CN102552145B - Preparation method of artificial liposome - Google Patents
Preparation method of artificial liposome Download PDFInfo
- Publication number
- CN102552145B CN102552145B CN201210023252.7A CN201210023252A CN102552145B CN 102552145 B CN102552145 B CN 102552145B CN 201210023252 A CN201210023252 A CN 201210023252A CN 102552145 B CN102552145 B CN 102552145B
- Authority
- CN
- China
- Prior art keywords
- protein
- liposome
- carrier
- spc
- epc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method of artificial liposome, belongs to the field of pharmacy, and relates to a preparation method of protein medicament liposome. In the preparation method, nano spherical grain yolk lecithin zirconium or soya bean lecithin zirconium is used as a carrier, and balsam pear protein MAP30, fleece-flower root protein GAP31, lipase CRL or bovine serum albumin (BSA) is used as a wrapping protein. The preparation method comprises the steps of: adding phosphate buffer solution to the carrier material, stirring under the conditions of the temperature being 4 DEG C and rotation rate being 3500rpm to obtain a colorless and transparent dispersion, dissolving the wrapping protein in a phosphate buffer solution, then slowly dropwise adding to the dispersion, quickly stirring in the low-temperature condition to form stable liposome, centrifugally separating out protein, repeatedly washing, centrifuging, and freeze-drying at low temperature to obtain a finished product. The method has the characteristics of simple process, mild condition, high repeatability, high protein adsorption capacity and stable liposome structure.
Description
Technical field
The invention belongs to pharmaceutical field, relate to a kind of preparation method of pharmaceutical grade protein liposome, be specifically related to a kind of preparation method of artificial liposome.
Background technology
Physics, physiology and pharmacology due to liposome uniqueness, as having, standby integrate hydrophilic and hydrophobic drug ability, biocompatibility is good, toxicity is low, without immune system response and in the target slow-release ability of action site active medicine etc., therefore, as therapeutic type or diagnosis type active agent, the research of liposome vectors is paid close attention to all the time widely.Since finding liposome, the size of its product can effectively be controlled from micron to nanometer; Part carrier system has carried out functional integration with polypeptide, protein and antibody after by surface modification.At present, FDA mechanism has ratified as amikacin/HSPC/CH/DSPG, muramyldipeptide/DSPC/PS(1:1) etc. 25 kinds of above Lipidosome medicines in clinical trial, Some Drugs has entered the clinical practice stage.
The technology of preparing of liposome is mainly taked following 4 kinds of approach: water stirring, ultrasonic technique (the ultrasonic dispersion technology of Probe Ultrasonic Searching and water-bath), inverted evaporation technology and lyophilization dry technology.Liposome medicament is the product that has hydrophilic group and hydrophobic group concurrently, by following 4 kinds of different approach, targeted cells is played a role respectively: in reticuloendothelium selectively acting, the phospholipid bilayer of the weak non-selective effects such as hydrophobicity (or static) of the endocytosis of macrophage and neutrophil cell, medicine and cell surface and medicine and cell surface component insert serum cell membrane and be discharged in Cytoplasm, liposome is transferred to cell inner membrance or subcellular organelle film in.In above situation, the action pathway of more difficult judgement competitive advantage, simultaneously not yet clear at present for a kind of above synergism process.
For therapeutic type and diagnosis type liposome medicament, although carried out research work extensively and profoundly.Yet in the Application and Development of concrete medicine, still there is obvious deficiency in liposome.For example, reticuloendothelium cell is very fast to the biodegradation rate of liposome, causes active medicine component be difficult to the slow release that reaches stable, lasting and transmit effect.In order to overcome above deficiency, in the structure of liposome medicament, successfully adopted the method for modifying of 2 Type of Collective materials: by hydrophilic polymeric material, existing surface of liposome is modified as polyethylene glycol (PEG); In addition, the drug-loaded liposome of parcel in advance can be incorporated in polymeric matrix system, thereby effectively control slow release and the transmission of liposome medicament.Yet in the compound structure of pre-parcel drug-loaded liposome and polymeric matrices, the discipline range relating to extensively demands strict technology, and needs pharmacy, biomaterial science, chemistry, molecule and the relevant research worker of cytobiology to work in concert and could realize simultaneously.
The deficiencies such as liposomal body objects system has poor stability, drug half-life is short and drug disposition clearance rate is fast, are used the pharmacological property that can obviously improve liposome after hydrophilic polymeric material modification.This method has had the advantage of drug-loaded liposome and parcel host material concurrently, and the stability of medicine improves, and simultaneously lasting drug delivery ability strengthens.Yet, use the disadvantage of hydrophilic polymeric material to be that biocompatibility is poor.For realizing the compound structure of drug-loaded liposome and polymeric material, liposome must be exposed to organic solvent and carry out heat ultrasonic, cause the biological activity of medicine obviously to decline.
Current, the technique for packing of the structure coupling polymeric matrices of liposome is the study hotspot of domestic and international pharmaceutical industry.This research is not only expected to improve medicine stability and lasting Controlled release, maintains drug disposition level and extends the interval of medication, reduces the toxic and side effects of medicine to tissues such as intestines and stomaches; Be expected to efficiently retain medicine vigor simultaneously, guarantee clinical therapeutic effect.For this reason, having built and developed different technology is used for drug-loaded liposome to wrap up in polymerism framing structure.All in all, this research is still in the basic exploratory stage, and the clinical successful Application of distance still has a large amount of technical barrier etc. to be solved.
The modification of existing liposome vectors is carried out to another study hotspot that technological innovation is pharmaceutical industry.Phospholipid (PC) liposome report is at home and abroad existing a lot, but PC causes the stability of the drug-loaded liposome that forms lower as Ovum Gallus domesticus Flavus lecithin (EPC) or soybean lecithin (SPC) have configuration changeable (taper and cylindrical isomery).In the common route of administration such as oral or intravenous drip, although the drug effect of liposome medicament is high, but body fluid physiological condition makes the poor stability of carrier as enzyme catalysis, directly caused maintaining and checkout time very short (conventionally maintaining a few hours) of levels of drugs, therefore at clinical middle needs, continued medication.Patent applicant was once hybridized EPC and SPC respectively with inorganic ions, the micelle nano material (referring to patent CN102107131A) that obtained good stability, biocompatibility is high, isoelectric point, IP is moderate, separation is simple and insoluble with purge process.With respect to other liposome system, the preparation of carrier material has the advantages such as condition is very gentle, technological process is simple, and product dispersibility is high, granularity is little (about 5nm has same order with the physical dimension of protein and drug molecule) and be evenly distributed.
Zr(EPC)2 (Zr (EPC) 2) and Zr(SPC)2 (Zr (SPC) 2) are as liposome vectors, have advantages of following: 1. liposome preparation condition as mild as a dove, avoid adopting organic solvent and heat ultrasonic, effectively prevented the change of configuration of protein molecule and vigor to decline; 2. the separation process of liposome medicament and substrate, substrate is simple, and centrifugal and washing operation can have been realized the purification of elaioplast nanometer particle easily; 3. stability height and the moderate carrier of mechanical strength are to guarantee that liposome medicament maintains stable prerequisite for conditions such as solvent and heating; 4. carrier is large to the adsorption capacity of pharmaceutical grade protein, and selects to carry out manual control by condition; 5. carrier is similar to the physical dimension of protein, and adsorption dynamics adsorption kinetics has concurrently initiatively and passive 2 processes simultaneously, and the manufacturing cycle of product is short.
For realizing effective transmission and the safe handling of medicine, use liposome to build coupling polymer backbone technique for packing development of new slow releasing pharmaceutical and have very important significance.Although liposome technology is applicable to the dynamic metabolisms such as the slow release of active medicine and transmission, yet still has obvious deficiency while being widely used in pharmaceutics, mainly concentrate on drug effect and two aspects of biocompatibility of liposome medicament.Be embodied in that plasma half-life is short, poor stability, vigor declines, toxic and side effects is strong and long-term slow release and to transmit control ability low etc.The surface modification of polymeric material or compound technique for packing can overcome the part shortcoming of liposome medicament; But the transformation of configuration (as the melting of pharmaceutical grade protein and fibrosis) and the vigor that have aggravated on the one hand packaging medicine decline, and on the other hand the cell of body tissue have been caused to certain metabolic burden.In addition, the specification requirement of the compound parcel of polymeric skeleton is high, needs relevant multidisciplinary research personnel to cooperate and just likely realizes.In research report few in number, the preparation technology of the liposome product of modification or parcel is numerous and diverse, condition is harsh and process cycle is long.
Summary of the invention
The preparation method that the object of this invention is to provide a kind of artificial liposome.
The present invention is achieved by the following technical solutions:
A preparation method for artificial liposome, with the Zr(EPC)2 Zr (EPC) of nanometer spherical granule
2or Zr(SPC)2 Zr (SPC)
2as carrier, using bitter melon protein MAP30, Radix Polygoni Multiflori Protein G AP31, lipase CRL or bovine serum albumin BSA as parcel albumen, wherein carrier is 74.3-77.7:22.3-25.7 with the mass ratio of parcel albumen, during preparation, at Zr(EPC)2 Zr (EPC)
2or Zr(SPC)2 Zr (SPC)
2carrier material in add phosphate buffered solution, in temperature, it is 4 ℃, rotating speed is to stir and obtain colourless, transparent dispersion liquid under the condition of 3500rpm, to wrap up protein dissolution in phosphate buffered solution, then slowly drop in dispersion liquid, under cryogenic conditions, rapid stirring is to forming stable liposome, after centrifugalize goes out protein, cyclic washing is also centrifugal, through frozen drying, obtain solid product, solid product cryopreservation or physiological saline solution are sealed up for safekeeping to be placed under cryogenic conditions and are stored, and get product.
Further, described carrier is 75-77:23-25 with the mass ratio of parcel albumen.
Zr(EPC)2 (Zr (EPC)
2) and Zr(SPC)2 (Zr (SPC)
2) as liposome vectors, having advantages of following: 1. liposome preparation condition as mild as a dove, avoids adopting organic solvent and heat ultrasonic, has effectively prevented the change of configuration of protein molecule and vigor to decline; 2. the separation process of liposome medicament and substrate, substrate is simple, and centrifugal and washing operation can have been realized the purification of elaioplast nanometer particle easily; 3. stability height and the moderate carrier of mechanical strength are to guarantee that liposome medicament maintains stable prerequisite for conditions such as solvent and heating; 4. carrier is large to the adsorption capacity of pharmaceutical grade protein, and selects to carry out manual control by condition; 5. carrier is similar to the physical dimension of protein, and adsorption dynamics adsorption kinetics has concurrently initiatively and passive 2 processes simultaneously, and the manufacturing cycle of product is short.
The protein of liposome is selected respectively therapeutic type natural medicinal plants, fat hydrolase and immune diagnostic reagent, and natural plant protein matter crude extract, respectively from Fructus Momordicae charantiae seed and Radix Polygoni Multiflori, is the cancer therapy drug of ribosome inactivation I-type; Lipase is used for being hydrolyzed Blood Cholesterol, glyceride and non-free fatty.The present invention exempts to use the parcel substrate of water-soluble polymeric skeleton, in liposome preparation, do not use any organic solvent, do not carry out the process conditions such as supersound process, build this medicinal liposome and there is important clinical, medical significance, there is development and application values widely.
The preparation method of liposome is taked aqueous-phase suspending adsorption technology, makes people's chemical product under low temperature and stirring condition, and carrier granularity is about 5nm, and protein size has the identical order of magnitude.Carrier granularity is little, causes in liposome protein content large; Meanwhile, in preparation process, there are active and passive 2 kinds of adsorptions.
Preparation process is implemented under certain adsorption dynamics adsorption kinetics condition, and technique is investigated in design, the factor of influence that orthogonal optimizing power are learned.Be conducive on the one hand obtain and form the liposome product stable, protein content is high; On the other hand, be conducive to investigate the material elements that affects pharmaceutical grade protein slow release.Liposome medicament after entering body fluid environment, the graded of salinity, pH and liposome (protein) concentration, medicine effectively discharges and transmits.Wherein, the dispose procedure of protein molecule presents the initiatively characteristic of diffusion; Meanwhile, stable physiology enzyme catalysis condition promotes the degraded of carrier material effectively, and drug release has the diffusion property of erosion simultaneously.
The inventive method has that technique is simple, mild condition, repeated high, and the bearer type of employing is Zr(EPC)2 Zr (EPC)
2or Zr(SPC)2 Zr (SPC)
2nanometer spherical granule, reproducible and the even particle size distribution of preparation, carrier has very high adsorption capacity to pharmaceutical grade protein, enzyme molecule, and in liposome, protein content is up to 22.3-25.7%, than the high 1-2 of a protein encapsulation efficiency order of magnitude of Common Artificial liposome, adopt as accompanying drawing
1configuration absorption, protein molecule is evenly distributed on the hydrophobicity special area of polymeric carrier, avoids forming protein aggregate structure, this location, configuration is distributed with and is beneficial to the bioavailable degree that improves protein in order; Liposome structure is stable, in conjunction with firmly, its storage-stable is for up to 6-10 month, to hot survivability temperature up to 60-80oC.
Accompanying drawing explanation
Fig. 1 is the configuration schematic diagram of artificial liposome complex;
Fig. 2 is that bitter melon protein matter medicine MAP30 is at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 3 is that bitter melon protein matter medicine MAP30 is at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 4 is that Radix Polygoni Multiflori pharmaceutical grade protein GAP31 is at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 5 is that Radix Polygoni Multiflori pharmaceutical grade protein GAP31 is at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 6 is lipase
candida Rugosa Lipase(CRL) at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 7 is lipase
candida Rugosa Lipase(CRL) at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 8 is that bovine serum albumin (BSA) is at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier;
Fig. 9 is that bovine serum albumin (BSA) is at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier.
Prepare bitter melon protein MAP30 and Zr(EPC)2 Zr (EPC)
2complex liposome: bitter melon protein (from seed) crude extract, through SDS-PAGE gel electrophoresis, confirm that molecular weight is 30 kDa, accurately weighing 6.8 mg MAP30 are dissolved in 1.0 ml phosphate buffers (pH 7.2,10 mM+100 mM NaCl) and save backup; Zr(EPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 201.6 μ g Zr (EPC) 2 particulate vectors, add 1.49 ml phosphate buffers (pH 7.2), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml MAP30 albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.5,1.0,4.0,7.0,10.0,12.0,15.0,18.0,20.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 201.6 μ g Zr (EPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 5.00,5.49,5.89,6.39,6.92,7.56,8.06,8.52,9.02 and 9.90) of 1.49 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml MAP30 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 15.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 12.6,37.8,63.0,75.6,100.8,126.0,138.6,165.0,189.0,201.6,226.8 and 252.0 μ g Zr (EPC)
2particulate vector, add 1.49 ml phosphate buffered solution (pH 8.52), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml MAP30 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 15.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 8.52) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
1artificial liposome product
Lyophilization obtains product for 3 days: white powder, water insoluble and polar organic solvent; The adsorption time that maximum protein content is corresponding is 15 hours, best pH 8.52, the maximum percentage composition 24.0 ± 1.0% of protein; After disperseing, obtain water white transparency suspension in injecting normal saline and phosphate buffer; Under solid phase condition, protein thermal desorption temperature is in 80 oC(5 hours); Under physiological condition outside analogue body, the time of liposome stable (protein desorption rate≤5%) reaches 9 months; Structure and exploitation for ribosome inactivation type Effective Anti cancer drug stabilizer type, can be used in as the treatment of I-type HIV.
Fig. 2 is that bitter melon protein matter medicine MAP30 is at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier; Experiment condition: T=4 oC wherein, stir speed (S.S.) 3500 rpm, mixing time 15 h, and phosphate buffered solution (pH 8.52,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
Prepare bitter melon protein MAP30 and Zr(SPC)2 Zr (SPC)
2complex liposome: accurately weigh bitter melon protein 6.8 mg and be dissolved in 1.0 ml phosphate buffers (pH 7.2,10 mM+100 mM NaCl) and save backup; Zr(SPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 201.6 μ g Zr (SPC)
2particulate vector, add 1.49 ml phosphate buffers (pH 7.2), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml MAP30 albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.5,1.0,4.0,7.0,10.0,12.0,15.0,18.0,20.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 201.6 μ g Zr (SPC)
2particulate vector, adding respectively pH is 5.00,5.49,5.89,6.39,6.92,7.56,8.06,8.52,9.02 and 9.90 phosphate buffers (1.49 ml), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml MAP30 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 15.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 12.6,37.8,63.0,75.6,100.8,126.0,138.6,165.0,189.0,201.6,226.8 and 252.0 μ g Zr (SPC)
2particulate vector, add 1.49 ml phosphate buffered solution (pH 9.02), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml MAP30 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 15.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 9.02) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
2artificial liposome product
Lyophilization obtains product for 3 days: white powder, water insoluble and polar organic solvent; In product, the time of protein adsorption maximum is 15 hours, best pH 9.02, the maximum percentage composition 24.4 ± 1.2% of protein; After disperseing, obtain water white transparency suspension in injecting normal saline and phosphate buffer; Under solid phase condition, protein thermal desorption temperature is in 72 oC(5 hours); Under physiological condition outside analogue body, the time of liposome stable (protein desorption rate≤5%) reaches 6 months; For structure and the exploitation of ribosome inactivation type Effective Anti cancer drug stabilizer type, for the treatment of I-type HIV.
Prepare Radix Polygoni Multiflori Protein G AP31 and Zr(EPC)2 Zr (EPC)
2complex liposome: Radix Polygoni Multiflori protein crude extract administration is from Radix Polygoni Multiflori, through SDS-PAGE gel electrophoresis, confirms that molecular weight is 31 kDa.Accurately weighing 5.8 mg GAP31 are dissolved in 1.0 ml phosphate buffers (pH 7.2,10 mM+100 mM NaCl) and save backup; Zr(EPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 201.6 μ g Zr (EPC)
2particulate vector, add 1.49 ml phosphate buffers (pH 7.2), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml GAP31 albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.5,1.0,4.0,7.0,10.0,12.0,15.0,18.0,20.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 201.6 μ g Zr (EPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 4.50,5.00,5.49,5.89,6.39,6.98,7.56,8.06,8.52,9.02 and 9.90) of 1.49 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml GAP31 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 20.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 12.6,37.8,63.0,75.6,100.8,126.0,138.6,165.0,189.0,201.6,226.8 and 252.0 μ g Zr (EPC)
2particulate vector, add 1.49 ml phosphate buffered solution (pH 5.00), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml GAP31 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 20.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 5.00) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
3artificial liposome product
Lyophilization obtains product for 3 days: white (yellowish) toner end, water insoluble and polar organic solvent; Protein is 20 hours in the stabilization time of carrier adsorption, the best pH=5.00 of buffer, the maximum percentage composition 23.5 ± 1.2% of protein in product; Must be without (pale yellow) color transperent suspension liquid after disperseing in injecting normal saline and phosphate buffer; Under solid phase condition, protein thermal desorption temperature is in 78 oC(5 hours); Under physiological condition outside analogue body, the time of liposome stable (protein desorption rate≤5%) reaches 10 months; For structure and the exploitation of ribosome inactivation type Effective Anti cancer drug stabilizer type, kill the virus as I-type HIV.
Accompanying drawing 4 is that Radix Polygoni Multiflori pharmaceutical grade protein GAP31 is at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier.Experiment condition: T=4 oC, stir speed (S.S.) 3500 rpm, mixing time 20 h, and phosphate buffered solution (pH 5.00,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
embodiment 4
Prepare Radix Polygoni Multiflori Protein G AP31 and Zr(SPC)2 Zr (SPC)
2complex liposome: Radix Polygoni Multiflori protein crude extract administration is from Radix Polygoni Multiflori, and SDS-PAGE gel electrophoresis confirms that its molecular weight is 31 kDa.Accurately weighing 5.8 mg GAP31 are dissolved in 1.0 ml phosphate buffers (pH 7.2,10 mM+100 mM NaCl) and save backup; Zr(SPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 201.6 μ g Zr (SPC)
2particulate vector, add 1.49 ml phosphate buffers (pH 7.2), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml GAP31 albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.5,1.0,4.0,7.0,10.0,12.0,15.0,18.0,20.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 201.6 μ g Zr (SPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 5.00,5.49,5.89,6.39,6.98,7.56,8.06,8.52,9.02 and 9.90) of 1.49 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml GAP31 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 15.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 12.6,37.8,63.0,75.6,100.8,126.0,138.6,165.0,189.0,201.6,226.8 and 252.0 μ g Zr (SPC)
2particulate vector, add 1.49 ml phosphate buffered solution (pH 5.89), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.01 ml GAP31 albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 15.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 5.89) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
4artificial liposome product
Lyophilization obtains product for 3 days: white (pale yellow) toner end, water insoluble and polar organic solvent; Protein is 15 hours in the stabilization time of carrier adsorption, the best pH=5.89 of buffer, the maximum percentage composition 23.8 ± 1.2% of protein in product; Must be without (pale yellow) color transperent suspension liquid after disperseing in injecting normal saline and phosphate buffer; Under solid phase condition, protein thermal desorption temperature is in 75 oC(5 hours); Under physiological condition outside analogue body, the time of liposome stable (protein desorption rate≤5%) reaches 8 months; For structure and the exploitation of ribosome inactivation type Effective Anti cancer drug stabilizer type, instil and kill as viruses such as I-type HIV.
Accompanying drawing 5 is that Radix Polygoni Multiflori pharmaceutical grade protein GAP31 is at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier.Experiment condition: T=4 oC, stir speed (S.S.) 3500 rpm, mixing time 15 h, and phosphate buffered solution (pH 5.89,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
embodiment 5
Prepare lipase CRL and Zr(EPC)2 Zr (EPC)
2complex liposome: lipase (
candida Rugosalipase, CRL) purchased from Sigma company, wherein protein content is 2.84%(mass percent), SDS-PAGE gel electrophoresis confirms that molecular weight is about 60 kDa.Accurately weigh the thick enzyme of 120.42 mg and be dissolved in phosphate buffer (pH 7.2,10 mM+100 mM NaCl), centrifugalize is also washed insoluble matter, and supernatant is collected and regulated cumulative volume is that 1.0 ml(CRL concentration are 3.42 mg/ml), save backup; Zr(EPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 900 μ g Zr (EPC)
2particulate vector, add 1.4 ml phosphate buffers (pH 7.40), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml CRL albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.6,1.5,3.5,7.5,11.0,14.0,19.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 540 μ g Zr (EPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 7.40,8.59,9.73 and 10.43) of 1.4 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml CRL albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 14.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 50,100,200,350,500 and 750 μ g Zr (EPC)
2particulate vector, add 1.4 ml phosphate buffered solution (pH 8.59), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, then adds 0.1 ml CRL albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 14.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 8.59) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
5artificial liposome product
Lyophilization obtains product for 3 days: white powder, water insoluble and polar organic solvent; In product, be 14 hours the stabilization time of protein adsorption, best pH 8.59, the maximum percentage composition 23.9 ± 1.2% of protein; Under solid phase condition, the protein thermal desorption temperature in liposome is in 64 oC(5 hours); In phosphate buffer solution, the time of liposome stable (protein desorption rate≤5%) reaches 6 months; Be used for structure and development of new, efficient enzyme, the reaction that catalysis fat splitting and ester exchange are synthetic.
Accompanying drawing 6 is lipase
candida Rugosa Lipase(CRL) at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier.Experiment condition: T=4 oC, stir speed (S.S.) 3500 rpm, mixing time 14 h, and phosphate buffered solution (pH 8.59,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
embodiment 6
Prepare lipase CRL and Zr(SPC)2 Zr (SPC)
2complex liposome: lipase (
candida Rugosalipase, CRL) purchased from Sigma company, the mass percent of protein is that 2.84%, SDS-PAGE gel electrophoresis confirms that molecular weight is about 60 kDa.Accurately weigh the thick enzyme of 120.42 mg and be dissolved in phosphate buffer (pH 7.2,10 mM+100 mM NaCl), centrifugalize is also washed insoluble matter, and supernatant is collected and regulated cumulative volume is that 1.0 ml(CRL concentration are 3.42 mg/ml), save backup; Zr(SPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 900 μ g Zr (SPC)
2particulate vector, add 1.4 ml phosphate buffers (pH 7.40), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml CRL albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.6,1.5,3.5,7.5,11.0,14.0,19.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 1.26 mg Zr (SPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 7.40,8.59,9.73 and 10.43) of 1.4 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml CRL albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 19.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 50,100,200,350,500 and 750 μ g Zr (SPC)
2particulate vector, add 1.4 ml phosphate buffered solution (pH 8.59), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, then adds 0.1 ml CRL albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 19.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 8.59) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
6artificial liposome product
Lyophilization obtains product for 3 days: white powder, water insoluble and polar organic solvent; In product, be 19 hours the stabilization time of protein adsorption, best pH 8.59, the maximum percentage composition 24.1 ± 1.2% of protein; Under solid phase condition, the protein thermal desorption temperature in liposome is in 60 oC(5 hours); In phosphate buffer solution, the time of liposome stable (protein desorption rate≤5%) reaches 6 months; Be used for structure and development of new, efficient biocatalyzer, promote fat splitting and the synthetic reaction of ester exchange.
Accompanying drawing 7 is lipase
candida Rugosa Lipase(CRL) at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier.Experiment condition: T=4 oC, stir speed (S.S.) 3500 rpm, mixing time 19 h, and phosphate buffered solution (pH 8.59,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
embodiment 7
Prepare bovine serum albumin BSA and Zr(EPC)2 Zr (EPC)
2complex liposome: bovine serum albumin is purchased from Sigma company, and molecular weight is 66.5 kDa, and protein content is 90%.Accurately weighing 3.80 mg BSA are dissolved in 1.0 ml phosphate buffers (pH 7.2,10 mM+100 mM NaCl) and save backup (BSA concentration is 3.42 mg/ml); Zr(EPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 1.1 mg Zr (EPC)
2particulate vector, add 1.4 ml phosphate buffers (pH 7.2), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml BSA albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.6,1.5,3.5,7.5,11.0,14.0,19.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 540 μ g Zr (EPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 7.40,8.59,9.73 and 10.43) of 1.4 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml BSA albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 19.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 40,80.5,161,282,402.5 and 603.8 μ g Zr (EPC)
2particulate vector, add 1.4 ml phosphate buffered solution (pH 8.59), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, then adds 0.1 ml BSA albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 19.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 8.59) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
7artificial liposome product
Lyophilization obtains product for 3 days: white powder, water insoluble and polar organic solvent; In product, the adsorption time of maximum protein content is 19 hours, best pH 8.59, the maximum percentage composition 24.5 ± 1.2% of protein; Under solid phase condition, protein thermal desorption temperature is in 70 oC(5 hours); Under physiological condition outside analogue body, the time of liposome stable (protein desorption rate≤5%) reaches 9 months; For building and development of new, stable immune diagnostic reagent, for biological and medical science detection.
Accompanying drawing 8 is that bovine serum albumin (BSA) is at Zr(EPC)2 (Zr (EPC)
2) the adsorption kinetic data result of nano-carrier.Experiment condition: T=4 oC, stir speed (S.S.) 3500 rpm, mixing time 19 h, and phosphate buffered solution (pH 8.59,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
embodiment 8
Prepare bovine serum albumin BSA and Zr(SPC)2 Zr (SPC)
2complex liposome: bovine serum albumin is purchased from Sigma company, and molecular weight is 66.5 kDa, and protein content is 90%.Accurately weighing 3.80 mg BSA are dissolved in 1.0 ml phosphate buffers (pH 7.2,10 mM+100 mM NaCl) and save backup (BSA concentration is 3.42 mg/ml); Zr(SPC)2 nano-carrier (5nm, spheroidal particle) is according to method preparation in patent (CN102107131A).Accurately weigh 1.1 mg Zr (SPC)
2particulate vector, add 1.4 ml phosphate buffers (pH 7.2), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml BSA albumen storing solution, stir respectively separation of supernatant after high speed centrifugation (12000 rpm) 20min 0.6,1.5,3.5,7.5,11.0,14.0,19.0 and 24.0 hour.Solid particle is used the washing of 0.1ml phosphate buffered solution rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines the optimal adsorption time.
Accurately weigh 1.26 mg Zr (SPC)
2particulate vector, add respectively the different pH phosphate buffers (pH 7.40,8.59,9.73 and 10.43) of 1.4 ml, high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, add again 0.1 ml BSA albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 24.0 hours.Solid particle is used the corresponding pH phosphate buffered solution washing of 0.1ml rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, determines optimal adsorption acidity.
Weigh respectively 40,80.5,161,282,402.5 and 603.8 μ g Zr (SPC)
2particulate vector, add 1.4 ml phosphate buffered solution (pH 8.59), high-speed stirred under 4 oC conditions (3500 rpm) is to the complete suspended dispersed of carrier, then adds 0.1 ml BSA albumen storing solution, stir separation of supernatant after high speed centrifugation (12000 rpm) 20min 24.0 hours.Solid particle is used 0.1ml phosphate buffered solution (pH 8.59) washing rear centrifugal, repeats 3 times, merges supernatant and measures residual protein quality, calculates protein adsorption capacity.
Example
8artificial liposome product
Lyophilization obtains product for 3 days: white powder, water insoluble and polar organic solvent; In product, protein adsorption stabilization time is 24.0 hours, best pH 8.59, the maximum percentage composition 24.2 ± 1.2% of protein; Under solid phase condition, protein thermal desorption temperature is in 68 oC(5 hours); Under physiological condition outside analogue body, the time of liposome stable (protein desorption rate≤5%) reaches 9 months; For building novel, stable biology and medical immunology diagnostic kit.
Accompanying drawing 9 is that bovine serum albumin (BSA) is at Zr(SPC)2 (Zr (SPC)
2) the adsorption kinetic data result of nano-carrier.Experiment condition: T=4 oC, stir speed (S.S.) 3500 rpm, mixing time 24 h, and phosphate buffered solution (pH 8.59,10mM).Ce and X represent that respectively supernatant protein residue concentration and protein adsorption quantity account for the ratio of total protein concentration.
Claims (2)
1. a preparation method for artificial liposome, is characterized in that the Zr(EPC)2 Zr (EPC) with nanometer spherical granule
2or Zr(SPC)2 Zr (SPC)
2as carrier, using bitter melon protein MAP30, Radix Polygoni Multiflori Protein G AP31, lipase CRL or bovine serum albumin BSA as parcel albumen, wherein carrier is 74.3-77.7:22.3-25.7 with the mass ratio of parcel albumen, during preparation, at Zr(EPC)2 Zr (EPC)
2or Zr(SPC)2 Zr (SPC)
2carrier material in add phosphate buffered solution, in temperature, it is 4 ℃, rotating speed is to stir and obtain colourless, transparent dispersion liquid under the condition of 3500rpm, to wrap up protein dissolution in phosphate buffered solution, then slowly drop in dispersion liquid, under cryogenic conditions, rapid stirring is to forming stable liposome, after centrifugalize goes out protein, cyclic washing is also centrifugal, through frozen drying, obtain solid product, solid product cryopreservation or physiological saline solution are sealed up for safekeeping to be placed under cryogenic conditions and are stored, and get product.
2. the preparation method of a kind of artificial liposome according to claim 1, is characterized in that described carrier and the mass ratio of parcel albumen are 75-77:23-25.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210023252.7A CN102552145B (en) | 2012-02-02 | 2012-02-02 | Preparation method of artificial liposome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210023252.7A CN102552145B (en) | 2012-02-02 | 2012-02-02 | Preparation method of artificial liposome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102552145A CN102552145A (en) | 2012-07-11 |
| CN102552145B true CN102552145B (en) | 2014-03-19 |
Family
ID=46399616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210023252.7A Expired - Fee Related CN102552145B (en) | 2012-02-02 | 2012-02-02 | Preparation method of artificial liposome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102552145B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908629A (en) * | 2012-10-16 | 2013-02-06 | 吕梁学院 | Lipid-based drug of serum degradable carrier and application method of lipid-based drug |
| CN104083326B (en) * | 2014-07-17 | 2016-07-06 | 沈阳药科大学 | A kind of preparation method of the liposome wrapping load protein medicaments |
| CN107213452A (en) * | 2017-02-16 | 2017-09-29 | 徐州市中心医院 | A kind of preparation of liposome comprising MAP30 albumen and contain method |
| CN106924747A (en) * | 2017-03-17 | 2017-07-07 | 临沂大学 | Imitative lipoprotein structure pharmaceutical carrier of a kind of nanometer and its preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102107131A (en) * | 2011-01-12 | 2011-06-29 | 乔元彪 | Method for preparing bio-based zirconium salt nanoparticle material |
-
2012
- 2012-02-02 CN CN201210023252.7A patent/CN102552145B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN102552145A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Chitosan mediated solid lipid nanoparticles for enhanced liver delivery of zedoary turmeric oil in vivo | |
| Qu et al. | Gas-shearing synthesis of core–shell multicompartmental microparticles as cell-like system for enzymatic cascade reaction | |
| CN102552145B (en) | Preparation method of artificial liposome | |
| Xu et al. | pH-responsive Astragalus polysaccharides-loaded poly (lactic-co-glycolic acid) nanoparticles and their in vitro immunogenicity | |
| CN109432498A (en) | A kind of bone repairing support and preparation method thereof for bone tuberculosis treatment | |
| Yang et al. | Biomimetic polysaccharide-cloaked lipidic nanovesicles/microassemblies for improving the enzymatic activity and prolonging the action time for hyperuricemia treatment | |
| ES2403544B2 (en) | NANOPARTICULAR SYSTEMS ELABORATED BASED ON SORBITAN ESTERS. | |
| CN108143719B (en) | Polypeptide-carrying nanoliposome and preparation method and application thereof | |
| CN102552293B (en) | Compound vitamin submicron emulsion lyophilized powder for injection and preparation method of compound vitamin submicron emulsion lyophilized powder | |
| CN109730966A (en) | A kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system and preparation method thereof of chitosan oligosaccharide modification | |
| CN102366410B (en) | Argatroban liposome injection | |
| CN102366408B (en) | Monosialotetrahexosyl ganglioside sodium liposome injection | |
| CN102716089B (en) | Gemcitabine hydrochloride liposome injection | |
| CN105997892A (en) | Preparation method of SOD active drug carrier wrapped with novel microsphere biological material | |
| CN104826090B (en) | Silkworm chrysalis anti-oxidation peptide liposome and preparation method thereof | |
| CN107669637B (en) | Artemether liposome for injection and preparation method and application thereof | |
| CN103006546A (en) | Preparation and application of carbon nano tube-containing thermo-sensitive type gel entrapping for indissolvable drug | |
| CN101708175A (en) | Sustained release microsphere medicinal preparation using PHBHHx as carrier and method for preparing same | |
| Ye et al. | Engineered self-healing single-cavity microcapsules for pulsatile release of drug delivery | |
| CN102579347B (en) | Thymalfasin liposome preparation for injecting | |
| CN106137975A (en) | A kind of preparation of phycocyanin microballoon | |
| CN101658493B (en) | Azithromycin nanostructured lipid carrier and preparation method thereof | |
| CN102988423B (en) | Compound ossotide liposome injection | |
| CN102552149A (en) | Calcium heparin liposome preparation for injection | |
| CN106727427A (en) | A kind of bovine serum albumin(BSA) coats the preparation method of taxusol-lipid nano particle preparations |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140319 Termination date: 20160202 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |