KR20140109027A - anti-cancer drug-loaded porous microspheres surface-modified by TRAIL and the method for preparing the same - Google Patents

anti-cancer drug-loaded porous microspheres surface-modified by TRAIL and the method for preparing the same Download PDF

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KR20140109027A
KR20140109027A KR1020130023140A KR20130023140A KR20140109027A KR 20140109027 A KR20140109027 A KR 20140109027A KR 1020130023140 A KR1020130023140 A KR 1020130023140A KR 20130023140 A KR20130023140 A KR 20130023140A KR 20140109027 A KR20140109027 A KR 20140109027A
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trail
porous
porous microparticles
present
microparticles
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윤유석
이강춘
김인수
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성균관대학교산학협력단
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Priority to KR1020130023140A priority Critical patent/KR20140109027A/en
Priority to PCT/KR2013/008332 priority patent/WO2014137050A1/en
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Abstract

TECHNICAL FIELD The present invention relates to a porous microparticle in which TRAIL is surface-modified and encapsulated with a chemical anticancer agent and a method for producing the microparticle, and more particularly, to a method for preventing or treating lung cancer, , Porous microparticles and a process for producing the same.

Description

TECHNICAL FIELD The present invention relates to porous microparticles having surface-modified trails and encapsulating chemical anticancer agents and methods for preparing the same,

TECHNICAL FIELD The present invention relates to a porous microparticle in which TRAIL is surface-modified and encapsulated with a chemical anticancer agent and a method for producing the microparticle, and more particularly, to a method for preventing or treating lung cancer, , Porous microparticles and a process for producing the same.

The lungs have a very wide surface area (~ 100 m 2 ), the thickness of the cells forming the alveolar sac (0.1-0.5 ㎛) is very thin and the density of the cells is lower than other cells. have. It has been known to be effective pathway for local diseases such as asthma / chronic bronchial obstruction because it is fast enough to reach the systemic circulation when the drug is delivered through the lungs and does not undergo liver first pass metabolism.

In addition, due to the above-described characteristics, not only the lung cells exhibit a high permeability to macromolecules but also the amount of bioenzymes present in the lung mucosa is relatively small. Therefore, most of them are injection-dependent Is known to have a great effect on the body's delivery pathway of proteins and peptide drugs (molecular weight of several thousands to tens of thousands Da). Indeed, several reports have reported that these drugs reach a peak blood concentration of up to 30 minutes and bioavailability relative to the subcutaneous route to 50% (Leuprolide). In addition, due to the improvement of patients' convenience that they can mediate themselves, studies on drug delivery systems and delivery media through the respiratory system are actively under way.

Research has been focused on the development of pulmonary inhalation formulations of protein and peptide drugs, taking advantage of these lungs. For example, attempts have been made to use a cationic nano-complex in which multiple cationic agents and drugs are ionically bound for pulmonary inhalation, but these nanoparticles have poor alveolar transport efficiency and are easy to control for sustained drug release (Nat. Rev. Drug Discov. 6: 67-74, 2007; US 2003-0068277 A1, 2003.04.10).

In another example, nanoparticles prepared from biodegradable natural or synthetic polymers of about 500 nm are prepared, and the drug is sealed / adhered to the alveolar nano particles (Nat. Rev. Drug Discov. 6: 67-74, 2007, US 2003-0068277 A1, 2003.04.10). However, in the case of particles smaller than 1 탆, which are used in the above method, it is generally disadvantageous in that not only the exhalation can be discharged but also a device having low portability such as a nebulizer is used for inhalation.

As an example of the development of a pulmonary inhalation type formulation using microparticles, it is possible to use drug particles having a particle diameter in the range of several micrometers by using micropulverizing or micromilling methods or adding a suspending agent (e.g., (Int. J. Pharm. 220: 101-110, 2001) has been reported. Particles of this size range are most effective for alveolar region delivery, but they have the disadvantage that the bioavailability can be drastically lowered by phagocytic action when the size of crystals is <5 μm. Therefore, the development of lung inhalation microparticles having a high bioavailability as a diameter of 5 占 퐉 or more, high lung cancer diagnosis effect, low density and immobilization in bronchi have been demanded.

Meanwhile, the TNF-related apoptosis inducing ligand (TRAIL) is a kind of tumor necrosis factor (TNF), and is a cell membrane protein involved in cell apoptosis. It is known that the extracellular portion from the arginine at position 114 to glycine at position 281 affects the self-destruction of the cell (MH Kim, et al., BBRC 2004, 321 , 930-935). It is also known that three trail molecules exhibit a structurally bound trimeric structure and that such a trail of trimeric structures binds to a receptor involved in cell death and induces apoptosis (FC Kimberley, et al. Cell Research 2004, 14, 359-372).

The most striking difference between the other TNF superfamilies, TNF and CD95L, is that they do not induce the death of normal tissue cells. Since TNF and CD95L also induce cell death, a variety of pharmaceutical applications have been attempted. These proteins such as TNF and CD95L induce the death of cancer cells and hyperactivated immune cells as well as affect normal cells. On the other hand, it is known that trail induces auto-apoptosis of various kinds of cancer cells and hyperactivated immune cells and does not affect general cells. This is known to be due to differences in the expression of trail receptor in each cell.

There are five types of tracer receptors so far. Representative apoptosis-related receptors include DR4 (TRAIL-R1) and DR5 (TRAIL-R2). When the trail binds to these receptors, the cell self-destroys through the activation of the death-related domain in the cell and various signal transduction systems. In addition, there are three receptors, DcR1, DcR2, and OPG (osteoprotegerin). These three receptors are known not to be involved in cell death. The difference in the degree of expression of DR4 and DR5 involved in apoptosis in normal cells and cancer cells is known to be insignificant. On the other hand, the other three receptors that are not related to apoptosis are well expressed in normal cells, but the expression level is low or not expressed in cancer cells. Therefore, in the case of trail, the binding of DcR1, DcR2 and OPG, which do not bind to the death-related domain, predominantly occurs in normal cells and does not induce apoptosis of cells. On the other hand, in cancer cells and hyperactivated immune cells, DR4 and DR5 It is known that self-apoptosis of the cell is induced by binding with Such selective cell death is considered to be a very useful property in the clinical application of trail.

Cancer cells, glioma cancer, lung cancer cells, prostate cancer cells, brain tumor and multiple myeloma have been reported as cancer cells which are observed by the trail-like cell death, and they exhibit excellent anticancer activity in animal experiments .

 Thus, trails can also be used to treat T-cell autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis and type 1 diabetes, as well as treatment of the various types of cancer mentioned above .

However, when only the protein of the natural trail is used for treatment, the cell can not bind properly to the receptor according to the low trimer formation ratio, and the cell death effect is delayed. In addition, there is a problem that not only toxicity can be shown to normal cells by using only the trail itself but also the half life is short. In the case of the trail, it has been reported that it has different half-life depending on the animal species used in the experiment, and it has been reported to have a half-life of about 30 minutes in rodents and in apes (H. Xiang, et al. Drug Metabolism and Disposition 2004, 32, 1230-1238).

Chemotherapeutic drugs are drugs that act on various metabolic pathways of cancer cells and exhibit cytotoxic or growth inhibitory effects. In particular, various anticancer drugs and compounds, including doxorubicin, which are used in chemotherapy for lung cancer, are injected mainly by intravenous injection. However, it has been reported that various side effects, which are represented by cardiovascular diseases, appear in chemotherapy using intravenous injection.

Under these circumstances, the inventors of the present invention have found that although they can be used for lung inhalation and have a target orientation to lung cancer, they have a higher anticancer effect than trail alone or a chemotherapeutic agent alone, and have a side effect that can be caused by the use of trail or chemotherapeutic agent alone As a result of intensive efforts to develop microparticles that can be reduced, porous microparticles surface-modified with trail proteins and encapsulated with chemotherapeutic agents have been developed. In addition, since the porous microparticles have a diameter of 5 μm or more and have high bioavailability, they have a low density and are not immersed in the bronchial tract. In addition, the dosage can be reduced by the combined administration to reduce side effects and delayed release, And is effective for the prevention or treatment of lung cancer, thus completing the present invention.

An object of the present invention is to provide a method for producing porous microparticles in which TRAIL is surface-modified and a chemical anticancer agent is encapsulated.

Another object of the present invention is to provide a porous microparticle in which TRAIL is surface-modified and encapsulated with a chemical anticancer agent.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating lung cancer, which comprises the porous microparticles.

As one aspect for solving the above problems, the present invention provides a method for producing porous microparticles in which TRAIL is surface-modified and a chemotherapeutic agent is encapsulated.

Specifically, the method comprises: (a) mixing an emulsion containing a chemotherapeutic agent and ammonium bicarbonate with a lactic acid-co-glycolic acid (PLGA), a poly (lactic acid) (PLA) Mixing the dispersions in combination with each other; (b) ultrasonically pulverizing the mixed solution of step (a); (c) adding and stirring the ultrasonic pulverized mixed solution to the continuous phase; (d) lyophilizing the mixed solution to obtain porous microparticles encapsulating a chemotherapeutic agent; And (e) adding the porous microparticles obtained in the step (d) to a sample containing a TRAIL protein.

The step (a) may be carried out by mixing a dispersion containing a chemical anticancer agent and an ammonium bicarbonate with a lactic acid-co-glycolic acid (PLGA), a poly (lactic acid) (PLA) Mixing the dispersion of the biodegradable polymer with an emulsion containing a chemical anticancer agent and a porogen.

Since the porous microparticles of the present invention enter in vivo through inhalation, the polymeric polymer used in the preparation should have biocompatibility and biodegradability. That is, it is preferable that the polymer should be safe for living cells, and can be decomposed by itself upon completion of the release of the trail protein and the chemotherapeutic agent.

The term "biodegradable polymer" in the present invention means a macromolecular material which is a main component constituting porous microparticles and which is metabolized by metabolism in at least one step of decomposition and changed into a low molecular weight compound. The biodegradable polymer that may be used in the method of the present invention may include poly (lactic acid-co-glycolic acid) (PLGA), poly (lactic acid) (PLA) co-glycolic acid (PLGA), but is not limited thereto. Considering the formation of microparticles having uniform porosity, the PLGA has a ratio of latic acid to glycolic acid of 40:60 to 60:40 on a mass basis and having a molecular weight of 5,000 to 50,000 And more preferably the ratio of latic acid to glycolic acid is 50:50 by mass and has a molecular weight of 10,000 but is not limited thereto.

The biodegradable polymer can be prepared in the form of a dispersion using a solution containing methylene chloride and deionized water, but is not limited thereto.

In the method of the present invention, the porous microparticles are prepared by mixing the biodegradable polymer dispersion with an emulsion containing a porous inducer such as ammonium bicarbonate and a chemotherapeutic agent.

The term "porogen" in the present invention means a pore inducing substance, for example, a gas bubble forming agent, such as ammonium bicarbonate. In the method of the present invention, ammonium bicarbonate was used among various porous inducers, and a process suitable for the preparation of porous microparticles encapsulating chemotherapeutic agents, especially doxorubicin, was developed. The ammonium bicarbonate used in the method of the present invention can produce microcapsules encapsulating chemical anticancer agents having density and porosity suitable for lung inhalation.

Specifically, the ammonium hydrogen carbonate is a foamable salt which decomposes when dissolved in water at an appropriate temperature to generate carbon dioxide (CO 2 ) via ammonia (NH 3 ) and carbonic acid. Therefore, when the ammonium hydrogen carbonate present in the aqueous phase of the mixture is distributed to the surface of the microparticles and continuously contacted with the water introduced through the water channel at an appropriate temperature, ammonia and carbon dioxide are generated. Holes through the surface of the microparticles. Therefore, when the emulsion containing the anticancer agent and the ammonium hydrogen carbonate and the dispersion of the biodegradable polymer are mixed with each other, the chemical anticancer agent is encapsulated in the microparticles formed of the biodegradable polymer and the hole is formed by the ammonium bicarbonate .

As used herein, the term "chemo-chemotherapeutic agent" is a substance capable of inhibiting proliferation or inducing death of cancer cells, examples of which include doxorubicin, paclitaxel, 5-fluorouracil, cisplatin The compounds of the present invention may be used in combination with other therapeutic agents such as cisplatin, carboplatin, oxaliplatin, tegafur, irinotecan, docetaxel, cyclophosphamide, gemcitabine, ifosfamide, mitomycin C, vincristine, etoposide, methotrexate, topotecan, tamoxifen, vinorelbine, camptothecin, but are not limited to, camptothecin, danuorubicin, chlorambucil, bryostatin-1, calicheamicin, mayatansine, levamisole, Mitoxantrone, nimustine, But are not limited to, doxifluridine, formestane, leuprolide acetate, megestrol acetate, carmofur, teniposide, bleomycin, But are not limited to, carmustine, heptaplatin, exemestane, anastrozole, estramustine, capecitabine, goserelin acetate, polysaccharide, It has been reported that the drug may be used in combination with other drugs such as polysaccharide potassuim, medroxypogexterone acetate, epirubicin, letrozole, pirarubicin, topotecan, altretamine ), Toremifene citrate, BCNU, taxotere, actinomycin D, Anasterozole, Belotecan, Imatinib, fluoxidane (Floxuridine), Gemcitabine But are not limited to, Gemcitabine, Hydroxyurea, Zoledronate, Vincristine, Flutamide, Valrubicin, Streptozocin, Silibinin, , Or polyethyleneglycol conjugated anticancer agents, as well as synthetic analogs thereof and materials which exhibit modified or equivalent efficacy. In the present invention, the chemical anticancer agent is preferably doxorubicin, but is not limited thereto.

The step (b) is a step of ultrasonic pulverizing the mixed solution of step (a), and the size of the microparticles can be controlled by applying ultrasonic waves to the mixed solution.

In the present invention, the ultrasonic pulverization in the step (b) may be performed at an output amplitude of 40% to 60% at a frequency of 20 kHz, and preferably at 50%. Those skilled in the art can arbitrarily vary the frequency and the amplitude as long as they correspond to the ultrasonic waves that can be generated at the above-mentioned amplitude at the frequency. In addition, it is preferable to apply ultrasonic waves for 20 seconds to 60 seconds under the above-described output conditions, and more preferably, ultrasonic waves can be applied for 30 seconds, but the present invention is not limited thereto. It is possible to appropriately determine the final size of the microparticles by adjusting the ultrasonic application conditions. For the purpose of the present invention, the porous microparticles should be of a size suitable for pulmonary inhalation, preferably of a size of 5 to 20 microns in diameter, more preferably of 8 to 15 microns in diameter, (Not more than 20 占 퐉) but not large enough to induce phagocytosis of macrophages (5 占 퐉 or more). Therefore, conditions of ultrasonic pulverization are important in the production of the porous microparticles of the present invention.

Also, in the present invention, it is preferable that the step (b) is operated in a container containing ice to prevent the occurrence of high temperature.

In the step (c), the ultrasonic pulverized mixture is added to a continuous phase and stirred, and the mixture is emulsified and methylene chloride is evaporated.

In the present invention, the stirring of step (c) may be carried out at 10 ° C to 40 ° C for 4 hours to 24 hours, preferably at 20 ° C for 5 hours.

In the present invention, the continuous phase may be an aqueous solution of poly (ethylene-alt-maleic anhydride) (PEMA) or polyvinyl alcohol, but is not limited thereto. In one embodiment of the present invention, a 0.5% PEMA aqueous solution was used as a continuous phase (Example 1).

In the step (d) of the present invention, the stirred mixture is lyophilized to obtain a porous microparticle having a chemotherapeutic agent encapsulated therein.

The porous microparticles prepared through the above steps (a) to (c) are dried, and the freeze-drying method is used in the method of the present invention.

The step (e) of the present invention is a step of adding the porous microparticles obtained as described above to a sample containing a TRAIL protein, wherein a porous microparticle is formed through ionic bonding between a TRAIL protein and the porous microparticles It is the step of modifying the trail protein on the surface.

The term "TNF-related apoptosis inducing ligand (TRAIL)" in the present invention is a kind of tumor necrosis factor (TNF), which refers to a cell membrane protein involved in cell apoptosis. For the purpose of the invention, the trail protein refers to a protein modified on the surface of porous microparticles, which can be modified on the surface of porous microparticles through ionic bonding to exhibit an anticancer effect, but the present invention is not limited thereto.

In addition, in the present invention, the trail protein may be a natural or recombinant trail obtained by genetic modification, and may include a zipper amino acid sequence leading to the formation of a trimer of trail or / and a terminal group which facilitates separation and purification Trail. The trail protein is preferably a human trail having 281 amino acid sequences. More preferably, the trail protein is a human trail having an amino acid sequence from amino acid 114 to glycine 281.

In addition, the trail protein can be obtained by attaching an isoleucine zipper to the N-terminal. It is easy to form a trail protein in the form of a trimer capable of effectively recognizing a trail protein having an isoleucine zipper at its N-terminus, so that it can exhibit an excellent anti-cancer effect as compared with a natural type trail.

In one embodiment of the present invention, doxorubicin is used as a representative chemical anticancer agent, and the toxin Rubin Shin and ammonium hydrogen carbonate as a porogen inducer are mixed with a PLGA solution, and then homogeneously mixed with an ultrasonic disintegrator. Then, it was added to a continuous 0.5% PEMA to be emulsified and stirred, methylene chloride was evaporated, and the porous microparticles containing the formed doxorubicin were washed and lyophilized. The lyophilized porous microparticles were then added to the trail-containing buffer to prepare trail-modified porous microparticles (Example 1 and Figure 1 b).

In another aspect, the present invention provides a porous microparticle in which TRAIL is surface-modified and encapsulated with a chemotherapeutic agent.

The above-mentioned trail and chemical anticancer agent are as described above.

In the present invention, the term "porous microparticles" refers to spherical particles having pores on the surface, and may be manufactured by the manufacturing method described in the first embodiment, but it is not limited thereto. For the purpose of the present invention, the porous microparticles preferably have a diameter of from 5 탆 to 20 탆 and a density of from 0.1 g / cm 3 to 0.4 g / cm 3, more preferably from 8 탆 to 15 탆. This can minimize the macrophage in the lungs by increasing the size of the particles. As the density becomes smaller due to the porosity, it can not be eroded into the bronchus, and thus it is possible to effectively deliver the drug through the alveoli.

In one embodiment of the present invention, treating TRAIL and doxorubicin together with a lung cancer cell line (H226) showed that the anticancer effect was further enhanced as compared with the case of treating each of trail and doxorubicin alone (Example 4). Therefore, the porous microparticles of the present invention contain both trail and doxorubicin, which are effective ingredients, so that they have remarkably improved anticancer effect through the combined administration of them, and the dose of the chemical anticancer drug is decreased so that strong systemic side effects and cardiovascular The disease can be significantly reduced.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating lung cancer comprising trabecular surface-modified porous microparticles encapsulated with a chemical anticancer agent.

The composition of the present invention is characterized by being used for prevention or treatment of lung cancer through anticancer activity against cancer cells and target orientation through lung inhalation by the combined administration of trail and chemotherapeutic cancer agent.

The composition may comprise an effective amount of the porous microparticles surface-modified with the trail of the present invention and encapsulated with a chemotherapeutic agent, and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, other adjuvant and / or carrier. The composition of the present invention can be formulated in the form of a dry powder for inhalation (freeze-dried) in a conventional manner, and various formulations for inhalation can be prepared according to techniques described in the literature well known in the art have.

Any additive that may be included in the composition may be any of those conventionally used in dry powders. For example, the base may comprise a mixture of inorganic salts such as sodium chloride or a mixture thereof, a mixture of saccharides such as mannitol, lactose, dextran, glucose, and mixtures of amino acids such as arginine. In addition, preservatives such as methylhydroxybenzoate and propylhydroxybenzoate or surfactants such as lecithin and nonionic surfactants can be added.

The thus prepared trail is surface-modified and the composition including the porous microparticles in which the chemical anticancer agent is encapsulated is delivered to the alveoli through lung inhalation to continuously release the drug, and has an increased anticancer effect and side effect reduction effect do.

In one embodiment of the present invention, a composition comprising a porous microparticle wherein the trail was surface-modified and encapsulated with doxorubicin was administered to a lung cancer-induced mouse, and as a result, it was confirmed that the composition was excellent in the treatment of pulmonary tumors 5).

The porous microparticles in which the trail of the present invention is surface-modified and the chemical anticancer agent is encapsulated are useful for lung inhalation and are useful for delayed release of the drug. The combined use of the two drugs exerts an elevated anticancer effect, Can be used to prevent or treat lung cancer.

FIG. 1 is a graph showing the results of microstructure analysis of microparticles encapsulating doxorubicin by using the chemical structure (a) of doxorubicin encapsulated in porous microparticles and the double emulsion method (W / O / W type emulsion preparation method) (B) showing a process of modifying the image of FIG.
2 is an electron micrograph of the porous microparticles prepared by the method of the present invention. a) is a photograph of the enlarged particle, and b) is a photograph showing a comparison of several particles in one dose.
FIG. 3 is a photograph showing the drug encapsulated and surface-modified in the porous microparticles of the present invention.
FIG. 4 shows the anticancer activity of trail and doxorubicin in the lung cancer cell line (H226). The green line in a) is the result of fixing 1 μg / ml of doxorubicin and changing the trail to 0.01-10 μg / ml. The green line of b) is fixed at 1 μg / ml of trail and 0.01-10 μg / ml of doxorubicin .
Fig. 5 shows the anticancer activity of the porous microparticles of the present invention against lung cancer. Fig. 5 (a) shows the apparent morphology and size of dissected mouse lungs and Fig. 5 (b) shows the weight of extracted lungs of mice.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Trail  Surface modified Doxorubicin Enclosed  Preparation of porous microparticles

 Porous microparticles using ammonium bicarbonate were prepared using a typical W / O / W double emulsion method.

Specifically, 150 mg of each PLGA polymer (Mw: 10,000; lactic acid: glycolic acid = 50: 50) was dissolved in 3.75 ml of methylene chloride and vigorously shaken and dissolved to prepare a dispersion. 1 mg of doxorubicin and 7 mg of ammonium bicarbonate were dissolved in 300 μl of deionized water and 100 μl of deionized water, respectively. Then, the mixture was mixed with PLGA solution, and the mixture was sonicated using an ultrasonic mill (Branson Sonifier 450, 20 kHz, UK) Ultrasonic waves were applied for 30 seconds to homogeneously mix. At this time, in order to prevent the occurrence of high temperature, it was put in a container containing ice. Thereafter, the whole amount was taken with a disposable syringe and emulsified for 2 minutes while being added to a continuous phase rotating at 4,000 rpm. 25 ml of a 0.5% aqueous solution of poly (ethylene-alt-maleic anhydride) (PEMA, Mw: 400,000, 1: 1) was used as a continuous phase. Thereafter, the emulsified solution was stirred at room temperature for 5 hours to evaporate the methylene chloride. The porous microparticles thus formed were centrifuged and washed three times by redispersing in deionized water. Thereafter, the precipitated particles were collected by centrifugation and lyophilized to obtain porous microparticles which were firstly encapsulated with doxorubicin. Thereafter, 10 mg of the particles were dispersed in 1 ml of a buffer containing 1 mg of zipper-free trail at the N-terminus, and surface adsorption was induced for 6 hours at room temperature. After centrifugation, the precipitated particles were collected and lyophilized to obtain final porous microparticles . A brief schematic diagram of the first embodiment is shown in FIG.

Example  2: Shape using scanning electron microscope ( Morphology ) Confirm

Specimen observations were made on a scanning electron microscope (JEOL, JSM7500F, Japan). The final porous microparticle powder prepared in Example 1 was attached to a sample magnet and coated under argon gas, and the observation result through a scanning electron microscope is shown in FIG.

As a result, as shown in FIG. 2, the microparticles of the present invention prepared by the method of Example 1 were porous and showed a diameter of 8 to 15 μm.

Example  3: Confocal  Confirmation of drug inclusion using laser scanning microscope

The doxorubicin encapsulated in the porous microparticles prepared in Example 1 and the surface-modified trail were observed using a confocal laser scanning microscope (Carl Zeiss, Meta LSM 510, Germany). Fluorescein isothiocyanate (FITC) was attached to the trail to identify the formula. Ten equivalents of FITC (0.6 mg) was added to a 10 mg trail and reacted at room temperature for 5 hours with shading. The unreacted FITC was then removed using a desalting column and the trail-FITC was concentrated to a final concentration of 1 mg / ml. Porous microparticles were prepared in the same manner as in Example 1, except that 1 ml of the above-mentioned 1 ml of Trail-FITC solution was used instead of 1 ml of the buffer containing 1 mg of the trail of Example 1.

Next, observation results of the prepared porous microparticles through a confocal laser scanning microscope are shown in FIG. Here, doxorubicin encapsulated inside the particles is red due to its own fluorescence, and the trail modified on the surface has a green color due to the fluorescent substance (FITC).

As a result, as shown in FIG. 3, the porous microparticles prepared using the method of the present invention showed doxorubicin in the inside thereof, and the trail was modified.

Example  4: Doxorubicin Trail  Identification of physiological activity through synergistic action

To investigate the anticancer activity of microparticles, H226 cells, lung cancer cells, were used for cytotoxicity experiments.

Specifically, 2x10 4 cells were dispensed into each well of a 96-well plate and cultured for 24 hours to induce cell stabilization. Then, each of the cells was treated with a final 0.01 to 10 μg / ml of trail (TRAIL) and doxorubicin, respectively.

On the other hand, to confirm synergism, separate well plates treated with 0.01 to 10 μg / ml of the drug were treated with doxorubicin and trail to 1 μg / ml, respectively, and then cultured for 24 hours. Next, 20 μl of MTT reagent at a concentration of 2.5 mg / ml was added to each well. After 2 hours, the absorbance was measured to confirm the activity of the drug. The results are shown in FIG.

As a result, as shown in FIG. 4, it was shown from the above results that treatment of trail and doxorubicin together had more elevated anticancer activity than treatment of each alone.

The above results suggest that the use of the porous microparticles containing the doxorubicin of the present invention and the trail-modified microparticles can provide an excellent anti-cancer effect while reducing the administration dose compared with the case of using each of the anticancer agent and trail to be.

Example  5: Confirmation of the effect of the porous microparticles of the present invention on the chemotherapy of lung cancer

The anticancer effect of the porous microparticles of the present invention was confirmed and evaluated by using 6 week old male BALB / c nu / nu mice.

Specifically, the adaptive environment of the mice was maintained for at least 1 week under constant conditions such as temperature (22 ± 3 ° C), humidity (55 ± 5%) and light (repeated bright and dark for 12 hours). Four weeks prior to dosing, 10 6 H226 cells per mouse were intravenously injected via the tail vein to induce lung cancer, randomly dividing 5 to 7 mice per group. For 4 weeks, induction of lung cancer by injected H226 cells was stabilized in group 1, in group 2, in group 2, in group 2, in group 3, in trabeculae. The dosage was divided into the microparticle dosing group (Group 4) of the present invention in which the trail prepared in Example 1 was modified and the doxorubicin was encapsulated for 4 weeks. Here, mice not injected with H226 cells were set as a control. As a result, the anticancer activity effect for Groups 1 to 4 was confirmed by comparative observation and histological staining of lung tissue after dissection, and the results are shown in FIG.

As a result, as shown in FIG. 5, in the case of Group 4 in which the chemical anticancer agent of the present invention was encapsulated and the trail-modified porous microparticles were treated, the apparent appearance of the lungs was significantly smaller than the other groups (Fig. 5 (a)). In addition, the weight of the lungs was significantly lower than that of the other groups, indicating that the porous microparticles of the present invention had an excellent anticancer effect, similar to that of the control group b).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (10)

(a) mixing an emulsion comprising a chemical anticancer agent and ammonium bicarbonate with a dispersion of poly (lactic acid-co-glycolic acid) (PLGA), poly (lactic acid) ;
(b) ultrasonically pulverizing the mixed solution of step (a);
(c) adding and stirring the ultrasonic pulverized mixed solution to the continuous phase;
(d) lyophilizing the mixed solution to obtain porous microparticles encapsulating a chemotherapeutic agent; And
(e) adding porous microparticles obtained in step (d) to a sample containing a TRAIL protein, wherein the TRAIL is surface-modified and the porous antimicrobial agent-containing porous microparticles are encapsulated. Way.
The method of claim 1, wherein the poly (lactic acid-co-glycolic acid) (PLGA) has a molecular weight of 5,000 to 50,000.
The method of claim 1, wherein the chemotherapeutic agent is doxorubicin.
The method according to claim 1, wherein the ultrasonic pulverization in the step (b) is performed by applying an ultrasonic wave for 20 to 60 seconds, wherein the frequency of the ultrasonic wave is 20 kHz and the output amplitude of the ultrasonic wave is 40% to 60% / RTI &gt;
The method according to claim 1, wherein the stirring of step (c) is performed at 10 캜 to 40 캜 for 4 hours to 24 hours.
Porous microparticles having a diameter of 5 mu m to 20 mu m and a density of 0.1 g / cm &lt; 3 &gt; to 0.4 g / cm &lt; 3 &gt;, wherein the trail TRAIL is surface-modified and the chemical anticancer agent is encapsulated.
The porous microparticle according to claim 6, wherein the chemical anticancer agent is doxorubicin.
The porous microparticle according to claim 7, wherein the porous microparticles are composed of poly (lactic acid-co-glycolic acid) (PLGA), poly (lactic acid) (PLA) or a combination thereof.
The porous microparticle according to claim 8, wherein the poly (lactic acid-co-glycolic acid) (PLGA) has a molecular weight of 5,000 to 50,000.
A pharmaceutical composition for preventing or treating lung cancer, comprising the porous microparticles of any one of claims 6 to 9.
KR1020130023140A 2013-03-05 2013-03-05 anti-cancer drug-loaded porous microspheres surface-modified by TRAIL and the method for preparing the same KR20140109027A (en)

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