DPPC) and dimyristoyl phosphatidyl choline (DMPC) is low transformation temperature phospholipid, combined preparation composite phospholipid liposome (having 4 kinds of combinations) in twos, under the situation that adds stealthy decorative material DSPE-PEG2000 relatively under various combination and the like combinations different composite phospholipid ratio to the medicine carrying effect of Propranolol and its stability (being the slip of Propranolol) in blood plasma relatively.Final determine that all good composite phospholipids of medicine carrying effect and stability are formed and ratio is used for the research of back liposome.Obtaining optimal proportion is: distearoyl phosphatidylcholine (DSPC), hydrogenated soya phosphatide (HSPC) are as high phase transition temperature phospholipid, mol ratio is 4:6, dipalmitoyl phosphatidyl choline (DPPC) and dimyristoyl phosphatidyl choline (DMPC) are low transformation temperature phospholipid, and mol ratio is 6:4.
Embodiment 3
The preparation of composite phospholipid liposome: adopt thin film-ultrasonic method preparation.Take by weighing phospholipid material 120mg and be dissolved in the 10mL chloroform, place 60 ℃ of decompression rotary evaporations formation thin film in the 50mL eggplant-shape bottle, take out eggplant-shape bottle, in vacuum drying oven, place 12h for 55 ℃.In eggplant-shape bottle, add ammonium sulfate 5mL, 60 ℃ of rotation hydration 40min, the gained liposome turbid liquor is in 60 ℃ of ultrasonic (400W of water-bath probe, 50 times), the 1000rpm low-speed centrifugal is removed titanium valve, adopts the pH7.4PBS dialysis to remove outer water ammonium sulfate, adds the Propranolol powder mixes, 60 ℃ of 20min, namely.Sephadex G on the drug-loaded liposome-50 post (internal diameter 1cm, length 27cm) is used the pH7.4PBS eluting, flow velocity 1mLmin
-1, collect liposome part and free fraction behind the upper prop, measure the concentration of Propranolol respectively, computational envelope rate (%)=liposome Chinese medicine content/(free drug content+liposome Chinese medicine content) * 100%.Calculating envelop rate is 81.2 ± 1.0%.
Stability study in the blood plasma: accurate draw 1mL sephadex column remove the Propranolol liposome of free drug, add and contain 9mL calf serum-PBS(1:1, v/v) in the release medium, mixing, constant temperature magnetic agitation under different temperatures, rotating speed is 500rpm, removes liposome in different time points sampling ultracentrifugation, gets and measures the wherein concentration of Propranolol after supernatant adds equal-volume acetonitrile vortex, high speed centrifugation.With the total amount of the Propranolol that the discharges cubage slip divided by Propranolol in the liposome.Calculating slip is 12.4 ± 0.5%.
Embodiment 4
The mensuration of phase transition temperature: adopt differential scanning calorimetric analysis (DSC) to measure the difference of medicine carrying front and back phase change temperature of liposome, investigate the variation of phase transition temperature in different body fluid.
DSC measures: take by weighing a certain amount of liposome turbid liquor and place in the stainless steel crucible, the PBS of equivalent is put in sealing in the reference crucible, in nitrogen atmosphere, is warming up to 60 ℃ with the speed of 1 ℃/min from 20 ℃, measures phase transition temperature.Attempt measuring the phase transition temperature of Propranolol liposome in blood, hemangioma tissue fluid, hemangioma culture fluid of endothelial cell on this basis, relatively its variation.
Find under study for action, Chen Jun etc. adopt the composite phospholipid liposome technology can the flexible phase transition temperature at a kind of liposome composite phospholipid that can regulate phase transition temperature of patent and in using, according to his research, as Fig. 2, we show the match between phase transition temperature (Tm) and the composite phospholipid ratio, there is tangible linear relationship between the inverse of the phase transition temperature that records (absolute temperature, unit are K) and the composite phospholipid ratio (the shared mol ratio of DPPC).
Prepare the composite phospholipid liposome of certain phase transition temperature according to the formula of match, adopt calcein to discharge and measure phase transition temperature, as can be seen from Table 1, very identical according to predictive value and practical measurement value that formula calculates.
The predictive value of table 1 phase transition temperature and practical measurement value
Embodiment 5
The preparation of Propranolol hidden liposome: be evaluation index with medicine carrying effect (envelop rate), relatively determining preparation method (membrane process, alcohol injection, reverse evaporation and dehydration-aquation method etc.) on the basis, adopt punctate opacity of the cornea-effect surface method preferred for preparation technology to determine to adopt above-mentioned membrane process, alcohol injection, the method that reverse evaporation and dehydration-the aquation method combines, concrete preparation technology sees it is embodiment 6.
Adopt glucosan G50 gel column to separate drug-loaded liposome and free clathrate, measure envelop rate, as evaluation index.
After determining preparation method, adopt punctate opacity of the cornea-effect surface method preferred for preparation technology, the factor that needs to investigate has: medicine fat ratio, phospholipid clathrate ratio, preparation temperature etc.
Embodiment 6
CPP modifies the investigation of the technology of hidden liposome: more different CPP comprise Arg8-12, Tat (49-57), Tat differences such as (48-60) is worn the film peptide that the hidden liposome membrane penetration effect is influenced difference; Estimate the technology that CPP modifies hidden liposome.
PNP-PEG-DPPE's is synthetic, gets the DPPE chloroformic solution, places round-bottomed flask, drips triethylamine, and (pNP) 2-PEG3400 is added in the above-mentioned mixed liquor, is blown into nitrogen, and airtight, lucifuge is in stirred overnight at room temperature.Evaporated under reduced pressure solvent, vacuum eliminate residual chloroform, add HCl solution, and supersound process forms transparent micellar solution.Sepharose separates, and removes the pNP of unreacted (pNP) 2-PEG3400 and release, collects the eluent that contains the pNP-PEG-DPPE micelle, and lyophilization is carried out qualitative examination to it.
CPP-PEG-DPPE's is synthetic, getting pNP-PEG-DPPE is dissolved in the chloroform, put in the eggplant type flask, chloroform is removed in decompression on Rotary Evaporators, forms adipose membrane, and vacuum concentration eliminates chloroform, with Arg8, Arg10, Tat (49-57), four kinds of CPP of Tat (48-60), be dissolved in respectively in the HCl solution, in incubated at room 30min, jog fully disperses adipose membrane.The Tris buffer 20mL that in suspension, adds 10mM (pH 9.0), mixing, nitrogen protection is in 4 ℃ of overnight incubation.With the bag filter dialysis buffer liquid of 5kD, reuse deionized water dialysis buffer liquid takes out solution in the bag filter, and lyophilization namely gets CPP-PEG-DPPE.
Form (DSPE-PEG2000 that contains 5% mol ratio) with selected composite phospholipid and make the Propranolol hidden liposome, temperature is bathed 2h in 37 ℃ of water-baths, CPP-PEG-DPPE directionally is inserted on the outer membrane of liposome, obtains the Propranolol hidden liposome that CPP modifies.Take the logarithm people's hemangioma endotheliocyte (HEC) of trophophase with l * 10
5/ porocyte is inoculated in the 24 porocyte culture bottles, inhale behind the 24h and remove complete medium, be changed to the RPMI1640 culture medium culturing 4h of serum-free, the Propranolol hidden liposome that adds CPP modification front and back is respectively hatched different time (0.5h, 1h, 2h, 4h, 8h) for 37 ℃, remove the not liposome of picked-up for 3 times with cold D-hanks liquid washing, with cell breakage, measure the interior drug level of cell and calculate ingestion efficiency.Determine optimum CPP according to ingestion efficiency.In liposome, account for the mol ratio difference of TL composition according to CPP, determine that best CPP-PEG-DPPE consumption is 6%.
Get the liposome after CPP modifies, ultra-filtration and separation is not attached to the CPP-PEG-DPPE in the liposome.Get ultrafiltrate 20 μ L in the 25mL measuring bottle, add 2 μ g/mL9, (9,10-phenanthrenequione is dissolved in dimethyl formamide to 10-phenanthrenequione fluorescence derivation reagent, fresh preparation) 2.5mL and 1mol/L sodium hydroxide are dissolved in 0.25mL, room temperature is placed 45min, adds 1mol/L hydrochloric acid 0.25mL, and water is settled to scale, (excitation wavelength 260nm on spectrofluorophotometer, wavelength of transmitted light 405nm) measure fluorescence intensity, carry out linear regression, the molar content of calculating CPP in the liposome is about 6%.
Embodiment 7
VEGFR-2 MONOCLONAL ANTIBODIES SPECIFIC FOR and modify being connected of stealthy liposome surface with medicine carrying CPP:
The VEGFR-2 MONOCLONAL ANTIBODIES SPECIFIC FOR, VEGFR-2 with purification is immunogen, the routine immunization BALB/c mouse, only carry out 3 inoculations, 30 μ g/, adopt the B lymphocyte hybridoma integration technology, merge under the 50%PEG mediation with the SP2/0 murine myeloma cell, prepare the hybridoma cell strain of mouse-anti human VEGFR-3-2 molecule monoclonal antibody with conventional hybridization tumor technology.The hybridoma of anti-VEGFR-2 is injected to the BALB/c mouse intraperitoneal, extracts ascites after 7-10 days, smudge cells, identify through Western blot identification antigen, measure the VEGFR-2 antibody activity, purification VEGFR-2 antibody protein finally obtains the VEGFR-2 monoclonal antibody.
The VEGFR-2 monoclonal antibody is modified medicine carrying CPP hidden liposome, adopt special-shaped bifunctional reagent 3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP) cross-linking antibody and liposome, Anti-human VEGFR-2 antibody is dissolved in the HEPES buffer (pH8.0), mix with the SPDP solution of 6.25mg/ml, reaction generates PDP-IgG.After fully reacting, centrifugal SPDP and the by-product that removes not coupling of ultrafiltration.Collect PDP-IgG, it is 50mmol/L that adding dithiothreitol, DTT (DTT) makes its final concentration, stirs 25 ℃ of reaction 25min, makes the PDP mercaptanization on the antibody, forms IgG-SH; IgG antibody-SH the solution of mercaptanization is mixed (mol ratio is about 1:1000), the dark place stirred overnight at room temperature with the Propranolol hidden liposome that CPP modifies.Ultrafiltration is centrifugal to remove unconjugated IgG-SH, and the long circulating liposomes that can be combined with the VEGFR-2 monoclonal antibody is the Propranolol hidden liposome of VEGFR-2 monoclonal antibody and CPP combined modification.
With fluorescent probe rhodamine-PHOSPHATIDYL ETHANOLAMINE (Rhodamine-PE, consumption be the phospholipid total mole number 0.1%) the labelling liposome, the VEGFR-2 monoclonal antibody is modified medicine carrying CPP hidden liposome in external and people's hemangioma endotheliocyte co-cultivation, adopt cells were tested by flow cytometry, fluorescence intensity with the cell combination is estimated modification effect, determines the most rational decorative material consumption.Adopt laser confocal microscope to observe in conjunction with effect.
The assay method of being combined with cell-targeting.People's hemangioma endotheliocyte (HEC cell) grown layer monolayer, after trypsinization, wash 1 time with fresh culture fluid, draw a certain amount of cell suspension and in 37 ℃ of water-baths, hatch 2h with the fluorescently-labeled composite phospholipid liposome (the decorative material consumption is different) that the VEGFR-2 monoclonal antibody is modified respectively, hatching the back that finishes washes 3 times with cold PBS, the rhodamine fluorescence intensity that cells were tested by flow cytometry is combined with cell (exciting light 540nm, emission light 625nm).
Laser confocal microscope is observed.Cell seeding is on coverslip, adherent growth to 50% is compiled, add the fluorescently-labeled composite phospholipid liposome (decorative material consumption difference) that the EGFR2 monoclonal antibody is modified, place 37 ℃, the interior 1h of cultivation of 5%CO2 incubator, use the PBS rinsing successively 3 times, 95% ethanol is fixed, 30%PBS glycerol mounting.Carry out graphical analysis with laser confocal microscope.
Embodiment 8
The pharmaceutics character of VEGFR-2 monoclonal antibody and CPP combined modification Propranolol hidden liposome is estimated:
Adopt the atomic force microscope observation form, adopt Zetasizer to measure particle diameter and current potential, adopt gel filtration chromatography to separate liposome and free clathrate and computational envelope rate, adopt dialysis to measure release, shelf stability is observed in long-term setting-out.
The observation of mode of appearance (atomic force microscope (AFM)): AFM experiment is carried out in Seiko SPM3800N atomic force microscope, mode of operation adopts formula (Tapping Mode) pattern of rapping, probe adopts silicon probe (NSG10, NT2MDT), its resonant frequency is approximately 300kHz, the preparation of AFM laboratory sample is adopted liposome turbid liquor is dropped on the mica sheet of newly peeling off, and at room temperature vacuum drying 24h detects.
The mensuration of particle diameter and surface zeta potential current potential: Zetasizer 3000HS analyser (Britain Ma Erwen Instr Ltd.), optical maser wavelength: He-Ne laser instrument, test angles: 90 degree, temperature: room temperature.
The mensuration of envelop rate.Adopting sephadex G 50 is filler, the dress post, employing PBS is eluent, flow velocity 1mL/min, collect liposome part and free clathrate part respectively, measure medicament contg, computational envelope rate (EE, encapsulation efficiency) EE%=liposome Chinese medicine content/(liposome Chinese medicine content+free drug content) * 100%.
The mensuration of release.Be release medium with the phosphate buffer that waits the pH7.4 ooze, drug-loaded liposome is placed bag filter, the constant temperature magnetic agitation is got medium in different time points and is measured drug level and replenish fresh medium simultaneously, measures the release percent of different time points.For the influence of evaluating blood composition to liposome stability, place bag filter to measure release isopyknic drug-loaded liposome and fresh rat blood plasma.The cumulative release degree of measuring 10h is 15.6 ± 0.4%.
The mensuration of setting-out stability.Liquid drug-loaded liposome is placed in 4 ℃ of lucifuges, measured the slip of placing different time points, slip is 11.5 ± 0.8%.
Embodiment 9
The targeting of VEGFR-2 monoclonal antibody and CPP combined modification Propranolol hidden liposome distributes and estimates:
Be animal model with lotus hemangioma nude mice, compare Propranolol solution, targeting efficient is calculated in influence to distributing in each tissue (especially hemangioma position) in the body after the Propranolol hidden liposome intravenous injection of not modified Propranolol hidden liposome and VEGFR-2 monoclonal antibody and CPP combined modification.Targeting efficient is 74.3 ± 2.8%.
Excision infant hemangioma tissue under aseptic condition is cut into the fritter of 5mm * 4mm * 3mm with tissue chopper, with UW liquid rinsing 3 times, inserts all two forelimb oxters of experiment nude mice through the skin otch respectively, in cervical region and the left buttocks subcutaneous tissue, and every 4 places.Inject estradiol 1 time in right leg muscle place, each 0.1mg for weekly after the transplanting animal.In inoculation grouping in back 45 days.Difference intravenous injection Propranolol solution, the Propranolol hidden liposome of not modified Propranolol hidden liposome and VEGFR-2 monoclonal antibody and CPP combined modification, get hemangioma and the heart, liver, spleen, lung, kidney, cerebral tissue after the administration, add normal saline and prepare homogenate, handle back mensuration then and organize Chinese medicine concentration, calculate targeting efficient, the targeting efficient that obtains is 86.3 ± 3.1%.
Embodiment 10
The pharmacodynamics evaluation of VEGFR-2 monoclonal antibody and CPP combined modification Propranolol hidden liposome: A. is external to people's hemangioma endotheliocyte inhibitory action and Mechanism Study; B. the dynamic (dynamical) research of medicine in people's hemangioma cell; C. to the pharmacodynamics evaluation of lotus hemangioma nude mice model; D. to the safety evaluatio of lotus hemangioma nude mice model (measuring physical signs such as liver, kidney, immune organ, blood).Below all with Propranolol solution, not modified Propranolol hidden liposome in contrast.
Adopting people's hemangioma endotheliocyte is model, cell is hatched 24h(37 ℃ in 96 orifice plates, 5%CO
2), in every hole, add Propranolol solution or Propranolol liposome then, airtight, at 37 ℃ or 42 ℃ of water-baths placement 15min, and then behind the cultured cell 24h, mtt assay is measured cell viability.Observe the mode of appearance of tumor cell by inverted microscope with method; Through Hoechst33258 and AO/EB dyeing, change by its karyomorphism of fluorescence microscope again; By the Change of Ultrastructure of its cell of transmission electron microscope observation, the ladder of agarose gel electrophoresis observation of cell DNA; Flow cytometry carries out mensuration and the cell cycle analysis of apoptosis rate.
Measure different time points (0.5,1,2,4,7, the 12h) distribution of medicine in people's hemangioma endotheliocyte.In the experiment cell with mixed by test preparation after, cultivate different time after, 10mL TBS (Tris-buffered saline) cleans cell, the centrifugal 5min of 1500 * g collects hepatoma carcinoma cell, precipitation adds 1mL TBS liquid suspendible, behind DL 15s on the microcentrifuge, carefully removes TBS liquid.Precipitation adds cold buffer A (10mM HEPES pH 7.9,10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, the 0.5mM PMSF) suspendible of 400 μ L, adds fully mixing 10s of 25 μ L NP-40 (0.5%) behind the ice bath 15min.Behind the centrifugal 30s of homogenate, get the drug level that supernatant is used for measuring cytoplasm.Get buffer C (20mM HEPES pH 7.9,400mM NaCl, 1mM each of DTT that precipitation adds 50 μ L ice baths, EDTA, EGTA and PMSF) suspendible, the centrifugal 5min of 11000 * g behind jolting 15min on the shaking table of rotation gets supernatant as endonuclear medicine solution to be measured.More than measure the wherein drug level of Propranolol after two kinds of solution-treated to be measured.Table 2 is the drug level of the Propranolol in cytoplasm and nucleus measured of different time points (0.5,1,2,4,7,12h).
The drug level of the Propranolol in cytoplasm and nucleus that table 2 different time points is measured.
| Time (h) | 0.5 | 1 | 2 | 47 | 12 |
| Cytoplasm drug level μ g/ml | 12.5±1.2 | 26.7±2.4 | 45.1±1.8 | 52.7±3.871.9±5.6 | 80.0±2.8 |
| Nucleus drug level μ g/ml | 0 | 8.1±2.0 | 11.2±7.1 | 20.6±1.125.6±2.5 | 29.3±2.4 |
Excision infant hemangioma tissue under aseptic condition, be cut into the fritter of 5mm * 4mm * 3mm with tissue chopper, with UW(the University of Wisconsin solution) liquid rinsing 3 times, insert all two forelimb oxters of experiment nude mice through the skin otch respectively, in cervical region and the left buttocks subcutaneous tissue, every 4 places.Inject estradiol 1 time in right leg muscle place, each 0.1mg for weekly after the transplanting animal.In inoculation grouping in back 45 days, the different Propranolol of intravenous injection are subjected to test preparation respectively, after administration, observe and record hemangioma form and size, every 2d administration 1 time, administration 15d, congested situation and the hemangioma volume of perusal every day local vascular tumor after the administration, and the variation of observing local vascular under stereomicroscope is to judge curative effect.
After finishing administration, get biology sample detection toxicity.Get blood system from serum, measure serum urea nitrogen (BUN), creatinine (Cr), total bilirubin (TBIL), total protein (TP), albumin (ALB), blood glucose (GLU), ALT (ALT), alkali phosphatase (ALP), aspartic acid aminotransferase (AST), T-CHOL biochemical indicators such as (CHOL).By body weight conversion liver, kidney device coefficient (the heavy g/100g body weight of internal organs), liver, kidney placed 10% formalin fixing, routine paraffin wax embedding, section, HE dyeing is carried out tectology and is observed, and lobules of liver slightly is hexagon, clear in structure.Hepatocyte does not have degeneration, necrosis.Around the central vein minute quantity cell infiltration is arranged, portal area NIP or proliferation of fibrous tissue, interlobular arteries, interlobular veins and interlobular bile duct do not have pathological changes.Tectology is observed and is found that liver and kidney all do not have pathological changes.
Embodiment 11
The safety evaluatio of VEGFR-2 monoclonal antibody and CPP combined modification Propranolol hidden liposome:
A. measure the anaphylaxis under the intravenous injection approach; B. measure hemolytic; C. measure long term toxicity.Below all with Propranolol solution, not modified Propranolol hidden liposome in contrast.
Injection anaphylaxis evaluation: the Cavia porcellus of getting body weight 250-300g, the next day intramuscular injection be subjected to test preparation, totally 3 times, animal is divided into 2 groups then, first group of intravenous injection in the 14th day after injection first is subjected to test preparation, observe and also to estimate anaphylaxis, second group after injection first same intravenous injection in the 21st day and observe.Observe no obvious allergic symptom.
Hemolytic is estimated: get 2% Sanguis Leporis seu oryctolagi red blood cell suspension and mix with the reagent liquid for the treatment of of different volumes, water-bath is placed 3h, perusal haemolysis situation for 37 ℃.100% and 0% haemolysis contrast liquid is respectively distilled water and normal saline.The result shows that haemolysis does not take place hidden liposome.
Long term toxicity is estimated: deliver medicine to rat by anti-effective dose tail vein injection, per 3 days 1 time, 3 totally months, during the administration and after the administration end, day by day observe the general situation of animal, weigh weekly 1 time, respectively at different time points after the administration, adopt the rat metabolic cage to collect rat urine, measure urine protein, glucose in urine; With urinalysis reagent paper qualitative analysis glucose in urine, urine ketone, urine bilirubin, urine protein, urobilinogen etc.Get blood system simultaneously from serum, measure serum urea nitrogen (BUN), creatinine (Cr), total bilirubin (TBIL), total protein (TP), albumin (ALB), blood glucose (GLU), ALT (ALT), alkali phosphatase (ALP), aspartic acid aminotransferase (AST), T-CHOL biochemical indicators such as (CHOL).Experiment finishes the back and puts to death animal, by body weight conversion liver, kidney device coefficient (the heavy g/100g body weight of internal organs).Place 10% formalin fixing in liver, kidney, routine paraffin wax embedding, section, HE dyeing is carried out tectology and is observed, and liver and kidney all do not have pathological changes.
Embodiment 12
The pharmacokinetics evaluation of VEGFR-2 monoclonal antibody and CPP combined modification Propranolol hidden liposome:
Compare Propranolol solution, not modified Propranolol hidden liposome and VEGFR-2 monoclonal antibody and the interior medicine dynamics behavior of CPP combined modification Propranolol hidden liposome intravenous injection behind rat.
The healthy SD rat, male and female half and half, 200～250g, tail vein injection give different Propranolol preparations, and different time points is got blood system from blood plasma before administration and after the administration, handles the back and measures drug level.Adopt DAS2.0 software to handle blood drug level-time data, the match compartment model also calculates corresponding pharmacokinetic parameter, the statistics comparative analysis.AUC
0-∞Be 17.61 ± 3.84 μ ghmL
-1, average residence time MRT is 3.12 ± 1.19h, clearance rate CL is 0.32 ± 0.06Lh
-1Kg
-1, apparent volume of distribution Vss is 0.49 ± 0.34Lkg
-1Statistics shows that relatively the Propranolol liposome of modification and not modified Propranolol liposome compare AUC, and MRT, CL all have significant difference (P＜0.05)
The above only is preferred implementation of the present invention; be noted that for those skilled in the art; under the prerequisite that does not break away from the principle of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1.VEGFR-2 the preparation method of the Propranolol hidden liposome of monoclonal antibody and CPP combined modification is characterized in that may further comprise the steps:
1) the liposome composite phospholipid is formed and determination of ratio;
2) the Propranolol hidden liposome preparation of CPP modification:
A) pNP-PEG-DPPE's is synthetic;
B) CPP-PEG-DPPE's is synthetic;
C) the Propranolol hidden liposome preparation of CPP modification;
3) the VEGFR-2 monoclonal antibody is connected the Propranolol hidden liposome that namely obtains VEGFR-2 monoclonal antibody and CPP combined modification with the Propranolol stealthy liposome surface that CPP modifies.
2. the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody according to claim 1 and CPP combined modification is characterized in that: described liposome composite phospholipid adopts distearoyl phosphatidylcholine, hydrogenated soy phosphatidyl choline as high phase transition temperature phospholipid; Adopting dipalmitoyl phosphatidyl choline and dimyristoyl phosphatidyl choline is low transformation temperature phospholipid, and the combined preparation composite phospholipid liposome has 4 kinds of combinations in twos.
3. the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody according to claim 1 and CPP combined modification, it is characterized in that: described liposome composite phospholipid consists of dipalmitoyl phosphatidyl choline and hydrogenated soy phosphatidyl choline.
4. the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody according to claim 3 and CPP combined modification, it is characterized in that: described dipalmitoyl phosphatidyl choline and hydrogenated soy phosphatidyl choline ratio are 6:4.
5. the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody according to claim 2 and CPP combined modification, it is characterized in that: the mol ratio of a kind of composite phospholipid of combined preparation and DSPE-PEG2000 is 95:5 in above-mentioned 4, obtains the DSPE-PEG2000 composite phospholipid.
6. the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody according to claim 4 and CPP combined modification, it is characterized in that: selected DSPE-PEG2000 composite phospholipid and the Propranolol that contains 5% mol ratio made the Propranolol hidden liposome, temperature is bathed 2h in 37 ℃ of water-baths, with step 2) in the synthetic CPP-PEG-DPPE that obtains directionally be inserted into according to 5% mol ratio on the outer membrane of Propranolol hidden liposome and obtain the Propranolol hidden liposome that CPP modifies.
7. the preparation method of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody according to claim 1 and CPP combined modification, it is characterized in that, the method of attachment of the Propranolol stealthy liposome surface that described step 3) VEGFR-2 monoclonal antibody and CPP modify is as follows: earlier the VEGFR-2 monoclonal antibody is dissolved in the HEPES pH8.0 buffer, mix with the SPDP solution of 6.25mg/ml, reaction generates PDP-IgG, fully after the reaction, the centrifugal removal of ultrafiltration is SPDP and the by-product of coupling not, collect PDP-IgG, it is 50 mmol/L that adding DTT makes its final concentration, stir 25 ℃ of reaction 25min, make the PDP mercaptanization on the antibody, form IgG-SH; IgG antibody-SH the solution of mercaptanization is mixed with the Propranolol hidden liposome that CPP modifies, molar ratio range is 1:1000-1:50, the dark place stirred overnight at room temperature, ultrafiltration is centrifugal to remove unconjugated IgG-SH, namely obtains the Propranolol hidden liposome of VEGFR-2 monoclonal antibody and CPP combined modification.
8. the VEGFR-2 monoclonal antibody that obtains of any described preparation method of claim 1-3 and the Propranolol hidden liposome of CPP combined modification, it is characterized in that: the envelop rate of the Propranolol hidden liposome of VEGFR-2 monoclonal antibody and CPP combined modification is 80%, the cumulative release degree of release: 10h is about 15%, setting-out stability: setting-out 70d is stable, targeting efficient: 60%.
9. the Propranolol hidden liposome of the VEGFR-2 monoclonal antibody that obtains of any described preparation method of claim 1-3 and CPP combined modification is for the preparation of the application on the treatment hemangioma medicine.
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| CN104059153A (en) * | 2013-09-13 | 2014-09-24 | 西安交通大学医学院第一附属医院 | Transmembrane antibody of targeting virus core protein and preparation method thereof |
| CN107375920A (en) * | 2017-06-23 | 2017-11-24 | 中农威特生物科技股份有限公司 | A kind of porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virosomes vaccine and its production and use |
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| CN102871956A (en) * | 2012-10-29 | 2013-01-16 | 中南大学湘雅医院 | Propranolol hydrochloride gel for treating infant superficial hemangioma |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104059153A (en) * | 2013-09-13 | 2014-09-24 | 西安交通大学医学院第一附属医院 | Transmembrane antibody of targeting virus core protein and preparation method thereof |
| CN107375920A (en) * | 2017-06-23 | 2017-11-24 | 中农威特生物科技股份有限公司 | A kind of porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virosomes vaccine and its production and use |
| CN107375920B (en) * | 2017-06-23 | 2020-08-11 | 中农威特生物科技股份有限公司 | Porcine reproductive and respiratory syndrome virus-swine influenza virus reconstituted virosome vaccine and preparation method and application thereof |
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