KR20100117948A - Porous microsphere for pulmonary administration and manufacturing method thereof - Google Patents

Abstract

PURPOSE: A porous microsphere for pulmonary administration and a method for manufacturing the same are provided to enable controlled release of physiologically active agent. CONSTITUTION: A porous microsphere for pulmonary administration contains micropores on the surface and inside of microsphere body of biodegradable polymer. The coefficient of variation of the microsphere and micropore is under 0.2. The diameter of the microsphere body and micropore is 1-300 um and 80 nm-1.5 um, respectively. The physiologically active agent is drug, gene, vaccine, peptide or mixture thereof.

Description

POROUS MICROSPHERE FOR PULMONARY ADMINISTRATION AND MANUFACTURING METHOD THEREOF

The present invention relates to a porous microsphere for pulmonary administration capable of sustained controlled release of a physiologically active agent and effectively deposited on alveoli and a method for preparing the same.

In general, drugs administered by various routes in vivo are unstable by different enzymes and macrophages, requiring excessive dosages and frequent frequency of administration. Therefore, a drug delivery system (DDS) for maintaining a therapeutic concentration of drugs in vivo has recently been an issue. It can deliver the drug to the treatment site stably and effectively to maintain the effective blood concentration for a long time to maximize the therapeutic effect and minimize the side effects of the drug.

As such, various drug delivery systems have been developed to solve the problems of efficacy and stability of drugs administered in vivo. For example, liposomes using biomaterials, sustained release drug carriers using hydrogels, irritant drug carriers using pH or temperature sensitive polymers, drug carriers for topical administration using fine particles, preparations for injection or inhalation using nanoparticles, Prodrugs in which drugs are bound to water-soluble polymers, and drug delivery systems using nano / microspheres using biodegradable polymers have been attempted.

The drug delivery system is classified into transdermal delivery, oral delivery, mucosal delivery, implant delivery, parenteral delivery and pulmonary delivery depending on the delivery method. In addition, various methods are being developed.

In particular, drug delivery through the lung is attracting attention due to various advantages. First of all, 180 cm ² Nasal surface area Alternatively, the lungs of an adult have a surface area of about 100 m ² , which is advantageous for drug absorption. Alveolar epithelium thickness and good vascularization of about 0.1-0.5 μm also aid rapid drug absorption. In addition, drug delivery through the lungs can prevent the first pass effect that occurs during oral administration, and lungs have the advantage of relatively low metabolic enzyme activity.

Pulmonary delivery systems have the advantages described above for ease of local and systemic administration, but have the disadvantage of being difficult to efficiently deposit drugs into the alveoli.

For example, an aerosol system for topical administration for the treatment of asthma is to prepare a drug in several micro sized formulations and deliver it to the alveoli, but requires frequent dosing due to enzyme instability and short drug duration. . In addition, corticosteroid-based drugs are widely used as inhalable formulations for the treatment of asthma, but the short half-life in vivo has limitations that make it difficult to maintain therapeutic blood levels.

In addition, biodegradable nanospheres having a size of several hundreds of nanometers show relatively high drug stability and sustained release behavior, but are prone to phagocytosis by macrophages, which is disadvantageous in terms of cytotoxicity.

On the other hand, microsphere (microsphere) has been used in radiodiagnostic drugs in the past, but recently attracted attention as a drug carrier. Such microspheres generally refer to a particulate system of 1 to 2000 μm in which the drug is dispersed in a dissolved or microcrystalline state in the polymer matrix.

Such microspheres have been developed for various purposes such as delivery of drugs, proteins, cells, delayed release of drugs, delivery to target sites, improvement of physical properties including fluidity (S. Freiberg, XX Zhu, Int. J. Pharm . 282, 1, 2004 ).

It has been found that porous microspheres can deliver drugs to the lungs more effectively than nonporous microspheres (DA Edwards et. Al., Science 276: 1868-1871, 1997 ). Microspheres with a porous structure have a smaller aerodynamic diameter compared to the geometric diameter (MA Matthew et. Al ., Journal of controlled release 121: 100-109, 2007 ), and thus drugs through the lungs. In terms of delivery, it can be effectively used to minimize phagocytosis by macrophages and to maximize the efficiency of deposition into good alveoli.

The method used for preparing such porous microspheres is typically a gas formation method using ammonium bicarbonate. However, there are disadvantages in that it is difficult to control the diameter and the size of the pores of the microspheres, and it is difficult to manufacture the microspheres having a uniform size of the body and the pores.

And, microspheres can be prepared by a double emulsification method, which is a kind of emulsification solvent evaporation. The double emulsification process involves two emulsification stages, and the porous structure can be introduced by changing the stability of the emulsion of the first emulsification stage. As an example, Yuki Kanai et al. Proposed a method of forming pores in chitosan microspheres by inducing osmotic pressure by adding NaCl to the aqueous phase in preparing chitosan microspheres through O / W / O type double emulsification. At this time, the diameter of the prepared microspheres was 143 ㎛, the pore size was 2 to 6 ㎛. However, the diameter of the porous microspheres prepared by the method can not achieve a stable alveolar deposition effect, the diameter of the body and pores is uneven, the size of the pores is large, there is a problem that the drug loading efficiency is low.

Therefore, it is possible to avoid enzymatic degradation and phagocytosis of macrophages, increase the proper diameter and drug loading efficiency to obtain stable and effective alveolar deposition effect, suppress initial excessive release, and size suitable for sustained release drug delivery. There is a need for development of a method for producing porous microspheres having pores and porous microspheres having uniform sized bodies and pores.

Therefore, the present inventors have made diligent efforts to improve the problems of the prior art, the diameter and pore size suitable for pulmonary administration when the osmotic pressure inducing agent is added to the inner aqueous phase when manufacturing the microspheres using the W / O / W type double emulsification method It was confirmed that the porous microspheres having the present invention was completed.

SUMMARY OF THE INVENTION An object of the present invention is a porous microsphere for pulmonary administration having appropriately sized diameter, high drug loading rate, initial excessive release that can achieve excellent alveolar deposition effect, and pores of size suitable for sustained release drug delivery. To provide.

Another object of the present invention is to provide a method for producing a porous microsphere that can easily produce a porous microsphere having a uniform size of the body and micropores by easily controlling the diameter and the pore size of the porous microsphere using an osmotic phenomenon. To provide.

In order to achieve the above object,

In a porous microsphere having micropores formed on the surface and inside of a body made of a biodegradable polymer, a bioactive agent is loaded and provides a porous microsphere for lung administration having a uniform size of the body and the micropore diameter.

Also,

S1) dispersing the internal aqueous phase in which the osmotic pressure agent is dissolved in water to prepare a W / O emulsion by dispersing the oil phase in which the biodegradable polymer is dissolved in the organic solvent, wherein the biologically active agent is added to the internal aqueous phase or the oil phase;

S2) dispersing the W / O type emulsion in an external water phase in which an emulsifier is dissolved in water to prepare a W / O / W type emulsion; And

S3) removing the organic solvent from the W / O / W type emulsion to obtain a porous microsphere

It provides a method for producing a porous microsphere comprising a.

Porous microspheres according to the present invention can avoid the enzymatic degradation and phagocytosis of macrophages, as well as have a diameter of the size that can obtain an excellent alveolar deposition effect, can suppress high drug loading rate, initial excessive release As well as pores of a size suitable for sustained release drug delivery. In addition, it is possible to easily control the diameter and the size of the pores of the porous microsphere through the manufacturing method according to the present invention, it is possible to manufacture a porous microsphere having a uniform body and pore size. Therefore, the porous microsphere according to the present invention can be usefully used as a drug delivery system for pulmonary administration.

Porous microspheres referred to throughout this specification refers to spherical solid particles having a diameter of several to several thousand micrometers in which micropores are formed on and in the surface of the spherical body.

The present invention is described in more detail below.

Porous microspheres for pulmonary administration of the present invention is characterized in that the body and the micropore diameter have a uniform size. Preferably the diameter of the body is 1 to 300 ㎛, the diameter of the micropores is 80 nm to 1.5 ㎛.

Here, the uniform diameter of the body and the micropores means that the distribution is narrow, and when expressed as a ratio of coefficient of variation, that is, the ratio of the standard deviation and the average, the value is 0.2 or less. Preferably the coefficient of variation is 0.20 or less for the body diameter and 0.20 or less for the micropore diameter.

At this time, the material of the microspheres is administered to the body and then decomposed by hydrolysis or enzyme, so as to include a biodegradable polymer having a characteristic that is absorbed or safely released into the body.

Such biodegradable polymers include collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, polyglycolic acid, poly (lactic acid-co-glycolic acid), poly-ε- (caprolactone), polyvalerolactone, Polyhydroxy butyrate, polyhydroxy valerate, polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly (ethylene oxide) -poly ( Propylene oxide) -poly (ethylene oxide) copolymers, copolymers thereof, mixtures thereof, and the like. Preferably polylactic acid, polyglycolic acid, poly (lactic acid-co-glycolic acid), poly-ε- (caprolactone), polyvalerolactone, polyhydroxy butyrate, polyhydroxy valerate, copolymers thereof And one polyester-based polymer selected from the group consisting of a mixture thereof.

The microspheres of the present invention preferably have a diameter of 1 to 300 µm, more preferably 1 to 20 µm, and preferably a diameter of micropores in consideration of deposition into the alveoli, loading rate and release behavior of the bioactive agent. Preferably it is 80 nm-1.5 micrometers, More preferably, it is 80 nm-1 micrometer.

At this time, if the size of the microsphere diameter is less than the above range, there is a risk of loss by the mucus ciliated or alveolar macrophages in the bronchus, on the contrary, if the range exceeds the above range, a stable alveolar deposition effect cannot be obtained. In addition, if the pore size is less than the above range, the porosity is lowered, so that it is difficult to be delivered to the depth of the lung. On the contrary, if the pore size is exceeded, the drug loading efficiency is lowered and the sustained release of the drug is difficult to be exhibited.

Since the porous microspheres can provide a large surface area and space for cell growth, they can be utilized as a cell support, and can be absorbed or adsorbed by capillary phenomena exhibited by micropores. Drugs can be loaded into the drug delivery system. In particular, the diameter and pore size of the porous microspheres of the present invention are suitable for pulmonary delivery for systemic and topical administration.

According to the alveolar deposition effect confirming experiments of Experimental Examples 3 to 4 of the present invention, the porous microspheres according to the present invention were generally deposited in the lungs, and quantitatively about 20 to 40% more than the non-porous microspheres. It was confirmed that the alveolar deposition efficiency was more than 60% by being deposited in the lungs. It can be seen that the diameter and pore size of the porous microspheres according to the present invention increases the porosity of the porous microspheres, and prevents loss by mucus ciliates or alveolar macrophages, thereby maximizing lung deposition effects. .

The physiologically active agent refers to a substance that produces a pharmacological or therapeutic effect by changing the action of cells, viruses, tissues, organs or organisms, drugs, genes, vaccines, prepared by all known chemical synthesis methods, Proteins, peptides, and the like are possible, but are not limited to these.

For example, drugs include corticosteroids, narcotic analgesics, non-narcotic analgesics, antipsychotics, antidepressants, anti-anxiety drugs, analgesics, antimicrobials, sedatives, anticonvulsants, Parkin's disease drugs, cholinergic drugs, adrenergic drugs, antihypertensive drugs, vascular drugs Dilators, peripheral vasodilators, local anesthetics, antiarrhythmics, cardiovascular agents, antiallergic drugs, antiulcers, prostaglandin homologues, antibiotics, antifungal agents, antibiotic drugs, repellents, antiviral drugs, anticancer drugs, hormone related drugs, Diabetes treatment, arteriosclerosis treatment, and diuretics. Preferably a corticosteroid.

The corticosteroid is budesonide, aldosterone, beclomethasone, betamethasone, ciclesonide, clopredol, cortisone, cortibazole, deoxycortone, desonide, desoxymethasone, dexamethasone, difluorocorkone Tolon, Flucloron, Flumethone, Flunisolide, Fluorcinolone, Fluorinolide, Fluorocortin Butyl, Fluorocortisone, Fluorocortolone, Fluorometholone, Flulandrenolone, Fluticasone, Halcy Nolnide, hydrocortisone, Icometasone, meprednisone, methylprednisolone, mometasone, paramethasone, prednisolone, prednisone, lopleponide, thixocortol, triamcinolone, beclomethasone dipropionate, beclomethasone Monopropionate, Dexamethasone 21-isonicotinate, Fluticasone Propionate, Icometason Enbutate, Thixocortol 21-P Rate, triamcinolone acetonide, and can be a mixture thereof.

The protein may be vascular endothelial growth factor (VEGF), growth hormone (GH), interferon (IFN), insulin, insulin-like growth factor (IGF), leukocyte proliferation factor. (granulocyte colony stimulation factor (G-CSF), erythropoietin (EPO), interleukin (IL), fibroblast growth factor (follicle stimulating hormone (FSH)), nerve growth factor (nerve growth factor) , NGF), octreotide, calcitonin, tumor necrosis factor (TNF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) ), Bone morphogenetic protein (BMP), tissue plasminogen activator (TPA), tissue growth factor (TGF), granulocyte macrophage colony stimulation factor (GMCSF), platelet formation Factors (TPO), mixtures thereof, and the like.

Porous microspheres according to the present invention are loaded with 48.8 to 86.6% bioactive agent. Porous microspheres of the present invention can reduce the loss of the bioactive agent in the manufacturing process compared to the conventional porous microspheres small pore size. This makes it possible to load a lot of bioactive agents into the porous microspheres.

At this time, the physiologically active agent is preferably coupled to the osmotic pressure inducing agent and electrostatic attraction. The osmotic agent is added during the manufacturing process of the porous microspheres to form micropores in the body and the surface of the microspheres, and some are present in the microspheres. Here, the osmotic pressure-inducing agent is a charged material, and when the oppositely charged bioactive material is loaded, they bind to each other by electrostatic attraction. In this case, not only the loading rate of the bioactive substance may be increased by the electrostatic attraction but also the initial excessive release may be suppressed and the sustained release drug release behavior may be induced.

Referring to Experiment 2 of the present invention, after preparing a porous microsphere using albumin as an osmotic agent and loading the angiogenesis factor (VEGF), a positive potential drug as a bioactive agent, and confirmed the drug release behavior, albumin And angiogenesis factors were coupled to each other by electrostatic attraction, it was confirmed that the initial release of the angiogenesis factor through the pores of the porous microsphere.

Such a porous microsphere of the present invention can be produced by the W / O / W type double emulsion method. Particularly, in the present invention, the porous microspheres are manufactured by the W / O / W type double emulsification method, and the micropores are formed on the body surface and inside of the microspheres by adding an osmotic agent to the inner aqueous phase. .

Hereinafter, a method of manufacturing a porous microsphere according to the present invention will be described in detail for each step.

S1) W / O type emulsion manufacturing step

First, an internal aqueous phase in which the osmotic agent is dissolved in water and an oil phase in which the biodegradable polymer is dissolved in an organic solvent are prepared. Thereafter, the internal aqueous phase is dispersed in an oil phase to prepare a W / O type emulsion. At this time, the internal water phases aggregate together in the oil phase to form droplets, thereby obtaining a W / O emulsion.

The emulsification method in preparing the emulsion is well known to those skilled in the art. For example, the emulsion may be prepared using an ultrasonic wave crusher, a homomixer, a stirrer, or the like.

When preparing a W / O emulsion, the volume ratio of the internal aqueous phase to the oil phase is 1: 2 to 1:10, preferably 1: 5. If the volume ratio of the internal aqueous phase to the oil phase is less than the above range, phase transition occurs under the same stirring conditions, so that it is difficult to expect to form a stable W / O emulsion. This is because the content of is so small that the yield and productivity decrease.

The osmotic agent may be any one as long as it can generate osmotic pressure in the inner aqueous phase. Representatively, albumin, casein, globulin, prolamin, gelatin and mixtures thereof may be used. Albumin may be negatively charged to form an electrostatic attraction with a bioactive agent and may also act as an emulsifier.

The osmotic agent is added at 5-30% (w / v) in the water phase. If the content of the osmotic agent is less than the above range can not form a pore of the appropriate size, on the contrary, if the amount exceeds the above range there is a problem that the size of the pore is too large to damage the spherical shape of the microspheres.

The organic solvent is not particularly limited in the present invention as long as it can dissolve the biodegradable polymer uniformly. Preferred organic solvents may be those which are hydrophobic and highly volatile that do not mix with the aqueous phase. For example, methylene chloride, chloroform, dimethylformamide, ethyl acetate, acetone, acetonitrile, tetrahydrofuran, dimethyl sulfoxide and mixed solvents thereof are possible.

At this time, the biodegradable polymer is added in 5 to 10% (w / v) in the oil phase. If the content of the biodegradable polymer is less than the above range it is not possible to obtain a microsphere having a diameter of the appropriate size, on the contrary, if it exceeds the above range there is a problem that the diameter of the microsphere is excessively increased.

In particular, the porous manufacturing method of the present invention can easily control the diameter and pore size of the finally obtained porous microsphere by adjusting the concentration of the osmotic agent and the biodegradable polymer.

According to Experimental Example 1 of the present invention, as a result of preparing a porous microsphere by fixing the concentration of the biodegradable polymer and changing the concentration of the osmotic agent, the average pore diameter of the porous microsphere increased as the concentration of the osmotic agent increased. The average diameter of the body was not significantly affected. On the contrary, the porous microspheres were prepared by fixing the concentration of the osmotic agent and changing the concentration of the biodegradable polymer. As the concentration of the biodegradable polymer increased, the average diameter of the porous microsphere increased, but the average diameter of the pores There was no significant change.

Meanwhile, when preparing a W / O type emulsion, various bioactive agents may be added to the internal aqueous phase or the oil phase, which may be loaded into a porous microsphere and expect a particular effect. If the bioactive agent is hydrophilic, it is dissolved in the inner water phase, and if hydrophobic, the oil phase, and finally a porous microsphere loaded with the bioactive agent is obtained.

S2) W / O / W emulsion preparation step

Subsequently, the W / O type emulsion is redispersed again in the external water phase to obtain W / O / W type. At this time, the inner aqueous phase is surrounded by an oil phase and agglomerated with each other to form droplets, thereby obtaining a W / O / W type emulsion.

The emulsification method in preparing the emulsion is as described in the W / O emulsion preparation step.

At this time, an osmotic pressure difference is generated between the inner aqueous phase and the outer aqueous phase by the osmotic agent, and water is introduced into the inner aqueous phase from the outer aqueous phase due to this difference. As a result, micropores are formed in the body and the surface of the finally obtained porous microspheres.

When preparing the W / O / W emulsion, the volume ratio of the W / O emulsion to the external water phase is 1: 2 to 1: 5, preferably 1: 5. If the volume ratio is less than the above range, it is difficult to expect a stable W / O / W type emulsion due to phase transition. On the contrary, if the volume ratio exceeds the above range, the content of the bioactive agent in the finally obtained W / O / W type emulsion is too high. It is difficult to expect high yield and productivity.

As the external aqueous phase, an aqueous solution containing a surfactant may be used. For example, an aqueous solution of polyvinyl alcohol, methyl cellulose, cetyltrimethyl ammonium bromide, sodium dodecyl sulfate or polyoxyethylene sorbitan monooleate is possible, and preferably an aqueous polyvinyl alcohol solution can be used. Here, the concentration of the aqueous solution may be 0.1 to 5% (w / v), preferably 0.4 to 4% (w / v).

S3) Obtaining Porous Microspheres

Thereafter, the biodegradable polymer is cured through evaporation and removal of the organic solvent in the oil phase to obtain a solidified porous microsphere. The porous microspheres can be worked up by other commonly known methods. For example, methods such as centrifugation, filtration, washing, drying and the like can be performed.

Porous microspheres for pulmonary administration obtained by the production method of the present invention has a diameter of 1 to 300 ㎛, preferably 1 to 20 ㎛, micropore diameter of 80 nm to 1.5 ㎛, preferably 80 nm to 1 μm. In addition, when expressed as a coefficient of variation of the body and pore diameter, that is, the ratio between the standard deviation and the mean, the value is 0.2 or less.

As described above, the diameter of the porous microsphere body and the pores obtained through the manufacturing method of the present invention can be easily controlled, and a porous microsphere having a uniform size of the body and the pore size can be obtained. As a result, additional operations such as classification are unnecessary, so that the yield and productivity are improved.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1 Preparation of PLGA Porous Microspheres

Porous microspheres were prepared by applying a solvent evaporation / extraction method based on water-in-oil-in-water (W / O / W) double emulsification.

Specifically, 0.3 g of bovine serum albumin (hereinafter referred to as 'BSA', Ameresco) was dissolved in deionized water to prepare 1 ml of an internal aqueous phase, and poly (lactic acid-co-glycolic acid) (hereinafter referred to as 'PLGA'). , RESOMER  0.5 g of RG 504H, Boehringer Ingelheim) was dissolved in methylene chloride (Wako Pure Chemical Industries) to prepare 5 ml of an oil phase. The internal and oil phases were emulsified at 50 W output for 10 seconds in an ice bath using a probe type sonicator (Branson Disital Sonifier).

The prepared W / O emulsion was poured into 25 ml of an aqueous aqueous 4% (w / v) polyvinyl alcohol (molecular weight 27,000-32,000, Sigma-Aldrich) solution to prepare a W / O / W type double emulsion. Homogenizer (Ultra-Turrax) T25 basic, IKA -Werke) was emulsified again at 6,000 rpm for 5 minutes.

The obtained double emulsion was poured into 300 ml of 0.4% (w / v) polyvinyl alcohol and thoroughly mixed at 600 rpm for 3 hours using a mechanical stirrer to evaporate / extract the organic solvent. The cured microspheres were centrifuged and washed five times with deionized water to remove polyvinyl alcohol. The washed microspheres were lyophilized for 3 days and then stored in a desiccator.

Examples 2-7 7: Preparation of PLGA Porous Microspheres

Examples 2 to 7 were prepared in the same manner as in Example 1, except for changing PLGA concentration and BSA concentration. The conditions according to each example are shown in Table 1.

division PLGA (%. W / v) BSA (%. W / v) Example 2 10 10 Example 3 10 20 Example 4 10 30 Example 5 5 30 Example 6 8 30 Example 7 10 30

Example 8 Preparation of Porous Microspheres Loaded with Budesonide

A porous microsphere loaded with budesonide was prepared in the same manner as in Example 1 except that 0.04 times the weight of PLGA budesonide was dissolved in methylene chloride to prepare an oil phase.

Examples 9 to 11: Preparation of porous microspheres loaded with angiogenesis factors

VEGF-loaded porous microspheres of Examples 9 to 11 were prepared in the same manner as in Examples 2 to 4, except that 1 µg of a vascular endothelial growth factor (hereinafter, referred to as 'VEGF') was dissolved in the inner aqueous phase. It was.

Examples 12-14: Preparation of Porous Microspheres Loaded with DiI

Examples 12 to 14 in the same manner as in Examples 5 to 8 except for using DiI (1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindo carbocynnine perchlorate, Invitrogen) instead of budesonide Porous microspheres loaded with DiI were prepared.

Comparative Example 1 Preparation of Nonporous Microspheres Loaded with Budesonide

Non-porous microspheres were prepared in the same manner as in Example 8, except that BSA was not used in the internal aqueous phase.

Observation of the prepared microspheres, it was confirmed that the diameter is 9.9 ± 2.5 ㎛.

Comparative Example 2: Preparation of Porous Microspheres Using Ammonium Bicarbonate

VEGF-loaded porous microspheres were prepared in the same manner as in Example 9, except that ammonium bicarbonate was used instead of BSA and 5% (w / v) citric acid was added to the outer aqueous phase. It was.

Comparative Example 3: Preparation of Nonporous Microspheres Loaded with Angiogenesis Factors

VEGF-loaded non-porous microspheres were prepared in the same manner as in Example 9, except that BSA was not used for the internal aqueous phase.

As a result of observing the prepared microspheres, it was confirmed that the diameter was 10.6 ± 2.1 μm.

Comparative Example 4: Preparation of Nonporous Microspheres Loaded with DiI

A non-porous microsphere was prepared in the same manner as in Example 8 except that BSA was not used for the internal water phase and DiI was used instead of budesonide.

Observation of the prepared microspheres, it was confirmed that the diameter is 11.9 ± 1.7 ㎛.

Experimental Example 1: Observation of Porous Microspheres

1-1. Scanning electron microscopy

The shape of the porous microspheres prepared in Examples 1 to 7 was examined using a scanning electron microscope (S-4800 UHR FE-SEM, Hitachi). A suspension of microspheres in deionized water was fixed on an aluminum holder at room temperature, dried overnight and then coated with platinum under vacuum. After freezing the slices of microspheres by cryostat, the shape of the cross section was observed.

1 is a photograph observing the elevation (A) and the cross-section (B) of the porous microsphere prepared in Example 1 with a scanning electron microscope.

2A to 2C are scanning electron microscope images of the porous microspheres prepared in Examples 2 to 4 of the present invention (scale bar = 10 μm).

3A to 3C are scanning electron micrographs of the porous microspheres prepared in Examples 5 to 7 of the present invention (scale bar = 5 μm).

1 to 3, it can be seen that micropores are formed on the body surface and the inside of the microspheres.

1-2. Diameter, pore size measurement

Isoton the porous microsphere suspension prepared in Examples 1 to 11 and Comparative Example 2 Diluted with II dilution, the average size of the porous microspheres was measured using a particle counting and size analyzer (Z2 Coulter, Beckman). Porous microsphere images were analyzed via Image Pro software to determine the average pore diameter.

The average diameter and pore size data of the measured microspheres are shown in Table 2 below and FIGS. 4 and 5. All data are presented as [mean ± standard deviation] and statistical analysis was performed by Student's t-test. ^* A value of P <0.05 was considered statistically significant.

division Body diameter (㎛) Pore diameter (㎛) Body variation coefficient Pore Variation Coefficient Example 1 13.9 ± 2.0 0.54 ± 0.11 0.14 0.20 Example 2 11.4 ± 1.4 0.18 ± 0.03 0.12 0.16 Example 3 11.3 ± 1.2 0.24 ± 0.03 0.11 0.13 Example 4 13.9 ± 1.9 0.83 ± 0.11 0.13 0.13 Example 5 2.9 ± 0.3 0.67 ± 0.10 0.10 0.15 Example 6 4.0 ± 0.8 0.59 ± 0.09 0.20 0.15 Example 7 13.9 ± 1.9 0.53 ± 0.11 0.13 0.20 Example 8 9.6 ± 1.8 0.67 ± 0.12 0.18 0.17 Example 9 2.9 ± 0.5 0.15 ± 0.02 0.17 0.13 Example 10 6.1 ± 1.1 0.22 ± 0.03 0.18 0.13 Example 11 12.9 ± 1.5 0.59 ± 0.11 0.11 0.19 Example 12 2.9 ± 0.2 0.79 ± 0.12 0.06 0.15 Example 13 6.5 ± 1.2 0.82 ± 0.11 0.18 0.13 Example 14 11.0 ± 1.8 0.81 ± 0.13 0.16 0.16 Comparative Example 2 13.3 ± 5.8 1.4 ± 0.7 0.43 0.50

Figure 4 is a graph showing the diameter (-◆-) and the size of the pores (-■-) of the porous microspheres prepared in Examples 2 to 4 of the present invention according to the BSA concentration.

5 is a graph showing the diameter (-◆-) and the size of the pores (-■-) of the porous microspheres prepared in Examples 5 to 7 of the present invention according to the PLGA concentration.

Referring to Table 2 and Figure 4, as a result of manufacturing a porous microsphere by varying the concentration of the osmotic pressure BSA in the inner aqueous phase, the average diameter of the pores of the PLGA microsphere increased as the concentration of BSA, the average diameter Was not significantly affected.

In addition, referring to Table 2 and Figure 5, as a result of producing a porous microsphere by varying the concentration of PLGA, a biodegradable polymer in the oil phase, as the concentration of the PLGA solution increases, the average of the porous microsphere without significant pore size change The diameter increased.

Through these results, it was confirmed that the control of the average diameter and the pore size is easy when the porous microsphere is prepared using the osmotic pressure inducing agent in the inner aqueous phase. In addition, the porous microsphere of Comparative Example 2 using ammonium bicarbonate has a coefficient of variation of body and pore of 0.43 and 0.50, respectively, and shows a wide size distribution, whereas the porous microsphere of the present invention has a diameter distribution coefficient of body and pore. It can be seen that the uniformity is very excellent because the variation of the size is small to 0.20 or less.

Experimental Example 2: Confirmation of sustained release drug release

2-1. Confirmation of Budesonide Emission Behavior in Vitro

In order to measure the content of encapsulated budesonide in the microspheres prepared in Example 8 and Comparative Example 1, the porous microspheres were dissolved in 1N NaOH solution and then neutralized with 1N HCl solution. The resulting solution was filtered through a 0.2 filter (Millipore, USA) and the budesonide content was measured using High Performance Liquid Chromatography (HPLC). The loading rate is expressed as the ratio between the actual budesonide content of the microspheres and the amount initially added to the microspheres and the results obtained are shown in Table 3.

On the other hand, in order to investigate the release behavior of the budesonide from the prepared microspheres, the porous microspheres of Example 8 and the non-porous microspheres of Comparative Example 1 were placed in a conical tube, Dulbecco phosphate buffer Saline (Dulbecco's PBS; DPBS) was added (200 rpm, 37, 5% C0 ₂ environment). At precomputed time intervals, the supernatant was removed and replaced with fresh Dulbecco's phosphate buffered saline. In the same manner, the amount of budesonide released through high performance liquid chromatography was measured, and the results obtained are shown in FIG. 6.

division Budesonide loading rate (%) Comparative Example 1 65.0 ± 7.2 Example 8 56.0 ± 7.2

Figure 6 is a graph showing the cumulative amount of budesonide discharge with time of Example 8 (-■-) and Comparative Example 1 (-●-) microsphere of the present invention.

Referring to Table 3 and FIG. 6, the loading rate of budesonide in the microspheres due to porosity was not significantly affected, but it was observed that the initial release of budesonide through the pores was increased.

2-2. Confirmation of In Vitro Endothelial Growth Factor (VEGF) Release Behavior

In vitro release was tested in the same manner as 2-1 to confirm the loading efficiency and release behavior of the VEGF-loaded microspheres of Examples 9 to 11 and Comparative Examples 2 to 3, and the results are shown in Table 4 and 7 is shown.

division VEGF loading rate (%) Example 9 70.6 ± 13.1 Example 10 74.6 ± 12.0 Example 11 72.5 ± 13.3 Comparative Example 2 45.0 ± 5.7 Comparative Example 3 89.4 ± 17.0

Referring to Table 4, although the loading rate is lower than the non-porous microspheres of Comparative Example 3, compared to the porous microspheres of Comparative Example 2 prepared by the gas formation method, the loading rate of the porous microspheres according to the present invention It can be confirmed that this is remarkably excellent.

FIG. 7 shows the time of Examples 9 (-▼-), 10 (-▲-), 11 (-●-) and Comparative Examples 2 (-◆-) and 3 (-■-) microspheres of the present invention. It is a graph showing the cumulative release amount of angiogenesis factors.

Referring to FIG. 7, it can be seen that the porous microspheres of Comparative Example 2 and Examples 9 to 11, which are porous microspheres prepared by a conventional gas forming method, exhibit different release behaviors. In particular, unlike Comparative Example 2 showed a strong initial release of VEGF, it was observed that the initial release is significantly suppressed in Examples 9 to 11. This is predicted to be caused by electrostatic attraction between the remaining BSA and the loaded VEGF during the preparation of the porous microspheres, and the BSA concentration appears to be an important factor in controlling positive potential drug release. This electrostatic attraction minimizes the loss of VEGF through the pores, thus increasing the loading rate.

Experimental Example 3: Confirmation of porous microsphere alveolar deposition effect

3-1. Porous Microsphere Suspension In Vivo Insertion

In vivo experiments using male balb / c mouse (19-22 g) were carried out using DiI (1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindo carbocynnine perchlorate, Invitrogen) of Example 14 and Comparative Example 4. Loaded microspheres were sprayed into the airways to deposit into the alveoli.

Specifically, a porous microsphere containing DiI was prepared and suspended in deionized water at a concentration of 0.5 μg / μl. 100 μl of the suspension was sprayed onto the mouse airways anesthetized with ketamine (43.75 mg / kg) and lumps (6.25 mg / kg) using a microspray (Penn Century, USA). After 24 hours, the mice were sacrificed to obtain lungs.

3-2. Qualitative evaluation of the invasion effect into the alveoli

Qualitative analysis was performed using the fluorescence difference of DiI contained in the porous microspheres. Image stations (Kodak) were used to observe porous microspheres containing DiI deposited throughout the lungs. In addition, after freezing the lungs with cryostat (Cryostat), the fluorescence of the lung cross-section was observed by fluorescence microscopy (Fluorescence spectroscopy, Molecular Device). The obtained results are shown in FIGS. 8 and 9.

8 is an image station image of the microspheres of Example 14 (A) and Comparative Example 4 (B) of the present invention deposited in the entire lung.

Figure 9 is a fluorescence micrograph of the microspheres of Example 14 (A) and Comparative Example 4 (B) of the present invention deposited in the lung cross-section.

8 and 9, the porous microspheres of Example 14 were excellently deposited throughout the lungs as compared to Comparative Example 4 which is a non-porous microsphere. In addition, it was confirmed that the porous microspheres were deposited about 1.5 times or more even in the closed cross-sectional sections.

3-3. Quantitative Evaluation of Invasive Effect into Alveoli

Quantitative analysis was performed using the fluorescence difference of DiI contained in the porous microspheres. Fluorescence intensity was analyzed based on the qualitative results of 3-2 to quantify the porous microspheres deposited in the lungs. Lyophilized lung tissue was rapidly cooled through liquid nitrogen and ground using a rod bowl. The pulverized lung tissue was placed in DMSO and suspended in a probe using a probe type sonicator (Branson Digital Sonifier). The suspension was shaken using a shaker for 24 hours to sufficiently dissolve the porous microspheres containing DiI in the suspension. After centrifugation, the supernatant was quantified by comparing the difference in fluorescence intensity at λ _ex = 549 nm and λ _em = 565 nm using a spectrophotometer (Spectrophotometer, SpectraMax M2 ^e ).

10 is a graph showing the deposition efficiency in the alveoli of Example 14 and Comparative Example 4 of the present invention (* P <0.05).

Referring to FIG. 10, it was confirmed that the porous microspheres of Example 14 were deposited in the lungs by about 20% more than the non-porous microspheres of Comparative Example 4. This was consistent with qualitative analysis and confirmed that the porous microspheres of the present invention are actually more efficient for delivery to the lungs.

Experimental Example 4: Comparison of lung deposition effect according to the diameter of the microsphere

DiI-loaded porous microspheres of Examples 12 to 14 deposited in the entire lung were observed using an image station (Kodak) in the same manner as in Experimental Example 3, and analyzed quantitatively.

11 is an image station image of the microspheres of Examples 12-14 of the present invention deposited in the entire lung.

12 is a graph showing the deposition efficiency in the alveoli of Examples 12 to 14 and Comparative Example 4 of the present invention.

Referring to Figures 11 and 12, the smaller the diameter size of the porous microsphere was deposited more effectively into the alveoli. In particular, in the case of porous microspheres having a diameter size of 2.9㎛, it can be confirmed that the deposition is almost twice as large as the non-porous microspheres.

Porous microspheres can reduce invasion in the airways or oral compared to non-porous during aerosols, and because of their low porosity, they can be delivered deep into the lungs. In addition, compared to nanospheres, porous microspheres can prevent loss of mucus cilia or alveolar macrophages in the bronchus when drugs are delivered to the lungs through the airways. In the present invention, a porous microsphere for inhalation type lung delivery was prepared and the deposition into the lungs could be maximized through easy diameter size and pore size control. This clearly suggests the possibility of long-acting carriers in pulmonary drug delivery systems.

Porous microspheres according to the present invention is excellent in alveolar deposition effect can be effectively applied to the lung drug delivery system.

1 is a photograph observing the elevation (A) and the cross-section (B) of the porous microsphere prepared in Example 1 of the present invention with a scanning electron microscope.

2A to 2C are scanning electron microscope images of the porous microspheres prepared in Examples 2 to 4 of the present invention (scale bar = 10 μm).

3A to 3C are scanning electron micrographs of the porous microspheres prepared in Examples 5 to 7 of the present invention (scale bar = 5 μm).

Figure 4 is a graph showing the diameter (-◆-) and the size of the pores (-■-) of the porous microspheres prepared in Examples 2 to 4 of the present invention according to the BSA concentration.

5 is a graph showing the diameter (-◆-) and the size of the pores (-■-) of the porous microspheres prepared in Examples 5 to 7 of the present invention according to the PLGA concentration.

Figure 6 is a graph showing the cumulative amount of budesonide discharge with time of Example 8 (-■-) and Comparative Example 1 (-●-) microsphere of the present invention.

FIG. 7 shows the time of Examples 9 (-▼-), 10 (-▲-), 11 (-●-) and Comparative Examples 2 (-◆-) and 3 (-■-) microspheres of the present invention. It is a graph showing the cumulative release amount of angiogenesis factors.

8 is an image station image of the microspheres of Example 14 (A) and Comparative Example 4 (B) of the present invention deposited in the entire lung.

Figure 9 is a fluorescence micrograph of the microspheres of Example 14 (A) and Comparative Example 4 (B) of the present invention deposited in the lung cross-section.

10 is a graph showing the deposition efficiency in the alveoli of Example 14 and Comparative Example 4 of the present invention (* P <0.05).

11 is an image station image of the microspheres of Examples 12-14 of the present invention deposited in the entire lung.

12 is a graph showing the deposition efficiency in the alveoli of Examples 12 to 14 and Comparative Example 4 of the present invention.

Claims (16)

In the porous microsphere formed with micropores on the surface and inside of the body made of biodegradable polymer, A porous microsphere for pulmonary administration in which a bioactive agent is loaded and the body and the micropore diameter have a uniform size. The method of claim 1, Porous microspheres for lung administration, characterized in that the coefficient of variation of the body and micropore diameter is less than or equal to 0.2. The method of claim 1, Porous microspheres for pulmonary administration, wherein the diameter of the body of the microsphere is 1 to 300 ㎛, the diameter of the micropores is 80 nm to 1.5 ㎛. The method of claim 1, The biodegradable polymers include polylactic acid, polyglycolic acid, poly (lactic acid-co-glycolic acid), poly-ε- (caprolactone), polyvalerolactone, polyhydroxy butyrate, polyhydroxy valerate, and air thereof Porous microspheres for pulmonary administration, characterized in that the one polyester-based polymer selected from the group consisting of a mixture and a mixture thereof. The method of claim 1, The diameter of the body of the microsphere is 1 to 20 ㎛ porous microspheres for pulmonary administration. The method of claim 1, The diameter of the micropores of the microspheres is 80 nm to 1 ㎛ porous microspheres for lung administration. The method of claim 1, The bioactive microspheres for lung administration, characterized in that the bioactive agent is one selected from the group consisting of drugs, genes, vaccines, peptides and mixtures thereof. The method of claim 1, The bioactive agent is a porous microsphere for lung administration, characterized in that coupled to the osmotic agent and electrostatic attraction. The method of claim 7, wherein The osmotic pressure inducing agent albumin, casein, globulin, prolamine, gelatin and porous microspheres for lung administration, characterized in that one selected from the group consisting of a mixture thereof. The method of claim 1, Alveolar deposition efficiency of the microspheres is characterized in that more than 60% porous microspheres for pulmonary administration. S1) dispersing the internal aqueous phase in which the osmotic pressure agent is dissolved in water to prepare a W / O emulsion by dispersing the oil phase in which the biodegradable polymer is dissolved in the organic solvent, wherein the biologically active agent is added to the internal aqueous phase or the oil phase; S2) dispersing the W / O type emulsion in an external water phase in which an emulsifier is dissolved in water to prepare a W / O / W type emulsion; And S3) removing the organic solvent from the W / O / W type emulsion to obtain a porous microsphere Method for producing a porous microsphere comprising a. The method of claim 11, The osmotic pressure-inducing agent is an albumin, casein, globulin, prolamine, gelatin and the production method characterized in that one selected from the group consisting of a mixture thereof. The method of claim 11, The osmotic pressure inducing agent is albumin. The method of claim 11, The osmotic pressure inducing agent is characterized in that the addition of 5 to 30% (w / v) in the aqueous phase. The method of claim 11, The biodegradable polymer is a manufacturing method, characterized in that added in 5 to 10% (w / v) in the oil phase. The method of claim 11, The bioactive agent is a manufacturing method, characterized in that one selected from the group consisting of drugs, genes, vaccines, peptides and mixtures thereof.

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