CN102885782A

CN102885782A - Growth hormone-containing nanometer medicament microspheres and preparation method thereof

Info

Publication number: CN102885782A
Application number: CN2012103615653A
Authority: CN
Inventors: 陈英辉; 袁伟恩; 施晓红
Original assignee: Jinshan Hospital of Fudan University
Current assignee: Jinshan Hospital of Fudan University
Priority date: 2012-09-26
Filing date: 2012-09-26
Publication date: 2013-01-23
Anticipated expiration: 2032-09-26
Also published as: CN102885782B

Abstract

The invention discloses growth hormone-containing nanometer medicament microspheres. The microspheres comprise a medicament, nanoparticles, a polymer and medicinal auxiliary materials. The invention further provides a preparation method of the microspheres. The method comprises the following steps of: preparing growth hormone and medicinal auxiliary materials into a nanometer medicament; adding the nanometer medicament into a polymer-containing organic solvent mixed solution for emulsifying; adding a nanometer medicament-in-oil mixed suspension into a water mixed suspension containing nanoparticles or containing nanoparticles and a surfactant for emulsifying to obtain an oil-in-nanoparticle mixed suspension-nanometer medicament-in-oil compound emulsion; and curing the obtained compound emulsion, and centrifugally collecting microspheres, wherein the obtained microspheres comprise the medicament, nanoparticles, the polymer and the medicinal auxiliary materials. An appropriate polymer material and an appropriate microsphere preparation method are selected, the prepared microspheres have high envelop rate, and a layer of self-assembled nanoparticles on the surfaces of the microspheres has the effects of improving cell adhesion and reducing inflammation and microencapsulation caused by local excessive acid and hydrophobic materials. The method disclosed by the invention can be applied to preparation of other medicament slow release or controlled release microspheres.

Description

Contain growth hormone Nano medication microsphere and preparation method thereof

Technical field

The present invention relates to the Nano medication microsphere, specifically, is to contain growth hormone Nano medication microsphere and preparation method thereof, belongs to the pharmaceutical technology field.

Background technology

Growth hormone (Growth Hormone) is the protein of animal adenopituicyte secretion, is a kind of peptide hormone, has the cell adhesion effect and promotes growth or delaying senility function.Because activity can be destroyed in gastrointestinal tract after the growth hormone oral administration, can not play a role, so can only adopt injection or heeling-in mode.Somatotropin preparation has buffer agent and two kinds of forms of slow releasing agent at present, and buffer agent is with bicarbonate buffer dissolving growth hormone lyophilized powder, and is now with the current, needs injection every day; Slow releasing agent is to adopt the measures such as embedding, absorption, fusion that the raised growth hormone is fixed in certain carrier space, makes it have the long-acting slow-release effect.

Chinese patent literature CN200910019437.9, open day on April 7th, 2010, a kind of calcium alginate-chitosan sustained-release microsphere carrying growth hormone and application thereof are disclosed, adopt the method for embedding that growth hormone is bound by in the calcium alginate microsphere, the recycling chitosan solution forms even semi permeability thin film at microsphere surface, obtains composite gel microsphere, the growth hormone envelop rate is at 70%-96%, microspherulite diameter is at 130 μ m-260 μ m, and the chitosan of microsphere surface has physiologically active, can be degraded and absorbed.

Adopt biodegradable poly lactic coglycolic acid to be coated with growth hormone, have slow release of active agent, prolong drug biological half-life, increase the advantage such as bioavailability.Employing emulsion-the solvent evaporations such as Lu Lifang are made long-acting injection microsphere with poly lactic coglycolic acid with growth hormone, the thus obtained microsphere mean diameter is about 30 μ m, the growth hormone envelop rate is up to more than 95%, drug release time reaches 30 days (to be seen for details: Lu Lifang, the research of biodegradable growth hormone sustained-release micro-spheres, Fudan University's Master's thesis in 2004).

With S/O, the S/O/W of routine and the medicine microspheres of S/O/O method preparation, because surface hydrophobicity cause easily the side effect such as in-vivo tissue microencapsulation and inflammation, and burst drug release easily causes the toxic and side effects of medicine itself.About nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) microsphere and preparation method thereof, yet there are no report.

Summary of the invention

The objective of the invention is for deficiency of the prior art; a kind of nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) microsphere is provided; not high with somatotropin preparation envelop rate in the solution prior art; incomplete release is released situation with prominent, and hydrophobic surface can cause the shortcoming of local microencapsulation and inflammation.

Again one purpose of the present invention is that the preparation method of a kind of nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) microsphere is provided.

For achieving the above object, the technical scheme taked of the present invention is:

A kind of growth hormone Nano medication microsphere that contains, the surface self-organization of described microsphere has one deck nano-particle, the percentage by weight of growth hormone is 0.01%-40% in the microsphere, the percentage by weight of nano-particle is 0.01%-96%, the percentage by weight of polymer is 3.65%-99.98%, the percentage by weight of pharmaceutic adjuvant is 0%-30%, and the particle diameter of microsphere is 1-500 μ m.

Described nano-particle is organic nanometer granule or inorganic nanoparticles, one or more in optional self-polystyrene nano-particle, cross-linking dextran nano-particle, nano SiO 2 particle, titania nanoparticles, hydroxyapatite nanoparticle, ferroferric oxide nano granules, ferric oxide particle, gold nano grain, aluminium sesquioxide nano-particle, calcium carbonate nano granule, calcium phosphate nanoparticles, magnesium carbonate nano-particle, magnesium hydroxide nanoparticles or the silver nano-grain.

Described polymer is selected from one or more in polycaprolactone, polylactic acid, poly lactic-co-glycolic acid, polylactic acid-polyglycol, PLGA-Polyethylene Glycol or the polycaprolactone-polyethylene glycol.

Described pharmaceutic adjuvant is the injection pharmaceutic adjuvant.

The particle diameter of described microsphere is 10-100 μ m.

A kind of preparation method that contains growth hormone Nano medication microsphere may further comprise the steps:

(1) growth hormone and pharmaceutic adjuvant are prepared into Nano medication, in the described Nano medication, the percentage by weight of growth hormone is 0.1%-90%, and the percentage by weight of pharmaceutic adjuvant is 0%-20 %;

(2) Nano medication that step (1) is prepared is dispersed in the organic solvent mixed solution that weight percent concentration is the 0.5%-80% polymer according to the weight ratio of 1:1-1:10, forms uniform suspension, i.e. oil bag Nano medication suspension;

(3) the oil bag Nano medication suspension that step (2) is formed joins that to contain percentage by weight be the aqueous suspension of 1%-80% nano-particle or to contain percentage by weight be that 1%-80% nano-particle and percentage by weight are in the aqueous suspension of 0.5%-5% surfactant, carry out emulsifying, form nano-particle suspension bag oil-oil bag Nano medication emulsion;

(4) described nano-particle suspension bag oil-oil bag Nano medication emulsion is transferred to contain in the aqueous solution that percentage by weight is the 1%-10% inorganic salt and solidified 1-4 hour;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and the washing thus obtained microsphere, afterwards lyophilizing, obtaining surface self-organization has nano-particle and the inner microsphere that contains the growth hormone Nano medication.

The preparation of the Nano medication described in the step (1) may further comprise the steps:

Growth hormone and pharmaceutic adjuvant are dissolved in the water, then add the porous nano granule, stir so that growth hormone and pharmaceutic adjuvant fully are adsorbed in the porous nano granule, centrifugal removal supernatant, more fully washing, then lyophilizing forms Nano medication; Or

Growth hormone and pharmaceutic adjuvant be dissolved in form pharmaceutical aqueous solution in the water, then pharmaceutical aqueous solution is transferred in the Aqueous Solutions of Polyethylene Glycol, fully behind the mixing in refrigerator pre-freeze, afterwards lyophilizing obtains Nano medication with dichloromethane dissolving Polyethylene Glycol and the centrifugal Polyethylene Glycol of removing again.

Also be added with Polyethylene Glycol or poloxamer that percentage by weight is 0.1%-20% in the organic solvent mixed solution described in the step (2).

Polymer weight percent concentration described in the step (2) is 5%-30%, and described organic solvent is selected from one or more in dichloromethane, ethyl acetate, acetonitrile, heptane, chloroform or the acetone.

Nano-particle weight percent concentration described in the step (3) is 20%-70%, and described surfactant is selected from one or more in polyvinyl alcohol, Polyethylene Glycol, polyvinylpyrrolidone, poloxamer, polysorbate, ethyl cellulose or the tween.

Beneficial effect of the present invention is:

1, the present invention has selected suitable polymeric material and has prepared the method for microsphere, the microsphere envelop rate of preparation is up to more than 80%, and this surface self-organization has the microsphere of one deck nano-particle to have to strengthen the effect of cell adhesion, and reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

2, adopt the microsphere of the inventive method preparation, its size can be regulated and control to 500 μ m from 1 μ m according to different needs, and preparation process is free from environmental pollution.

3, the microsphere of the inventive method preparation, greatly reduce burst drug release, medicine almost completely discharges, can reach zero level discharges, the Nano medication that discharges can be absorbed by sick cell by local, thereby reduce the toxic and side effects of medicine itself, can make medicine keep high activity in whole preparation process and therapeutic process simultaneously is non-inactivation.

4, the microball preparation that adopts the inventive method to make, the smooth rounding of its microparticle surfaces, granule regularizing is without adhesion, its freeze dried powder is fine and smooth, the loose powder body of white, can not subside, adhesion, redispersibility is good, can apply in the preparation of other medicines slow release or control-release microsphere.

Description of drawings

Accompanying drawing 1 is scanning electron microscope (SEM) photo of the embodiment of the invention 1 preparation thus obtained microsphere.

Accompanying drawing 2 is release in vitro curves of the embodiment of the invention 1 preparation thus obtained microsphere.

Accompanying drawing 3 is drug action curves of the embodiment of the invention 1 preparation thus obtained microsphere.

Accompanying drawing 4 is the embodiment of the invention 1 preparation thus obtained microsphere and the in-vivo tissue compatibility situation for preparing microsphere with the S/O/W method.

The specific embodiment

Below in conjunction with specific embodiment and with reference to accompanying drawing the present invention is elaborated.

Should be appreciated that these embodiment only are used for explanation the present invention, and are not intended to limit the scope of the invention.In actual applications improvement and the adjustment made according to the present invention of those skilled in the art still belongs to protection scope of the present invention.

Need to prove; in the technical solution of the present invention; described nano-particle is organic nanometer granule or inorganic nanoparticles, specifically refers to be selected from polystyrene nanoparticles, cross-linking dextran nano-particle, nano SiO 2 particle, titania nanoparticles, hydroxyapatite nanoparticle, ferroferric oxide nano granules, ferric oxide particle, gold nano grain, aluminium sesquioxide nano-particle, calcium carbonate nano granule, calcium phosphate nanoparticles, magnesium carbonate nano-particle, magnesium hydroxide nanoparticles or the silver nano-grain etc. one or more.

Described polymer is selected from one or more in polycaprolactone (PCL), polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), polylactic acid-polyglycol (PLA-PEG), PLGA-Polyethylene Glycol (PLGA-PEG) or the polycaprolactone-polyethylene glycol (PCL-PEG).

Described pharmaceutic adjuvant is the injection pharmaceutic adjuvant, can be that little saccharide is (such as sucrose, trehalose, glucose, maltose or lactose etc.), the polyhydroxy compounds is (such as mannitol, sorbitol, glycerol, 1,2-PD, erythritol, Polyethylene Glycol, polyvinyl alcohol, poly(ethylene oxide) or polypyrrole alkane ketone etc.), polysaccharide compound is (such as glucosan, sodium alginate, chitosan, starch, cellulose or cyclodextrin etc.), amino-acid compound is (such as glycine, lysine, arginine, glutamic acid or histidine etc.) or the inorganic salts material (such as zinc salt, calcium salt, mantoquita, magnesium salt or molybdenum salt etc.) in a kind of or combination in any.

In the step (2) of preparation microsphere, also be added with the Polyethylene Glycol that percentage by weight is 0.1%-20% (PEG) or poloxamer (Poloxamer) in the described polymer organic solvent solution.

Described organic solvent is selected from one or more in dichloromethane, ethyl acetate, acetonitrile, heptane, chloroform or the acetone, and wherein one or more organic solvents in dichloromethane, ethyl acetate or the acetonitrile are as good.

In the step (3) of preparation microsphere, described surfactant is selected from one or more in polyvinyl alcohol (PVA), Polyethylene Glycol (PEG), polyvinylpyrrolidone (PVP), poloxamer (Poloxamer), polysorbate, ethyl cellulose (EC) or the tween.

In the step (2) of preparation microsphere, described dispersing mode can be selected emulsifying, vortex or ultrasonic etc., and jitter time is preferably 1-5 minute.

In the step (3) of preparation microsphere, described adding mode can be selected droppings, disposable adding, spray pattern adding or pour into etc.; Described emulsifying manner can be selected emulsifying, vortex or ultrasonic etc., and emulsification times is 0.1-5 minute.

In the step (4) of preparation microsphere, described inorganic salt can be selected from sodium chloride, potassium chloride, potassium nitrate or sodium carbonate etc.; Described branch mode can be dropping, disposable adding, spray pattern adds or pour into etc.

In the step (5) of preparation microsphere, can adopt water, ethanol or alcohol-water mixed liquor to wash during washing 3-5 time.

Embodiment 1

Be loaded with the preparation of poly lactic-co-glycolic acid (PLGA) microsphere of growth hormone Nano medication, comprise the steps:

(1) gets in the water that 10 mg growth hormone and 10 mg glucosans are dissolved into 0.4 ml and form pharmaceutical aqueous solution, then mentioned solution being transferred to 3.2 ml concentration is 5%(w/w) Polyethylene Glycol (PEG8000) aqueous solution in, abundant mixing, then-80 ℃ of refrigerator pre-freezes 12 hours, use again the freeze dryer lyophilizing, then obtain the growth hormone Nano medication with dichloromethane dissolving PEG and the centrifugal PEG of removing;

(2) be above-mentioned growth hormone Nano medication and concentration 30%(w/w) the acetonitrile solution of PLGA be that 1:8 mixes according to weight ratio, and formed uniform suspension in ultrasonic 1 minute, i.e. oil bag growth hormone Nano medication (N/O) suspension;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 40 ml concentration is 20%(w/w) the hydroxyapatite nanoparticle aqueous suspension in and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in ultrasonic 0.1 minute;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to concentration is 10%(w/w) sodium-chloride water solution in solidified 4 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and with washing with alcohol 5 times, obtain being loaded with poly lactic-co-glycolic acid (PLGA) microsphere of growth hormone Nano medication after the lyophilizing.

In the obtained microsphere, the percentage by weight of medicine is 0.17% in the present embodiment, and the percentage by weight of nano-particle is 84.45%, and the percentage by weight of polymer is 15.17%, and the percentage by weight of pharmaceutic adjuvant is 0.21%.

The poly lactic-co-glycolic acid that is loaded with growth hormone (PLGA) microsphere to the present embodiment preparation carries out morphology characterization, the release profiles test, efficacy testing reaches in vivo tissue compatible property testing, and (see for details: Li Zhiping with its drug effect and histocompatibility and with the S/O/W method, Li Yunfu, Zhang Zhen is inferior, Liu Yan, Qu Yanyan, prunus mume (sieb.) sieb.et zucc. is made the country prosperous, preparation and the factors influencing of interferon A-2b sustained-release micro-spheres, institute of Military Medical Science Institute periodical, 2007,31 (5): 451-455) microsphere of preparation compares, wherein the efficacy testing condition is: single administration, accumulated dose and matched group aqueous solution group totally 15 days accumulated dose once a day is identical.

Fig. 1 is scanning electron microscope (SEM) photo of poly lactic-co-glycolic acid (PLGA) microsphere that is loaded with the growth hormone Nano medication in the present embodiment, wherein, A is the scanning electron microscope (SEM) photograph of microsphere, B is the surperficial enlarged drawing of microsphere, as can be seen from the figure, prepared microsphere form is good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 10-120 μ m.Fig. 2 is the release in vitro curve of poly lactic-co-glycolic acid (PLGA) microsphere that is loaded with the growth hormone Nano medication in the present embodiment, as can be seen from the figure, prepared microsphere almost reaches 100% drug release rate, dashing forward, it is very little to release, almost completely discharge, substantially (each bar curve represents the microsphere of different PLGA materials preparations can to reach zero level release, different release times that can regulating medicine, wherein each PLGA is respectively 2A50/50: hydroxyacetic acid-lactic acid=1:1 and molecular weight are 20000-30000Da, 2A65/35: hydroxyacetic acid-lactic acid=0.65:0.35 and molecular weight are 20000-30000Da, 3A50/50: hydroxyacetic acid-lactic acid=1:1 and molecular weight are 30000-47000Da, 3A65/35: hydroxyacetic acid-lactic acid=0.65:0.35 and molecular weight are 30000-47000Da), its release in vitro performance meets the requirements.Growth hormone is that the 92.0%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).Fig. 3 is the drug action curve of poly lactic-co-glycolic acid (PLGA) microsphere that is loaded with the growth hormone Nano medication in the present embodiment, the microsphere group is the microsphere of present embodiment method preparation, matched group is the microsphere with the preparation of S/O/W method, blank group is not for containing the microsphere of medicine, can find out, use the prepared microsphere of present embodiment, the body weight that body weight increases the contrast group increases soon, and drug effect is better.Fig. 4 is the tissue compatible implementations of poly lactic-co-glycolic acid (PLGA) microsphere that is loaded with the growth hormone Nano medication in the present embodiment, as can be seen from the figure, the compatibility contrast group of prepared microsphere (N/O/N group) good, during treating, microencapsulation or the fibrosis of injection site do not appear in animal tissue.

Microsphere envelop rate with the preparation of present embodiment method is high, minimumly can reach 80%, dashing forward, it is very little to release, almost completely discharge, substantially can reach zero level and discharge, simultaneously owing to nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make biopharmaceutical macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.And this surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

The biopharmaceutical macromolecular drug microsphere of present embodiment method preparation can needing be used for the disease of frequent drug administration by injection, long-term treatment, especially needs the treatment of the major diseases such as anemia that the disease of topical therapeutic such as tumor, the various cause of disease cause, dwarf.

Embodiment 2

Be loaded with the preparation of polycaprolactone (PCL) microsphere of growth hormone Nano medication, comprise the steps:

(1) getting 20 mg growth hormone is dissolved in the water of 0.5 ml, then stirred 24 hours with poriferous titanium dioxide nano-particle 20 mg, so that growth hormone fully is adsorbed in the titania nanoparticles of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 0.5%(w/w) the ethyl acetate solution of PCL mix according to weight ratio 1:10 and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 5 minutes;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 50 ml and contains 10%(w/w) silver nano-grain and 1%(w/w) in the aqueous suspension of polyvinyl alcohol (PVA) surfactant, stirs and formed nano-particle suspension bag oil-oil in 5 minutes and wrap growth hormone Nano medication (N/O/N) emulsion;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 1%(w/w) potassium chloride solution in solidified 3 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with polycaprolactone (PCL) microsphere of growth hormone Nano medication after the lyophilizing.

In the microsphere of present embodiment gained, the percentage by weight of medicine is 0.47%, and the percentage by weight of nano-particle is 83%, and the percentage by weight of polymer is 16.53%, and the percentage by weight of pharmaceutic adjuvant is 0%.

Polycaprolactone (PCL) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 10-100 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 93.5%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 3

Be loaded with the preparation of polylactic acid (PLA) microsphere of growth hormone Nano medication, comprise the steps:

(1) getting 20 mg growth hormone is dissolved in the water of 0.5 ml, then stirred 24 hours with porous silicon dioxide nano granule 20 mg, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 20%(w/w) the dichloromethane solution of PLA mix according to weight ratio 1:9 and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 5 minutes;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 50 ml concentration is 10%(w/w) the silver nano-grain aqueous suspension in, stirs and formed nano-particle suspension bag oil-oil in 5 minutes and wrap growth hormone Nano medication (N/O/N) emulsion;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 5%(w/w) sodium-chloride water solution in solidified 2 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with polylactic acid (PLA) microsphere of growth hormone Nano medication after the lyophilizing.

In the prepared microsphere of present embodiment, the percentage by weight of medicine is 0.35%, and the percentage by weight of nano-particle is 96%, and the percentage by weight of polymer is 3.65%, and the percentage by weight of pharmaceutic adjuvant is 0%.

Polylactic acid (PLA) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 1-130 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 92.9%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 4

Be loaded with the preparation of polylactic acid-polyglycol (PLA-PEG) microsphere of growth hormone Nano medication, comprise the steps:

(1) gets 10 mg growth hormone and 10 mg cellulose dissolutions form pharmaceutical aqueous solution in the water of 0.4 ml, then mentioned solution being transferred to 3.2 ml concentration is 5%(w/w) Polyethylene Glycol (PEG8000) aqueous solution in, abundant mixing, then-80 ℃ of refrigerator pre-freezes 12 hours, use again the freeze dryer lyophilizing, then obtain the growth hormone Nano medication with dichloromethane dissolving PEG and the centrifugal PEG of removing;

(2) be above-mentioned growth hormone Nano medication and concentration 5%(w/w) the dichloromethane solution of PLA-PEG be that 1:5 mixes according to weight ratio, and formed uniform suspension in ultrasonic 1 minute, i.e. oil bag growth hormone Nano medication (N/O) suspension;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 40 ml and contains 20%(w/w) hydroxyapatite nanoparticle and 0.5%(w/w) in the aqueous suspension of Polyethylene Glycol (PEG) surfactant and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in ultrasonic 0.1 minute;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to concentration is 5%(w/w) sodium-chloride water solution in solidified 1 hour;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and with ethanol water washing 5 times, obtain being loaded with polylactic acid-polyglycol (PLA-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the obtained microsphere, the percentage by weight of medicine is 0.17% in the present embodiment, and the percentage by weight of nano-particle is 74.15%, and the percentage by weight of polymer is 25.47%, and the percentage by weight of pharmaceutic adjuvant is 0.21%.

Polylactic acid-polyglycol (PLA-PEG) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 2-280 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 94.7%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 5

Be loaded with the preparation of PLGA-Polyethylene Glycol (PLGA-PEG) microsphere of growth hormone Nano medication, comprise the steps:

(1) 5 mg growth hormone and 5 mg glycine are dissolved in the water of 0.2 ml and form pharmaceutical aqueous solution, then the concentration of mentioned solution being transferred to 3.2 ml is 5%(w/w) Polyethylene Glycol (PEG8000) aqueous solution in, abundant mixing, then-80 ℃ of refrigerator pre-freezes 12 hours, use again the freeze dryer lyophilizing, then obtain the growth hormone Nano medication with dichloromethane dissolving PEG and the centrifugal PEG of removing;

(2) be above-mentioned growth hormone Nano medication and concentration 80%(w/w) the acetonitrile solution of PLGA-PEG be that 1:1 mixes and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 1 minute according to weight ratio;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 4 ml concentration is 20%(w/w) the titania nanoparticles aqueous suspension in and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in ultrasonic 2 minutes;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 10%(w/w) sodium-chloride water solution in solidified 4 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 4 times, obtain being loaded with PLGA-Polyethylene Glycol (PLGA-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.51% in the present embodiment, and the percentage by weight of nano-particle is 89.89%, and the percentage by weight of polymer is 9.04%, and the percentage by weight of pharmaceutic adjuvant is 0.56%.

PLGA-Polyethylene Glycol (PLGA-PEG) microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has the layer of titanium dioxide nano-particle, and particle diameter is at 1-300 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 96.0%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with titania nanoparticles in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 6

Be loaded with the preparation of polycaprolactone-polyethylene glycol (PCL-PEG) microsphere of growth hormone Nano medication, comprise the steps:

(1) 5 mg growth hormone and 5 mg glycerol are dissolved in the water of 0.2 ml and form pharmaceutical aqueous solution, then the concentration of mentioned solution being transferred to 3.2 ml is 5%(w/w) Polyethylene Glycol (PEG8000) aqueous solution in, abundant mixing, then-80 ℃ of refrigerator pre-freezes 12 hours, use again the freeze dryer lyophilizing, then obtain the growth hormone Nano medication with dichloromethane dissolving PEG and the centrifugal PEG of removing;

(2) be above-mentioned growth hormone Nano medication and concentration 10%(w/w) the n-heptane solution of PCL-PEG be that 1:8 mixes and formed uniform suspension in ultrasonic 1 minute according to weight ratio, namely oil bag growth hormone Nano medication (N/O) suspension also contains 0.1%(w/w in the n-heptane solution) poloxamer;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 4 ml and contains 50%(w/w) titania nanoparticles and 5%(w/w) in the aqueous suspension of polyvinylpyrrolidone surfactant and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in ultrasonic 2 minutes;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 8%(w/w) sodium-chloride water solution in solidified 4 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and with washing with alcohol 4 times, obtain being loaded with polycaprolactone-polyethylene glycol (PCL-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.51% in the present embodiment, and the percentage by weight of nano-particle is 49.89 %, and the percentage by weight of polymer is 49.04 %, and the percentage by weight of pharmaceutic adjuvant is 0.56%.

Polycaprolactone-polyethylene glycol (PCL-PEG) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has the layer of titanium dioxide nano-particle, and particle diameter is at 1-370 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 95.8%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with titania nanoparticles in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 7

Be loaded with the preparation of polycaprolactone (PCL) and polylactic acid (PLA) microsphere of growth hormone Nano medication, comprise the steps:

(1) 5 mg growth hormone and 5 mg glucosans are dissolved in the water of 0.2 ml and form pharmaceutical aqueous solution, then the concentration of mentioned solution being transferred to 3.2 ml is 5%(w/w) Polyethylene Glycol (PEG8000) aqueous solution in, abundant mixing, then-80 ℃ of refrigerator pre-freezes 12 hours, use again the freeze dryer lyophilizing, then obtain the growth hormone Nano medication with dichloromethane dissolving PEG and the centrifugal PEG of removing;

(2) be above-mentioned growth hormone Nano medication and concentration 10%(w/w) PCL and 10%(w/w) chloroformic solution of PLA is that 1:9 mixes and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 1 minute according to weight ratio;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 5 ml concentration is 20%(w/w) the hydroxyapatite nanoparticle aqueous suspension in and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 1%(w/w) sodium-chloride water solution in solidified 1 hour;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with polycaprolactone (PCL) and polylactic acid (PLA) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.30% in the present embodiment, and the percentage by weight of nano-particle is 71.79%, and the percentage by weight of polymer is 27.55%, and the percentage by weight of pharmaceutic adjuvant is 0.36%.

The polycaprolactone (PCL) and polylactic acid (PLA) the microsphere form that are loaded with the growth hormone Nano medication in the present embodiment are good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 1-500 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 93.2%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 8

Be loaded with the preparation of polycaprolactone (PCL) and PLGA-Polyethylene Glycol (PLGA-PEG) microsphere of growth hormone Nano medication, comprise the steps:

(1) 5 mg growth hormone and 5 mg sorbitol are dissolved in the water of 0.2 ml and form pharmaceutical aqueous solution, then the concentration of mentioned solution being transferred to 3.2 ml is 5%(w/w) Polyethylene Glycol (PEG8000) aqueous solution in, abundant mixing, then-80 ℃ of refrigerator pre-freezes 12 hours, use again the freeze dryer lyophilizing, then obtain the growth hormone Nano medication with dichloromethane dissolving PEG and the centrifugal PEG of removing;

(2) be above-mentioned growth hormone Nano medication and concentration 20%(w/w) PCL and 60%(w/w) the acetone soln of PLGA-PEG be that 1:2 mixes and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 2 minutes according to weight ratio;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 5 ml and contains 40%(w/w) hydroxyapatite nanoparticle and 5%(w/w) in the aqueous suspension of poloxamer surfactants and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 1%(w/w) sodium-chloride water solution in solidified 2 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with polycaprolactone (PCL) and PLGA-Polyethylene Glycol (PLGA-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.36% in the present embodiment, and the percentage by weight of nano-particle is 41.79%, and the percentage by weight of polymer is 57.49%, and the percentage by weight of pharmaceutic adjuvant is 0.36%.

The polycaprolactone (PCL) and PLGA-Polyethylene Glycol (PLGA-PEG) microsphere form that are loaded with the growth hormone Nano medication in the present embodiment are good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 10-100 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 89.8%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 9

(2) be above-mentioned growth hormone Nano medication and concentration 20%(w/w) the acetonitrile solution of PCL be that 1:9 mixes and formed uniform suspension in ultrasonic 5 minutes according to weight ratio, namely oil bag growth hormone Nano medication (N/O) suspension also contains 20%(w/w in the acetonitrile solution) Polyethylene Glycol;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 1 ml concentration is 70%(w/w) the hydroxyapatite nanoparticle aqueous suspension in and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and with washing with alcohol 3 times, obtain being loaded with polycaprolactone (PCL) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.70% in the present embodiment, and the percentage by weight of nano-particle is 44.03%, and the percentage by weight of polymer is 54.72%, and the percentage by weight of pharmaceutic adjuvant is 0.55%.

Polycaprolactone (PCL) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 1-450 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 91.8%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 10

(2) be above-mentioned growth hormone Nano medication and concentration 20%(w/w) dichloromethane-ethyl acetate (1:1 of PLGA, v/v) solution is the 1:9 mixing according to weight ratio and formed uniform suspension in ultrasonic 5 minutes, namely oil bag growth hormone Nano medication (N/O) suspension also contains 0.1%(w/w in dichloromethane-ethyl acetate solution) Polyethylene Glycol;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 1 ml and contains 20%(w/w) hydroxyapatite nanoparticle and 2%(w/w) in the aqueous suspension of polysorbate surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with poly lactic-co-glycolic acid (PLGA) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 1.70% in the present embodiment, and the percentage by weight of nano-particle is 43.03%, and the percentage by weight of polymer is 54.12%, and the percentage by weight of pharmaceutic adjuvant is 1.15%.

Poly lactic-co-glycolic acid (PLGA) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 5-410 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 89.9%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 11

(1) getting 20 mg growth hormone is dissolved in the water of 0.5 ml, then stirred 24 hours with 20 mg porous silicon dioxide nano granules, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 20%(w/w) the dichloromethane solution of PLGA-PEG mix according to weight ratio 1:9 and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 5 minutes;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 50 ml concentration is 10%(w/w) the silver nano-grain aqueous suspension in and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with PLGA-Polyethylene Glycol (PLGA-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.35% in the present embodiment, and the percentage by weight of nano-particle is 96%, and the percentage by weight of polymer is 3.65%, and the percentage by weight of pharmaceutic adjuvant is 0%.

PLGA-Polyethylene Glycol (PLGA-PEG) microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 1-500 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 92.9%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 12

(1) gets in the water that 45 mg growth hormone are dissolved into 0.5 ml and form pharmaceutical aqueous solution, then stirred 24 hours with 20 mg porous silicon dioxide nano granules, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration that the dichloromethane solution of 20% PLGA-PEG mixes according to weight ratio 1:10 and formed uniform suspension in ultrasonic 5 minutes, namely oil bag growth hormone Nano medication (N/O) suspension also contains 20%(w/w in the dichloromethane solution) poloxamer;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 50 ml, to contain concentration be 10%(w/w) silver nano-grain and 5%(w/w) in the aqueous suspension of ethyl cellulose surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

In the thus obtained microsphere, the percentage by weight of medicine is 14.5% in the present embodiment, and the percentage by weight of nano-particle is 60.18%, and the percentage by weight of polymer is 25.32%, and the percentage by weight of pharmaceutic adjuvant is 0%.

PLGA-Polyethylene Glycol (PLGA-PEG) microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 1-450 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 92.6%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 13

(2) be above-mentioned growth hormone Nano medication and concentration 30%(w/w) the dichloromethane solution of PCL-PEG be that 1:9 mixes and formed uniform suspension, i.e. oil bag growth hormone Nano medication (N/O) suspension in ultrasonic 1.5 minutes according to weight ratio;

(3) oil bag growth hormone Nano medication (N/O) suspension of step (2) being added drop-wise to 1 ml concentration is 60%(w/w) hydroxyapatite nanoparticle and 1%(w/w) in the aqueous suspension of tween surfactants and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(4) the nano-particle suspension bag of step (3) gained oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 5%(w/w) potassium chloride solution in solidified 2 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 5 times, obtain being loaded with polycaprolactone-polyethylene glycol (PCL-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 1.29% in the present embodiment, and the percentage by weight of nano-particle is 51.72%, and the percentage by weight of polymer is 46.99%, and the percentage by weight of pharmaceutic adjuvant is 0%.

Polycaprolactone-polyethylene glycol (PCL-PEG) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 10-290 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 94.1%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation is given birth to.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 14

(1) 5 mg growth hormone and 10 mg glucosans are dissolved in the water of 0.2 ml and form pharmaceutical aqueous solution, then porous alchlor nano-particle 20 mg are added in the mentioned solution and stirred 24 hours, so that growth hormone and glucosan fully are adsorbed in the alchlor nano-particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) above-mentioned growth hormone Nano medication and to contain concentration be 15%(w/w) the dichloromethane solution of PCL-PEG be that 1:1 mixes and formed even suspension in ultrasonic 1.5 minutes according to weight ratio, namely oil bag growth hormone Nano medication (N/O) suspension also contains 10%(w/w in the dichloromethane solution) poloxamer;

(3) oil bag growth hormone Nano medication (N/O) suspension of step (2) being added drop-wise to 1 ml, to contain concentration be 80%(w/w) cross-linking dextran nano-particle and 0.5%(w/w) in the aqueous suspension of polyvinyl alcohol (PVA) surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(4) step (3) gained nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion being added drop-wise to 1000 ml concentration is 5%(w/w) sodium-chloride water solution in solidified 2 hours;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and with washing with alcohol 5 times, obtain being loaded with polycaprolactone-polyethylene glycol (PCL-PEG) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 6.15% in the present embodiment, and the percentage by weight of nano-particle is 61.54%, and the percentage by weight of polymer is 7.69%, and the percentage by weight of pharmaceutic adjuvant is 24.62%.

Polycaprolactone-polyethylene glycol (PCL-PEG) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck cross-linking dextran nano-particle, and particle diameter is at 15-360 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 91.8%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 15

Be loaded with the preparation of poly lactic-co-glycolic acid (PLGA) and polylactic acid (PLA) microsphere of growth hormone Nano medication, comprise the steps:

(2) be above-mentioned growth hormone Nano medication and concentration 12.5%(w/w) the dichloromethane solution of PLGA be that 1:4 mixes and stirred 2.5 minutes according to weight ratio, form uniform suspension, be 1.6 ml concentration 12.5%(w/w again) the ethyl acetate solution of PLA be added in the above-mentioned suspension, restir formed even suspension in 2 minutes, i.e. oil bag growth hormone Nano medication (N/O) suspension;

(3) oil bag growth hormone Nano medication (N/O) suspension of step (2) being added drop-wise to 2 ml, to contain concentration be 10%(w/w) hydroxyapatite nanoparticle and 2%(w/w) in the aqueous suspension of polyglycol surfactants and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in ultrasonic 0.5 minute;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and wash with water 3 times, obtain being loaded with poly lactic-co-glycolic acid (PLGA) and polylactic acid (PLA) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 1.58% in the present embodiment, and the percentage by weight of nano-particle is 60.20%, and the percentage by weight of polymer is 38.22%, and the percentage by weight of pharmaceutic adjuvant is 0%.

The polycaprolactone-polyethylene glycol (PCL-PEG) and polylactic acid (PLA) the microsphere form that are loaded with the growth hormone Nano medication in the present embodiment are good, and its surface self-organization has one deck hydroxyapatite nanoparticle, and particle diameter is at 1-350 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 93.8%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.Because nano-particle can react with hydroxyapatite nanoparticle in the acid that material degradation produces, thereby neutralizing acid, relatively stable with the interior environment that guarantees microsphere, can make macromolecular drug in whole preparation process and therapeutic process, keep high activity, i.e. non-inactivation.

Embodiment 16

(1) gets in the water that 0.5 mg growth hormone is dissolved into 0.5 ml and form pharmaceutical aqueous solution, then stirred 24 hours with 20 mg porous silicon dioxide nano granules, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 20%(w/w) the dichloromethane solution of PLGA mix according to weight ratio 1:9 and formed uniform suspension in ultrasonic 5 minutes, namely oil bag growth hormone Nano medication (N/O) suspension also contains 10%(w/w in the dichloromethane solution) Polyethylene Glycol;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 50 ml and contains 10%(w/w) silver nano-grain and 2%(w/w) in the aqueous suspension of polyvinylpyrrolidone surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

In the thus obtained microsphere, the percentage by weight of medicine is 4.17% in the present embodiment, and the percentage by weight of nano-particle is 35.71%, and the percentage by weight of polymer is 60.12%, and the percentage by weight of pharmaceutic adjuvant is 0%.

Poly lactic-co-glycolic acid (PLGA) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 12-280 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 90.9%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 17

(1) gets in the water that 0.01 mg growth hormone is dissolved into 0.1 ml and form pharmaceutical aqueous solution, then stirred 24 hours with 20 mg porous silicon dioxide nano granules, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 10%(w/w) the dichloromethane solution of PLGA mix according to weight ratio 1:10 and formed uniform suspension in ultrasonic 5 minutes, namely oil bag growth hormone Nano medication (N/O) suspension also contains 1%(w/w in the dichloromethane solution) Polyethylene Glycol;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 0.1 ml concentration is 1%(w/w) silver nano-grain and 2%(w/w) in the aqueous suspension of ethyl cellulose surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

(5) step (4) gained sample is carried out centrifugal, collect microsphere, and with washing with alcohol 3 times, obtain being loaded with poly lactic-co-glycolic acid (PLGA) microsphere of growth hormone Nano medication after the lyophilizing.

In the thus obtained microsphere, the percentage by weight of medicine is 0.01% in the present embodiment, and the percentage by weight of nano-particle is 0.01%, and the percentage by weight of polymer is 99.98%, and the percentage by weight of pharmaceutic adjuvant is 0%.

Poly lactic-co-glycolic acid (PLGA) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 10-100 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 92.7%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 18

(1) gets in the water that 4 mg growth hormone are dissolved into 0.2 ml and form pharmaceutical aqueous solution, then stirred 24 hours with 20 mg porous silicon dioxide nano granules, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, centrifugal removal supernatant, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 1%(w/w) the acetonitrile solution of PLGA mix according to weight ratio 1:1 and formed uniform suspension in ultrasonic 5 minutes, namely oil bag growth hormone Nano medication (N/O) suspension also contains 1%(w/w in the acetonitrile solution) poloxamer;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension being added drop-wise to 0.1ml concentration is 4%(w/w) silver nano-grain and 1%(w/w) polyvinyl alcohol (PVA) surfactant and 1%(w/w) in the aqueous suspension of polysorbate surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

In the thus obtained microsphere, the percentage by weight of medicine is 40% in the present embodiment, and the percentage by weight of nano-particle is 40%, and the percentage by weight of polymer is 20%, and the percentage by weight of pharmaceutic adjuvant is 0%.

Poly lactic-co-glycolic acid (PLGA) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 12-495 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 94.1%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

Embodiment 19

(1) gets in the water that 3 mg growth hormone and 3 mg Polyethylene Glycol are dissolved into 0.2 ml and form pharmaceutical aqueous solution, then stirred 24 hours with 20 mg porous silicon dioxide nano granules, so that growth hormone fully is adsorbed in the nano SiO 2 particle of porous, the centrifugal supernatant that goes, fully wash 3 times, then lyophilizing forms the growth hormone Nano medication again;

(2) be above-mentioned growth hormone Nano medication and concentration 5%(w/w) the dichloromethane solution of PLGA mix according to weight ratio 1:3 and formed uniform suspension in ultrasonic 5 minutes, namely oil bag growth hormone Nano medication (N/O) suspension also contains 5%(w/w in the dichloromethane solution) Polyethylene Glycol;

(3) step (2) gained oil bag growth hormone Nano medication (N/O) suspension is added drop-wise to 0.1 ml and contains 20%(w/w) silver nano-grain and 2%(w/w) in the aqueous suspension of polyvinyl alcohol (PVA) surfactant and stir and formed nano-particle suspension bag oil-oil bag growth hormone Nano medication (N/O/N) emulsion in 5 minutes;

In the thus obtained microsphere, the percentage by weight of medicine is 30% in the present embodiment, and the percentage by weight of nano-particle is 20%, and the percentage by weight of polymer is 20%, and the percentage by weight of pharmaceutic adjuvant is 30%.

Poly lactic-co-glycolic acid (PLGA) the microsphere form that is loaded with the growth hormone Nano medication in the present embodiment is good, and its surface self-organization has one deck silver nano-grain, and particle diameter is at 5-475 μ m.The drug release in vitro rate of microsphere almost reaches 100%, prominent releases very littlely, almost completely discharges, and substantially can reach zero level and discharge, and its release in vitro performance meets the requirements.Growth hormone is that the 92.9%(computational methods are with respect to the envelop rate of its original dosage in the microsphere: actual packet is enclosed in the envelop rate of the dose of the medicine of microsphere/input * 100%=medicine).The drug effect of prepared microsphere is better, during treating, microencapsulation or the fibrosis of injection site do not occur in animal tissue.This surface has the biopharmaceutical macromolecular drug microsphere of nano-particle, because the histocompatibility of surface hydrophilicity material is better than the hydrophobic material, has and strengthens cell adhesion, reduces inflammation that local overacidification and hydrophobic material cause and the effect of microencapsulation.

The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

Claims

1. one kind contains growth hormone Nano medication microsphere, it is characterized in that, the surface self-organization of described microsphere has one deck nano-particle, the percentage by weight of growth hormone is 0.01%-40% in the microsphere, the percentage by weight of nano-particle is 0.01%-96%, the percentage by weight of polymer is 3.65%-99.98%, and the percentage by weight of pharmaceutic adjuvant is 0%-30%, and the particle diameter of microsphere is 1-500 μ m.

2. the growth hormone Nano medication microsphere that contains according to claim 1; it is characterized in that; described nano-particle is organic nanometer granule or inorganic nanoparticles, optional self-polystyrene nano-particle; the cross-linking dextran nano-particle; nano SiO 2 particle; titania nanoparticles; hydroxyapatite nanoparticle; ferroferric oxide nano granules; ferric oxide particle; gold nano grain; the aluminium sesquioxide nano-particle; the calcium carbonate nano granule; calcium phosphate nanoparticles; the magnesium carbonate nano-particle; in magnesium hydroxide nanoparticles or the silver nano-grain one or more.

3. the growth hormone Nano medication microsphere that contains according to claim 1, it is characterized in that described polymer is selected from one or more in polycaprolactone, polylactic acid, poly lactic-co-glycolic acid, polylactic acid-polyglycol, PLGA-Polyethylene Glycol or the polycaprolactone-polyethylene glycol.

4. the growth hormone Nano medication microsphere that contains according to claim 1 is characterized in that described pharmaceutic adjuvant is the injection pharmaceutic adjuvant.

5. the growth hormone Nano medication microsphere that contains according to claim 1 is characterized in that, the particle diameter of described microsphere is 10-100 μ m.

6. a preparation method that contains growth hormone Nano medication microsphere claimed in claim 1 is characterized in that, may further comprise the steps:

7. preparation method according to claim 6 is characterized in that, the preparation of the Nano medication described in the step (1) may further comprise the steps:

8. preparation method according to claim 6 is characterized in that, also is added with Polyethylene Glycol or poloxamer that percentage by weight is 0.1%-20% in the organic solvent mixed solution described in the step (2).

9. preparation method according to claim 6, it is characterized in that, polymer weight percent concentration described in the step (2) is 5%-30%, and described organic solvent is selected from one or more in dichloromethane, ethyl acetate, acetonitrile, heptane, chloroform or the acetone.

10. preparation method according to claim 6, it is characterized in that, nano-particle weight percent concentration described in the step (3) is 20%-70%, and described surfactant is selected from one or more in polyvinyl alcohol, Polyethylene Glycol, polyvinylpyrrolidone, poloxamer, polysorbate, ethyl cellulose or the tween.