CN104146961B - A kind of film evaporation method prepares the method for carrying cell factor and SPP1 microballoon - Google Patents

Abstract

Cell factor and the preparation method of bone morphogenetic protein microspheres are carried the invention discloses a kind of, is comprised the following steps：Membrane material dispensing, injection lecithin, cholesterol, octadecylamine or vitamin E are matched by charge ratio；Microballoon film forming, the membrane material raw material of proportioning is mixed in organic solvent, be added in the eggplant shaped reaction bottle of Rotary Evaporators, film is formed with stationary temperature, vacuum and rotary speed；Protein solution to be wrapped, Cytokine protein and bone morphogenetic protein are added into cushioning liquid, obtain protein solution；Aquation, add protein solution hydrated films；High-pressure homogenization, the rough microballoon for having wrapped up albumen is subjected to high-pressure homogenization and obtains liposome solutions；Freeze-drying, after adding freeze drying protectant, liposome solutions are placed on first precooling in refrigerator, then be dried in vacuo, prepare the dried frozen aquatic products of microballoon；It is rehydrated, the microballoon lyophilized product for carrying albumen are added into water for injection, concussion hydration obtains milky white solution.

Description

A kind of film evaporation method prepares the method for carrying cell factor and SPP1 microballoon

Technical field

The invention belongs to biomedicine field, more precisely prepared the present invention relates to a kind of film evaporation method carry cell because The method of son and bone morphogenetic protein lipid microsphere.

Technical background

The lipid microsphere for carrying medicine is the development with biomaterial and material processing equipment, with reference to pharmacy and clinical doctor The discipline development such as learn to get up, recent two decades develop and are applied.Lipid microsphere can be by drug powder or solution bag Be embedded in a diameter of nano level particulate, this particulate can have class eucaryotic cell structure, into human body in mainly by reticular endothelium system System phagocytosis and activate the autoimmune function of body, and change be encapsulated medicine inside be distributed, make medicine mainly liver, spleen, Put aside in the histoorgan such as lung and marrow, so as to improve the therapeutic index of medicine, reduce the therapeutic dose of medicine and reduce medicine Toxicity.Simultaneously drug bearing microsphere be applied in biomaterial, can by the biomaterial of bioengineered tissue further with human body group Fusion is knitted, the application of organizational engineering can be promoted significantly.

Lipid microsphere can wrap up fat-soluble and water-soluble two types medicine or albumen, and it is that have determining for multiple functions The pharmaceutical carrier of tropism.It has following features：1. preparation technology is simple, general medicine is all easier to be encapsulated in lipid microsphere In；2. water-soluble and fat-soluble two types medicine can be all wrapped in same lipid microsphere, the envelop rate of medicine is mainly and medicine Thing lipid in itself and membrane material property are relevant；3. lipid microsphere is small to human toxicity in itself, and lipid microsphere pair Human body is without immunosuppressive action；Essential characteristic when 4. natural targeting is lipid microsphere intravenously administrable.This is due to that lipid is micro- Ball enters caused by the natural tendency just swallowed in vivo by macrophage as external foreign matter, is treatment liver parasite, Li Shiman The preferable pharmaceutical carrier of the reticuloendothelial cell systemic disease such as disease, and the ideal carrier of immunoactivator；Exempt from 5. can be made Epidemic disease lipid microsphere, lipid microsphere is transported in specific tissue according to target molecule characteristic, with antibody or receptor acting release medicine 6. medicine is wrapped in lipid microsphere thing is non-Covalent bonding together, and some pharmaceutical carriers are to be combined with covalent bond with medicine into people It is not easy to depart from carrier by the medicine of Covalent bonding together after in vivo, influences drug effect, and enter in human body can for the medicine in lipid microsphere Discharged completely in appointed part；7. during lipid microsphere intravenously administrable, leave the chance very little that blood vessel enters people's cell interstitial；Through In subcutaneous or intraperitoneal injection the main Jin Ren regional nodeses of lipid microsphere, only the less lipid microsphere of particle diameter is just from devices such as livers The sinusoid of the discontinuous blood vessel of official enters cytoplasm.The lipid microsphere entered in human body is mainly by the macrophage of reticuloendothelial system Monocyte phagocytosis in cell and blood.8. medicine is encapsulated in lipid microsphere, drug toxicity can be reduced, enhancing pharmacology is made With.9. lipid microsphere preparation can reduce the supersession rate of medicine, extend drug treating time, increase outer stable inside medicine Property.

The main bottleneck of bone tissue engineer is that still there is a big difference with natural physiological bone for the tissue engineered bone to be formed.May The main reason for be the neuralization for being not carried out tissue engineered bone.Growth factor plays a very important role in bon e formation process, this Mainly by stimulating differentiation of the mescenchymal stem cell to sclerous tissues direction.In the past few years, a variety of growth factors have Osteanagenesis potentiality, including fibroblast growth factor, platelet derived growth factor, IGF, conversion growth because Son, vascular endothelial growth factor, and BMP (BMP).

Cell factor is that a kind of activating cell by hemopoietic system, immune system or inflammatory reaction produces, and can be adjusted thin Born of the same parents' differentiation and proliferation and inducing cell play function, multi-functional polypeptide, protein or the glycoprotein with high activity.More than 10 kinds Cell factor is produced and clinical practice by research by technique for gene engineering.

Nerve growth factor (Nerve Growth Factor, NGF), belongs to one of member of neurotrophins. NGF is a kind of small molecule class secretory protein, and the growth to certain form of neuronal cell, is maintained and survival has important work With.Growth factor of human nerve (human nerve growth factor, abbreviation hNGF) is a kind of thin to normal neuronal in human body The nutritious effect of born of the same parents, repairing function to injured nerve has the bioactie agent of adjustment effect, and it can maintain sympathetic nerve and sense Feel the existence of nerve, promote the differentiation of nerve cell, determine the direction of extension of aixs cylinder.Development to promoting brain, nervous system Growth, the regeneration of injured nerve and functional rehabilitation have decisive role.Especially NGF following biology performance is such as to embryo The importance of the survival of tire puberty neuron, the growth for promoting postnatal development neuron and the survival for maintaining mature neuron Effect, can make it be applied in biological bone tissue engineer.Promote biological bone and human body intervention after for bone tissue around The development and influence of nerve.

Quasi-insulin growthing factor I GF-I, it is with the development of molecular biology technology, people in 1978 have purified two kinds The NSILA (I, II) of form simultaneously has found that its structure is similar to proinsulin, is respectively designated as insulin-like growth factor-Ⅰ, II (IGF I, II) is to emphasize the homology of they and insulin structure." sulphation factor " is confirmed simultaneously and " propagation, which stimulates, lives Property " with IGF be same polypeptide family member.IGFs is different from other growth factors, in extracellular fluid, cell culture All combine with specific associated proteins (Binding Proteins, BPs) in liquid, exist with inactive composite form. IGFs biological function is not limited solely to mitosis stimulation, and they can also induce differentiation or promote the table of differentiation function Reach.Its accurate biological effect depends on the presence of the state and other hormones or growth factor of cell development.Especially exist Different tissues, different growth and development stages, IGF- I and IGF- II effect and level have quite poor different.IGF- I is depended on GH, the various kinds of cell of in vitro culture can be promoted to breed, promote protein and DNA synthesis.The many histocytes of body can divide certainly Secrete and paracrine IGF- I.And IGF- II is referred to as antenatal essential growth factors, growth hormone regulation is not required to, at a variety of groups Knit in organ and express.In addition, research recently shows：GH not growths institute in itself is directly required, all to be described as by GH Caused height growth is essentially all to be completed by IGF- I.IGFs research is the focus in current cell biological field, increasingly It is taken seriously.It will likely turn into the important breakthrough that the mankind explore life secret.IGF starts to individual growth to send out with human embryos Educating has substantial connection.But effects of the IGF to many system organizations is also only experiment in vitro and results of animal, therefore is also had Many relevant IGFs work need to be furtherd investigate further.

Interferon (fibroblast interferon- β, IFN-β) β has antiviral and immunoregulation effect, can block The synthesis of IFN-γ, suppress the release of the cell factor of other infringement oligodendroglias, reduce the hyperplasia of T cell, increase The activity of strong rejection capability T cell, while the expression of the ajor histocompatibility compound protein II with albuminous cell surface is prevented, Clinically it is usually used in treating multiple sclerosis (multiple sclerosis, MS) and the nervous system disease.MS be it is a kind of compared with For common autoimmune disease, central nervous system white matter is attacked by immune-mediated specificity, causes nervous centralis System white matter demyelinate.In all treatment MS medicine, IFN-β interferon portion has exceeded 70%, and IFN-β not only may be used To reduce the acute episode frequency of MS patient, moreover it is possible to the effectively course advancement of delay MS patient.We have found the albumen for the first time There is the speed for being obviously promoted biological bone tissue and cell fusion in organizational project bone material culture is applied to, if parcel arrives Original biological bone material degradation excessive velocities or excessively slow can be further overcome the shortcomings of in lipid microsphere, for organizational project It is significant.

Bone morphogenetic protein (bone morphogentetic protein, BMP) is also known as bone growth factor, Neng Gouzeng Add marker enzyme (alkaline phosphatase) and the isogenic expression of osteocalcin of bone cell differentiation, promote formation and the layer of new bone tissue Secondaryization, the growth for bone play an important role.U.S. FDA in 2002 have approved OP-1 (i.e. BMP-7) and be used for bone defect Clinical treatment.At present both at home and abroad for BMP classes material especially BMP-2 and BMNP-7 for bone inductive effect clinical medical The evaluation of affirmative is all given in effect.But its be applied to bone tissue application when the shortcomings that be that albumen is easily diluted and tissue melts Close, make its bioavilability very low.Suitable carrier is needed so that so that BMP slowly discharges, Chinese patent CN101816634B is public A kind of microballoon prepared with spray technique of cloth, but still have in parcel during due to spraying that protein loss is more, simultaneously spray The protein encapsulation rate of microballoon caused by mist is not easy measure (independent of the measure of encapsulation ratio and release rate in embodiment), microballoon Controlled release degree it is unstable the problems such as.

The content of the invention

It is an object of the invention to provide a kind of load cell factor and the preparation method of bone morphogenetic protein microspheres, including with Lower step：

(1) membrane material dispensing, injection lecithin, cholesterol, octadecylamine or vitamin E are matched by charge ratio；

(2) microballoon film forming, the membrane material raw material of proportioning is mixed in organic solvent, the eggplant shape for being added to Rotary Evaporators is anti- Answer in bottle, film is formed with stationary temperature, vacuum and rotary speed；

(3) protein solution to be wrapped is prepared, Cytokine protein and bone morphogenetic protein are added into cushioning liquid

(4) aquation, the film that the hydration step of cushioning liquid containing albumen 2 prepared obtains is added in step 3, through concussion, water Bath ultrasound obtains the rough microballoon for having wrapped up albumen；

(5) high-pressure homogenization, the rough microballoon for having wrapped up albumen is subjected to high-pressure homogenization, the liposome for obtaining parcel albumen is micro- Ball emulsion；

(6) it is freeze-dried, freeze drying protectant is added to the cushioning liquid prepared to step 5, is then sub-packed in cillin bottle, Cillin bottle is placed on first precooling in refrigerator, then is dried in vacuo, prepares the dried frozen aquatic products of microballoon；

(7) it is rehydrated, the microballoon lyophilized product for carrying albumen are added into water for injection, concussion hydration obtains milky white solution；

(8) envelop rate and protein content of lyophilized lipid microsphere albumen are detected, milky white solution plus water are subjected to aquation, used Gel column, lipid microsphere and non-encapsulated albumen are separated, and the protein content in the second peak is determined by Lowry methods.

Further, a kind of preparation method for carrying cell factor and bone morphogenetic protein microspheres of the present invention include with Lower step：

(1) membrane material dispensing, injection lecithin, cholesterol and octadecylamine (or vitamin E) are pressed into charge ratio 3~7:1~ 3:1 weighs, and is then dissolved in final concentration of 0.5~1.5% dichloromethane or chloroform；

(2) microballoon film forming, the membrane material of proportioning in Rotary Evaporators with the 40-60 DEG C of rotation of 60rpm speed controls temperature, directly Lipid membrane is formed on to bottle wall；

(3) protein solution to be wrapped is prepared, Cytokine protein and bone morphogenetic protein are added into cushioning liquid；Its Described in Cytokine protein be selected from NFG, insulin-like growth factor and recombinant human interferon alpha 2 IFN β 1a In one or more, the bone morphogenetic protein is one or more in BMP-2, BMP-7 and BMP-1；

(4) aquation, the protein liquid aquation lipid membrane for adding step (3) to prepare, makes the content of membrane material in 0.5%-1.0% models In enclosing, add small bead and acutely shake, after film is completely fallen off from bottle wall, water bath sonicator about 20~40min, liquid in bottle Body is in light cloud, obtains rough microballoon；

(5) high-pressure homogenization, with high pressure homogenizer it is homogeneous in 15Kpa~30Kpa pressure limits in pressure by rough microballoon To 200~400 nanometers of particle diameter, lipid microsphere solution is obtained；

(6) it is freeze-dried, trehalose (final concentration of 1%-3%) is added to the solution prepared to step 5, is placed on after packing First precooling in refrigerator, then the dried frozen aquatic products that vacuum drying obtains microballoon is carried out, it is divided in 1~2m1/ bottles in cillin bottle；

(7) it is rehydrated, add 1.0ml-2.0ml waters for injection to carry out aquation lyophilized microglobulin, concussion hydration obtains Milky white solution；

(8) envelop rate and protein content of lyophilized lipid microsphere albumen are detected, milky white solution plus water are subjected to aquation, used Sephdex G-100 gel columns, lipid microsphere and non-encapsulated albumen are separated using PBS as eluent, and surveyed by Lowry methods The envelop rate of protein content and calculating albumen in fixed second peak.

The lipid microsphere albumen that the present invention is obtained adds 1~2m1 PBS or physiology before tissue bone material is applied to Salt solution, which is shaken to light cloud, can add or wrap up in biological bone material.

Further, the final concentration of protein of NFG solution is 50- in step described in the inventive method (4) 100ng/ml；Quasi-insulin growthing factor I GF-I final concentration of protein is 120-200ng/ml；Recombinant human interferon alpha 2 IFN β 1a's Final concentration of protein is 60-120ng/ml；BMP-2 final concentration of protein is 50-100ng/ml；BMP-7 final concentration of protein is 50- 100ng/ml；BMP-1 final concentration of protein is 50-100ng/ml.Wherein, the albumen of preferably NFG solution is whole Concentration is 80ng/ml；Quasi-insulin growthing factor I GF-I final concentration of protein is 150ng/ml；Recombinant human interferon alpha 2 IFN β 1a Final concentration of protein be 100ng/ml；BMP-2 final concentration of protein is 100ng/ml；BMP-7 final concentration of protein is 100ng/ ml；BMP-1 final concentration of protein is 100ng/ml.

Further, precooling temperature is -50 DEG C in step described in the inventive method (6)；Vacuum drying pressure is 20Pa, First paragraph temperature control is dried at -50 DEG C~-60 DEG C, 4~5 hours duration；Second segment temperature control is held at 28~32 DEG C The continuous 42 hours time.

Further, the amount of the rehydrated addition water for injection in step described in the inventive method (7) and lyophilized microballoon The ratio of amount is 1:1-1:5.

Further, the gel of the gel column in step described in the inventive method (8) also includes being selected from Sephadex G- 50, Sephadex G-75, SepharoseCL-2B and SepharoseCL-6B.

The preparation method of lipid microsphere of the present invention simply and stably, by controlling Rotary Evaporators and high pressure homogenizer Parameter, make individually or various combination cell factor and bone morphogenetic protein by parcel enter lipid microsphere in, system Standby microsphere volume is uniform, and reaches nanoscale.The load proteolipid microballoon of preparation can slowly discharge albumen, beneficial in biology The metabolic balance of new bone tissue formation and biological bone is preferably carried out during the intervention of bone tissue, lipid microsphere greatly improves parcel The bioavilability of albumen, and commercial application can be carried out.

Brief description of the drawings

Fig. 1 is SepharoseCL-2b, SepharoseCL-6b chromatography figures.

Fig. 2 is the grain size distribution determined after prepared by 20130402 batches of lipid microspheres.

Fig. 3 is the grain size distribution determined after prepared by 20130501 batches of lipid microspheres.

Fig. 4 is three batches of lipid microspheres (NGF albumen and BMP-7 albumen) average grain diameter study on the stability.

Fig. 5：Three batches of lipid microsphere (NGF albumen and BMP-7 albumen) span study on the stability.

Fig. 6：Three batches of lipid microsphere (NGF albumen and BMP-7 albumen) lipid microsphere film oxidation index study on the stability.

Fig. 7：Three batches of lipid microsphere (NGF albumen and BMP-7 albumen) envelop rate stability.

Embodiment

Below will be so that the present invention is described in detail in conjunction with the embodiments, it is impossible to be not understood as limitation of the present invention.It is real Apply material used in example, reagent, instrument unless otherwise specified, be common commercially available.

【Embodiment 1】The optimization of membrane material composition

Lipid microsphere membrane material optimum formula is determined using interactive orthotropic test design is not considered, selectes lipid The optimal proportion formula of three kinds of compositions such as injection lecithin, cholesterol, octadecylamine among microballoon membrane material.Its preparation method is Using optimization come film evaporation method, uniformly crushed using super-high-pressure homogenization instrument, then carry out lipid microsphere correlation again Granularmetric analysis, envelop rate, the index such as stability, set according to each single index and beaten from difference to outstanding 5 ranks, according to rank Divide (1~5,5 score values), optimum formula is finally drawn according to variance analysis.

(1) selected results of optimal membrane material are shown in Table 1

Table 1：Influence of three kinds of different phosphatide to lipid microsphere

From the point of view of experiment process, make lipid microsphere in the phosphatide produced with sigma, be particularly easy to occur in preparation process The change of color, in addition during high-pressure homogeneous, the triumphant phosphatide of ring is particularly easy to crush, and its solution is easier into translucent Homogeneous solution.Study on the stability behind is interim, the gradual yellow of lipid microsphere color prepared by sigma phosphatide.We are comprehensive Result above is closed, we use material by the phosphatide optimal as us of selected injection phosphotide.

(2) optimum results of membrane material combination

Using Orthogonal Method experimental result, we have obtained the comparatively ideal ratio of three kinds of Main Ingredients and Appearances in membrane material, these three The mol ratio of composition is (3~7：1~3：1) it is last we determined that a kind of membrane material formula by variance analysis within the scope of (OP-A) lipid microsphere, prepared with the formula quite stable under solution state, after 4 DEG C are stored 8 months, is visually observed without bright Significant difference is different.Its particle diameter distribution does not also change significantly.From the point of view of this data, the lipid microsphere membrane material formula after optimization, system There is good quality stability for the lipid microsphere gone out, even this explanation is in a liquid state, 4 DEG C preserve, and this batch of lipid is micro- The grain size stability of ball at least at 8 months, is traced it to its cause, it should be membrane material institute band electric charge and solution in protein The electric charge of institute's band, after integration so that lipid microsphere particle carries certain electrostatic charge, this electrostatic interaction so that lipid is micro- Ball keeps good stability.

【Embodiment 2】The selection of solvent

It is compared using influence of four kinds of different solvents to the lipid microsphere after preparation, the results are shown in Table 2：

Table 2：The comparison (0 month) of influence of four kinds of different solvents to the lipid microsphere after preparation

Note：★ oxidation index values are smaller, and the oxidized degree of phosphatide is smaller

^★★Span value is smaller, particle diameter convergence normal distribution, and lipid microsphere particle is more homogeneous

Influence of four kinds of different solvents to the lipid microsphere grain size stability after preparation is shown in Table 3：

Table 3：Influence of four kinds of different solvents to the lipid microsphere grain size stability after preparation

The solvent of membrane material is in preparation process, influences lipid microsphere form and a key factor of envelop rate, meanwhile, it is molten The temperature that the species of agent and related physicochemical property (such as boiling point size, and to membrane material fusing speed) are had influence in preparation process Degree, also determine lipid microsphere production technology can large-scale mass production the problem of；Residual of four kinds of solvents in lipid microsphere All meet corresponding limit value regulation, table 2, table 3 are measurement result of four kinds of solvents to each index of lipid microsphere after preparation.

Solvent is extremely crucial in the preparation process of lipid microsphere, but the selection of solvent is to a certain extent by membrane material Property pins down, in addition the selection of solvent to consider the various phosphatide of membrane material or auxiliary ingredients solvent solubility, must also examine Consider solvent and obtain boiling point size, this not only has influence on the phase transition temperature of membrane material, also has influence on the energy consumption and residual for preparing lipid microsphere The removal of organic solvent, meanwhile, temperature height, also cause oxidation Decomposition speed of the phospholipid molecule in evaporation process, therefore, oxygen Change the index balance index larger as one in selected solvent species.In the present embodiment, prepared due to solvent combination A Lipid microsphere, it is preferable in lipid microsphere form, oxidation index, grain size stability, it is comprehensive although the index of envelop rate is not optimal Close and consider to still fall within optimal sample, and the less stable of other several lipid microspheres, especially grain size stability.

【Embodiment 3】The preparation of lipid microsphere

It is as follows that film evaporation method prepares lipid microsphere step：1. film forming：Membrane material dispensing：By injection lecithin, cholesterol Charge ratio 3~7 is pressed with octadecylamine (or vitamin E):1~3:1 weighs, and is then dissolved in final concentration of 0.5~1.5% dichloro In methane or chloroform, the liquid is placed in the eggplant type bottle of Rotary Evaporators, 60 DEG C of water-baths make as evaporating temperature, rotation Solvent evaporates, and final stage dries up solvent with nitrogen, makes liposome film forming in eggplant type bottle.2. aquation：Add cell factor weight Group people's IFN β 1a (final concentration of 100ng/ml) and bone morphogenic protein BMP-2-2 (final concentration of 80ng/ml) aqueous phase solution About 50ml (membrane material is 1% in the concentration of solution), adds several small bead, acutely concussion, treats that film is completely de- from bottle wall Fall behind, water bath sonicator about 30min, liquid is in light cloud in bottle.3. high-pressure homogenization：Rough lipid microspheres high pressure homogenizer is existed Pressure is in 15Kpa~30Kpa pressure limits, and by condensed water, the temperature control that refiner is exported is at 30 DEG C~37 DEG C, Particle diameter of the matter to 200~400nm.

Freeze-drying (FDM), which is combined, prepares lipid microsphere, then increases following steps on the basis of the above：4. freezing is dry It is dry:By the liposomal mixtures after homogenate, concussion, adding appropriate trehalose makes wherein final concentration of 1.5%, and liquid is being pressed By the leaf filter that aperture is 0.45um under power, aseptic process is carried out, while the pore size control of liposome can also be existed It below 450nm, can also further make the particle diameter of liposome more homogeneous, take the sample after filtering aseptically to divide Dress, 2m1/ bottles, is divided in cillin bottle；5. freezing, dry.Lipid microsphere albumen freezes sample in use, certain body can be added Long-pending PBS or physiological saline (2m1), shake to light cloud.

【Embodiment 4】The separation of lipid microsphere and floating preteins

A) centrifuge test result：

In the present embodiment, with reference to pertinent literature, we centrifuge the lipid microsphere of parcel albumen using following methods And floating preteins, it the results are shown in Table 4.

Table 4：Centrifuge test result

It is 40,000g when we use centrifugal force, centrifuge obtains rotating speed and has arrived at 22,000rpm, but does not reach Say that free medicine and lipid microsphere are separated to us, if again lifted speed, this is unfavorable for the technique and obtained on a large scale Production, because energy consumption is too big, therefore we have given up this separation method.

B) column chromatography for separation method：

Because centrifugal process can not be kept completely separate the lipid microsphere and free drug of packaging medicine, therefore we select gel layer Analysis method separates, it is contemplated that cell factor (NFG NGF；Recombinant human interferon alpha 2 IFN β 1a；Insulin-like growth Factor IGF-I) and bone morphogenic protein BMP-2-2；BMP-7；BMP-1 molecular weight ranges are 13-20kDa, are selected Two kinds of gels of SepharoseCL-2B, SepharoseCL-6B, it separates tomographic map and sees Fig. 1.

From the point of view of separating spectrum, first peak is lipid microsphere peak, is inferred according to flowing elution speed, lipid microsphere peak Substantially just eluted from a void volume, if pertinent literature is also considered as lipid microsphere particle with molecular weight to weigh, all More than 300KD again, this conclusion is almost without big biased；SepharoseCL-6b can no doubt be separated, but just for 1.6 × 14cm obtains splitter, time-consuming oversize, therefore in view of separative efficiency and the balance of separating effect, is coagulated using SepharoseCL-2B Glue is as optimal separating gel, and for this method when being amplified to 2.6 × 20, separating effect is almost unchanged.

【Embodiment 5】The granularmetric analysis of lipid microsphere

Malvern Mastersizer2000 laser diffraction particle size analyzers to be produced with Britain micro- to obtained lipid at room temperature The particle diameter of ball is analyzed, determines its particle diameter distribution and average grain diameter.The automatically derived lipid microsphere of Particle Size Analyzer software kit Grain size distribution and the correlation values such as average grain diameter.Measurement result shows that particle size is changed into 306nm from 291nm, span from 1.134 are changed into 1.085 (see Fig. 2,3), and the mainly particle diameter distribution that diminishes of span levels off to normal distribution, plus maximum particle diameter and The distance between minimum grain size does not change, so numerical value diminishes, it may also be appreciated that the lipid microsphere film used in immunization experiment Material is lipid microsphere prepared by the formula.

【Embodiment 6】The morphological observation of lipid microsphere

Because the lipid microsphere particle diameter in this research is all between 100~1000um, therefore, common microscope can not be made For the instrument of the morphologic observation of lipid microsphere, transmission scanning electron microscope mainly is used in our current research, is seen using negative staining The form of lipid microsphere is examined, in research process we have found that the form of lipid microsphere is with the pH value and dyeing liquor of dyeing liquor Concentration, Color change.

Electron microscopic observation：Take the solution of lipid microsphere appropriate, with the PBS for preparing its aqueous phase solution same concentrations and ionic strength Buffer solution dilutes 10 times, drips to and is placed on the copper mesh on filter paper, and the 1% of a drop same pH is added dropwise according to the pH of PBS Phosphotungstic acid, low-temperature heat drying, remove the copper mesh, with transmission electron microscope observing and take pictures.

【Embodiment 7】The measure of lipid microsphere envelop rate

Determine protein content Content Method (Lowry methods)

The lyophilized lipid microsphere albumen for taking the above method to prepare, add 1.0ml water to carry out aquation, coagulated with sephdex G-100 Glue post (1.6 × 20.0cm), lipid microsphere and non-encapsulated albumen are separated by the use of PBS as eluent, by Lowry methods [measure the Protein content in two peaks.Then calculated by following equation:

A) reagent preparation

1. 4% sodium carbonate liquor weighs sodium carbonate 4g, add appropriate purified water to dissolve and be diluted to 100ml.

2. 0.8% sodium hydroxide solution weighs sodium hydroxide 0.8g, add appropriate purified water to dissolve and be diluted to 100ml.

3. 0.04mol/L copper-baths weigh copper sulphate 1.0g, add appropriate purified water to dissolve and be diluted to 100ml.

4. 0.1mol/L potassium tartrate solution weighs potassium tartrate 2.35g, add appropriate purified water to dissolve and be diluted to 100ml。

5. alkaline copper solution：Take before use reagent 1., 2., 3., 4. with 50：50：1：1 ratio mixed preparing forms.

6. phenol reagent (1N)

7. standard protein：Standard protein solution takes a human serum albumins standard items, with containing 0.02%NaN₃Note Penetrate and 50ml volumetric flasks are all moved into after being dissolved with water 1ml, and use 0.02%NaN₃Water for injection be accurately settled to 50ml, This solution is as standard protein stock solution.Accurate measuring standard protein stock solution in right amount in 25ml volumetric flasks, with containing 0.02%NaN₃Water be diluted to scale, every 1ml is contained 100 μ g standard proteins, as standard protein solution.

B) making of standard curve：

Accurate measuring standard protein solution (100ug/ml) 0.0,0.2,0.4,0.6,0.8,1.0ml are respectively placed in test tube In, 1ml is added water to, adds alkaline copper solution 5ml, shakes up, room temperature is placed 10 minutes, is rapidly joined phenol reagent (1N) 0.5ml, is shaken Even, room temperature is placed 30 minutes, and after colour developing, according to UV-VIS spectrophotometry, absorbance (colour developing is determined at wavelength 650nm Afterwards, it is as muddy in found, after per minute 3000 leave the heart 15 minutes, take supernatant to determine).With the protein concentration pair of standard curve Its absorbance is answered, seeks linear regression equation.

C) measure of sample：

Second peak solution is not diluted, prepare lipid microsphere by the use of albumen aqueous phase as testing sample, sample is diluted to About 50 μ g/ml or so, precision measure test sample dilution 1ml and put in test tube, add alkaline copper solution 5ml, shake up, and room temperature places 10 Minute, phenol reagent 0.5ml is rapidly joined, is shaken up, room temperature is placed 30 minutes, after colour developing, according to UV-VIS spectrophotometry, Absorbance is determined at wavelength 650nm (after colour developing, such as to find muddiness, after per minute 3000 leave the heart 15 minutes, take supernatant to survey It is fixed).

D) calculate:

Software is operated using the computer interconnection of Shimadzu spectrophotometer, standard protein solution is determined into A one by one₆₅₀, automatically Standard curve is obtained, then testing sample is determined into corresponding A₆₅₀, the concentration of the automatically derived testing sample of spectrophotometer software, The related extension rate of sample and the sample concentration drawn are substituted into the formula of envelop rate, draw envelop rate.

The selection of the assay method of envelop rate

By using the result of the method measure envelop rate of lowry methods measure protein content, the result of two methods measure There is certain difference, be shown in Table 5；By 1 in table 5^#、2^#、3^#Three batches of samples are determined three times respectively with two methods, and it the results are shown in Table 6。

The envelop rate result of lipid microsphere prepared by table 5, distinct methods (data source is also shown in Table 7)

6, three batches of samples of table determine envelop rate result three times respectively

From the point of view of the result of above-mentioned two table, the active method for measuring that interferon is determined by HIV suppression method draws bag Data of the data of envelope rate generally than protein content measure envelop rate are low, and this species diversity has significant difference, to find out its cause, this Be because be related in preparation process rotary evaporation, ultrasonication, homogenizing method, during freeze-drying etc., lead The inactivation of Partial Protein is caused, and the method for determining the protein quantity can not then determine the activity change situation of albumen, therefore we are most After weigh the pros and cons, the method for finally selecting protein active determines envelop rate, compared with the feelings that can truly reflect in preparation process Condition changes.

【Embodiment 8】Lipid microsphere study on the stability

Lipid microsphere is prepared using film evaporation method, takes 300mg injections lecithin, 150mg cholesterol, 50mg vitamins E mixing is dissolved in 100ml chloroforms, and the liquid is placed in the eggplant type bottle of Rotary Evaporators, and 60 DEG C of water-baths are as evaporation temperature Degree, rotating speed are set to 50rpm, and rotation evaporates solvent, and final stage dries up solvent with nitrogen, make liposome in eggplant type bottle into Film.Then add nerve growth factor (final concentration of 100ng/ml) and bone morphogenic protein BMP-2-7 is (final concentration of The about 50ml of aqueous phase solution 80ng/ml), several small bead is added, acutely concussion, after film is completely fallen off from bottle wall, Water bath sonicator about 30min, liquid is in light cloud in bottle.By rough lipid microspheres with high pressure homogenizer pressure be 15Kpa~ In 30Kpa pressure limits, by condensed water, by the temperature control of refiner outlet at 30 DEG C~37 DEG C, homogeneous to 200~ 400nm particle diameter.By the liposomal mixtures after homogenate, concussion, adding appropriate trehalose makes wherein final concentration of 1.5%, Liquid is passed under pressure through into the leaf filter that aperture is 0.45um, carries out aseptic process, while also can be by the hole of liposome Footpath is controlled in below 450nm, can also further make the particle diameter of liposome more homogeneous, takes the sample after filtering in sterile bar Dispense, 2m1/ bottles, be divided in cillin bottle under part；Freezing, dry.Lipid microsphere albumen freezes sample in use, one can be added Determine the PBS or physiological saline (2m1) of volume, shake to light cloud.

Three batches of lyophilized lipid microspheres of trial-production are placed in 4-8 DEG C by us.Every month carries out a study on the stability, mainly Investigate following index：Mode of appearance, granularmetric analysis, envelop rate, phospholipid oxidation index, investigate 11 months altogether, three lot samples The detection of product outward appearance all without significant change, is injected after water for injection, is dissolved into homogeneous solution rapidly, more homogeneous after vibration, quiet Only it is visible by naked eyes sedimentary within 30 minutes.Three batches of proteolipid microballoons prepared by lyophilized aquation method, its average grain diameter stability See that Fig. 4, span change are shown in Fig. 5, the oxidation index change of film is shown in that Fig. 6, envelop rate change are shown in Fig. 7.

【Embodiment 9】The release rate detection of lipid microsphere albumen

The detection of main supplement interferon

Take and prepare the microballoon lyophilized product 1 containing cell factor IFN β 1a, whole experiment process is sterile working, is used After the sterile PBS dissolvings of 1ml, it is placed in bag filter (bag filter interception is 30,000 Da, does aseptic process), bag filter is put in 9ml In the centrifuge tube of PBS (pH 7.4) solution, it is put into the shaking table that rotating speed is 60rpm, 37 degrees Celsius of constant temperature, was taken after the tenth day PBS solution outside 1ml bag filters, carry out interferon activity detection.The microballoon lyophilized product conduct after crushing after dissolving is taken simultaneously Control comparisons product.

Test sample Activity determination, when the WISH cell is grown to 80% degree of converging in complete medium, discard culture Liquid, PBS use trypsin digestion cell after washing, and every 1ml is configured to containing 2.5 × 10 with complete culture solution⁵~3.5 × 10⁵Individual cell it is thin Born of the same parents' suspension, it is inoculated in 96 porocyte culture plates, per the μ l of hole 100.Cultivated 4~6 hours under 37 DEG C, 5% carbon dioxide conditions. It will prepare in the culture plate of the interferon beta 1a standard solutions completed and sample solution access containing WISH cell, added per hole 100μl.Cultivated 18~24 hours under 37 DEG C, 5% carbon dioxide conditions.Discard nutrient solution.By the vesicular stomatitis disease of preservation Poison (VSV, -70 DEG C preservation) is diluted to about 100CCID50 with attacking malicious nutrient solution, per the μ l of hole 100.In 37 DEG C, 5% carbon dioxide Culture 24 hours.Then the supernatant in Tissue Culture Plate is discarded, the μ l of violet staining liquid 50 are added per hole, room temperature places 30 points Zhong Hou, dyeing liquor is carefully washed away with flowing water, and blot residual moisture, the μ l of destainer 100 are added per hole, room temperature places 3~5 points Clock.After mixing, with ELIASA using 630nm as reference wavelength, absorbance is determined at wavelength 570nm, measurement result is calculated And compared with control comparisons product, it is shown in Table 7.

7. 3 batches of samples of table determine release rate result three times respectively

Claims (1)

1. a kind of prepare the method for carrying cell factor and bone morphogenetic protein microspheres, comprise the following steps：

(1) membrane material dispensing, injection lecithin, cholesterol, octadecylamine or vitamin E are pressed 3~7:1~3:1 charge ratio is entered Row proportioning；

(2) microballoon film forming, the membrane material raw material of proportioning is mixed in organic solvent, is added to the eggplant shaped reaction of Rotary Evaporators In bottle, film is formed with stationary temperature, vacuum and rotary speed, the organic solvent is dichloromethane or chloroform；

(3) protein solution to be wrapped is prepared, Cytokine protein and bone morphogenetic protein are added into cushioning liquid；Its In, the Cytokine protein is final concentration of 100ng/ml recombinant human interferon alpha 2s IFN β 1a, and the bone morphogenetic protein is Final concentration of 80ng/ml BMP-2；

(4) aquation, step is added（3）The hydration step of cushioning liquid containing albumen of middle preparation（2）The lipid membrane of acquisition, makes film The content of material obtains the rough microballoon for having wrapped up albumen through concussion, water bath sonicator in the range of 0.5% ~ 1.0%；

(5) high-pressure homogenization, the rough microballoon for having wrapped up albumen is subjected to high-pressure homogenization, obtains the liposome microballoon of parcel albumen Emulsion；

Wherein, it is in 15Kpa~30Kpa pressure limits in pressure with high pressure homogenizer by rough microballoon, will be even by condensed water The temperature control of pulp grinder outlet is at 30 DEG C~37 DEG C, the particle diameter of homogeneous to 200~400nm；

(6) it is freeze-dried, by step（5）The emulsion of preparation is placed on first precooling in refrigerator, then is dried in vacuo, and prepares micro- The dried frozen aquatic products of ball；

Wherein the liposomal mixtures after homogenate are shaken, 1%~3% trehalose is added, cillin bottle is divided in 1~2m1/ bottles In, the cillin bottle equipped with solution is placed on first precooling in refrigerator, then carry out the dried frozen aquatic products that vacuum drying obtains microballoon；

(7) it is rehydrated, the microballoon lyophilized product for carrying albumen are added into water for injection, concussion hydration obtains milky white solution；

(8) envelop rate and protein content of lyophilized microglobulin are detected, milky white solution plus water are subjected to aquation, used SepharoseCL-2B gel columns, microballoon and non-encapsulated albumen are separated, and determine the protein in the second peak by Lowry methods and contain Amount.