KR20090117331A - Pharmaceutical composition for lung-targeting - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for lung-targeting containing a surface-modified nanoparticle is provided to deliver drug without drug electric charge. CONSTITUTION: A pharmaceutical composition for lung-targeting contains a surface-modified nanoparticle which is coated with chitosan on the surface. The nanoparticle further contains polylactide or its copolymer and active ingredient. The copolymer of polylactide contains lactide and glycolide in 50:50-90:10. The active ingredient is anticancer drug or therapeutic agent of tuberculosis.

Description

Pharmaceutical composition for lung-targeting

The present invention relates to a pharmaceutical composition for lung-targeting, and more particularly, surface-modified obtained by coating the surface of the nanoparticles containing the active ingredient, namely drug, with chitosan. It relates to a pharmaceutical composition for lung-target orientation using nanoparticles.

Most anticancer drugs used in the clinic for chemotherapy have a low selectivity to cancer cells and accumulate in normal tissues, causing serious side effects. In addition, since most of the anticancer drugs show rapid disappearance when administered in the body, when the dose is increased for a sufficient therapeutic effect, side effects also increase, resulting in a lower treatment coefficient than other drugs. In order to improve this problem, various researches on targeting to cancer cells or cancer tissues have been conducted.

Meanwhile, Wang et al. Reported the microparticles encapsulated with paclitaxel using a copolymer of polylactide and glycolide (poly (lactic-co-glycolic acid), PLGA) (Wang, YM, Sato, H). ., Dachi, I., and Horikoshi, I., Preparation and characterization of poly (lactic-co-glycolic acid) microspheres for targeted delivery of novel anticancer agent, taxol.Chem. Pharm. Bull., 44, 1935-1940, (1996), Fonseca et al. Reported PLGA nanoparticles encapsulated with paclitaxel using nanoprecipitation (Fonseca, C., Simoes, S., and Gaspar, R., Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity.J. Control.Release, 83, 273-286, 2002).

Mo and Lim have reported PLGA nanoparticles encapsulated with paclitaxel modified with surface germ agglutinin (WGA) (Mo, Y. and Lim, LY, Paclitaxel-loaded PLGA nanoparticles: potentiation of anticancer activity by surface conjugation with wheat germ agglutinin.J. It has been reported that active targeting is possible by an endocytosis mechanism.

Recently, Nafee et al. Have disclosed chitosan-coated PLGA nanoparticles for DNA / RNA transport (Noha Nafee, Sebastian Taetz, Marc Schneider, Ulrich F. Schaefer, Claus-Michael Lehr, Chitosan-coated PLGA nanoparticles for DNA). / RNA delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides, Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 173-183). According to Nafee et al., PLGA is dissolved in PVA and chitosan in the oil phase, and then the oil phase is added to the aqueous phase to form nanoparticles by elulsion-diffusion-evaporation to impart a positive charge to the surface of the nanoparticles. Oligonucleotides such as and the like are attached to the surface using electrostatic attraction to produce chitosan-coated PLGA nanoparticles for DNA / RNA transport. The nanoparticles have a reduced surface charge as the concentration of the oligonucleotide increases, but the zeta potential of the nanoparticles remains positive because the surface charges of the nanoparticles are not completely canceled out. This is due to blocking of further binding of oligonucleotides due to steric hindrance. Nafee et al. Report in vitro testing that can increase cellular uptake by cancer cells due to the surface positive charge of these nanoparticle complexes.

However, the chitosan-coated PLGA nanoparticles reported by Nafee et al. Convert the surface charges of the nanoparticles into positive charges with chitosan and then form complexes with oligonucleotides such as DNA and RNA having negative charges. There is a problem that can not be applied. Nafee et al. Also disclose that in vitro testing of cellular uptake by cancer cells (eg, A549 human lung tumor cells) can increase the uptake of nanoparticle complexes with surface positive charges. However, the test relates to an endocytosis uptake test by an electrostatic mechanism and does not teach whether the nanoparticles can be targeted to specific organs or cells when introduced into the systemic bloodstream. . Furthermore, given that the cellular membranes of various cells in the body are negatively charged, when the positively charged particles are introduced into the bloodstream, the membranes of all the negatively charged cells are not specific target organs or cells. Is expected to show affinity for).

The present inventors have conducted various studies to develop a drug transporter for targeting. As a result, when a drug is encapsulated inside a nanoparticle made of a polymer such as PLGA and the surface of the nanoparticle is coated with chitosan, a surface-modified nanoparticle obtained is produced. It has been found that the transport of cationic drugs as well as anionic drugs is possible without. In particular, surprisingly, when such surface-modified nanoparticles are introduced into the systemic blood flow, the nanoparticles introduced into the blood stream form transient aggregates with components in the plasma, which aggregate It was found to migrate selectively along the lung tissue.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for lung-target orientation comprising surface-modified nanoparticles obtained by coating the surface of a nanoparticle containing an active ingredient, i.e., a drug, with chitosan.

According to an aspect of the present invention, a surface-modified nanoparticle having chitosan coated on a surface of a nanoparticle consisting of polylactide or a copolymer thereof and an active ingredient, wherein the active ingredient is formed inside the nanoparticle. Provided is a pharmaceutical composition for lung-targeting, comprising surface-modified nanoparticles encapsulated therein.

The copolymer of polylactide is a copolymer of D, L-lactide and glycolide, a copolymer of L-lactide and glycolide, a copolymer of D, L-lactide and L-lactide, At least one selected from the group consisting of a copolymer of L-lactide with trimethylene carbonate, a copolymer of L-lactide with ε-caprolactone, and a copolymer of glycolide with L-lactone The polylactide copolymer may be a polylactide copolymer (poly (d, l-lactide-) having a copolymerization ratio of lactide and glycolide in a range of 50:50 to 90:10. co-glycolide).

The active ingredient includes, without limitation, drugs useful for the prophylaxis and / or treatment of lung disease, and may be, for example, an anticancer agent such as paclitaxel or a pulmonary tuberculosis agent such as opfloxacin.

The chitosan may have a weight average molecular weight of 10 to 150 kDa, the content of chitosan may be 0.1 to 5% by weight relative to the total weight of the nanoparticles.

The surface modified nanoparticles contained in the pharmaceutical composition for lung-targeting according to the present invention are designed such that the drug is encapsulated inside the surface instead of the surface of the nanoparticles, thereby irrespective of the charge of the drug, that is, cationic as well as anionic Drug transport is possible. It has also been discovered by the present invention that the surface-modified nanoparticles form transient aggregates with components in plasma when introduced into the systemic blood stream. The association increases the particle size by the self amplifying mechanism, and can also increase the safety by avoiding the occurrence of embolism in the permanent or semi-permanent transport (eg permanent microsphere) have. That is, the transient aggregates are capable of pulmonary-target orientation by selectively shifting to pulmonary vessels while moving along the bloodstream without causing embolism. In particular, surface-modified nanoparticles reached into the lung can be useful for treating lung tumors because of the overexpression of anionic phospholipids, glycoproteins, and proteoglycans, thus enabling additional cellular uptake to negatively charged lung cancer cells. . Therefore, the pharmaceutical composition according to the present invention can be usefully used for the treatment of lung diseases including various lung tumors, pulmonary tuberculosis and the like.

As used herein, the term "nanoparticle" refers to a drug carrier encapsulated with an active ingredient (ie, a drug) having a size of about 1000 nm or less. The average particle diameter of the nanoparticles is of a size which allows the formation of transient aggregates with components in the plasma in the systemic blood stream, for example 10 to 1,000 nm, preferably 50 to 500 nm, more preferably 200 To 300 nm.

"Surface-modified" refers to a state in which the surface of a nanoparticle is coated with chitosan, wherein the coating is a physical layer formation or chemical interaction, for example nanoparticles. It includes a variety of physicochemical interactions, including binding to the surface, adsorption, association, and the like.

The present invention provides a surface-modified nanoparticle having a chitosan coated on the surface of a nanoparticle consisting of polylactide or a copolymer thereof and an active ingredient, the active ingredient being encapsulated inside the nanoparticle. It provides a pharmaceutical composition for pulmonary-targeting, comprising.

As the polymer used to form the nanoparticles, polylactide (for example, poly (D, L-lactide), poly (L-lactide), etc.) or a copolymer of polylactide may be preferably used. As the copolymer of the polylactide, a copolymer of D, L-lactide and glycolide, a copolymer of L-lactide and glycolide, a copolymer of D, L-lactide and L-lactide , Copolymers of L-lactide with trimethylene carbonate, copolymers of L-lactide with ε-caprolactone, copolymers of glycolide with L-lactone, and the like. Specific examples are shown in Table 1. In Table 1, the composition ratios are described as examples, and those skilled in the art will understand that the composition ratios may be changed.

Polymer type Composition ratio (weight ratio) Poly (D, L-lactide-co-glycolide) 50: 50 Poly (D, L-Lactide) 100: 0 Poly (D, L-lactide-co-glycolide) 75: 25 Poly (L-Lactide-Co-D, L-Lactide) 70: 30 Poly (L-Lactide-Co-D, L-Lactide) 92: 8 Poly (L-lactide-co-trimethylene carbonate) 70: 30 Poly (L-Lactide-Co-Glycolide) 82: 18 Poly (L-Lactide-Co-Glycolide) 85: 15 Poly (L-lactide-co-ε-caprolactone) 70: 30 Poly (L-lactide) 100: 0 Poly (glycolide-co-L-lactone) 90: 10

More preferably, a polylactide copolymer [poly (d, l-lactide-co-glycolide) having a copolymerization ratio of lactide and glycolide in the range of 50:50 to 90:10 may be used. In particular, a polylactide copolymer [poly (d, l-lactide-co-glycolide]) having a copolymerization ratio of lactide and glycolide in the range of about 50:50 may be used. The amount of the polylactide or its copolymer is not particularly limited and may be selected by those skilled in the art in consideration of the content of the active ingredient and the desired particle size. With respect to the total weight (1 part by weight), it may be used in the range of 20 to 40 parts by weight, but is not limited thereto.

As the active ingredient contained in the pharmaceutical composition of the present invention, a drug useful for the prevention and / or treatment of lung disease may be applied without limitation, and an anticancer agent or a pulmonary tuberculosis therapeutic agent useful for the prevention or treatment of lung cancer may be preferably applied. Examples of the anticancer agents include irinotecan, oxaliplatin, oxaliplatin, melphalan, chlororambucil, teniposide, paclitaxel, docetaxel, docetaxel, uracil, 5 5-Fluorouracil, Tegafur, Vinorelbine bitartrate, Methotrexate, Carboplatine, Cisplatin, Doxorubicine HCl (Doxorubicin HCl) ), Cytocine arabinoside HCl (Cytocine arabinoside HCl), Mitoxantrone HCl (Mitoxantrone HCl), Tamoxifen Citrate, Nimustine HCl (Nimustine HCl), Daunorubicin HCl (Daunorubicin HCl), Epirubicin Shin HCl (Epirubicin HCl), Idarubicin HCl (Idarubicin HCl), Doxifludine, Dacarbazine, Thioguanine, Thioguanine, Formestane, Leupreline Acetate (Leupr) orelin acetate, tretinoin, vinblastine sulfate, vincristine sulfate, teniposide, etoposide HCl, carmustine (BCNU), Romustine, esturamustine, ranimustine, cymide, cyclophosphamide, pocarbazine (Porcarbarzine), gemcitabine, docetaxel, capecitabine, imatinib mesylate, rituximab ( Rituximab), Doxifluridine, Toremifene citrate and the like. Examples of pulmonary tuberculosis therapeutics also include oploxacin, isoniazid, ethambutol, rifampicin, rifabutin, ciprofloxacin, levofloxacin, capreomycin, cycloserine, and the like.

The nanoparticles encapsulated with the drug may be prepared according to conventional nanoparticle manufacturing methods such as a solvent evaporation method. For example, in the case of preparing nanoparticles encapsulated with a drug by a solvent evaporation method, after dissolving a polymer such as PLGA and a drug in a suitable organic solvent, if necessary, after adding a surfactant such as polyvinyl alcohol, It can be prepared by centrifugation at 35,000-45,000 g. In addition, if necessary, the method may include removing the used surfactant by centrifugation at 800 to 950 g, and the obtained nanoparticles may be dried by lyophilization or the like.

The weight average molecular weight of chitosan used for the surface-modification of the nanoparticles may be in the range of 10 to 150 kDa, more preferably in the range of 30 to 60 kDa. The surface-modification can be carried out by conventional coating methods. For example, chitosan may be dissolved in a solvent such as acetic acid to prepare a chitosan solution, and then the drug-encapsulated nanoparticles may be dispersed and then centrifuged at 85,000 to 95,000 g to obtain surface-modified nanoparticles. The obtained particles can be dried by conventional drying methods such as, for example, vacuum drying and lyophilization. For the surface-modified nanoparticles, the chitosan content can be controlled by varying the concentration of the chitosan solution. The content of chitosan coated on the nanoparticles may range from 0.1 to 5% by weight, more preferably from 2 to 3% by weight, based on the total weight of the nanoparticles.

The surface-modified nanoparticles can be formulated in the form of a solid powder for preparation, ie injectable powder, when used, including conventional excipients such as lactose, sucrose, maltose and the like, if necessary, conventional stabilizers, preservatives And the like. The solid powder preparation for use may be prepared in parenteral forms, such as intravenous injection, intravenous infusion, by dispersing in conventional diluents, for example, diluents such as injectable distilled water, phosphate buffered saline. The diluent may include a analgesic agent, a buffer, an isotonic agent, and the like as necessary.

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

Example

1. Test Method

(1) Test Material

Materials used and where to buy for the following examples are as follows. Paclitaxel was purchased from Taihua National Plant Pharmaceutical Corporation (Shan Xi, China) and has a 50:50 ratio of PLGA (MW = 40,000-75,000), polyvinyl alcohol (PVA, MW = -67,000), chitosan (molecular weight: about 50,000). , Coumarin 6 and RPMI 1640 (cell culture medium), and phosphate buffered saline (PBS, pH 7.4, 0.01 M) were purchased from Sigma Aldrich (St. Louis, MO, USA). Human lung cancer cell line A549 was purchased from Korea cell line bank (Seoul, Korea) and dichloromethane (extra pure) from Daejeong Chemical (Kyeongnam, Korea). Other chemicals were also used commercially.

(2) Preparation of Surface-Modified PLGA Nanoparticles

PLGA nanoparticles were prepared according to the solvent evaporation method using paclitaxel as a model drug. 47.5 mg of PLGA and 2.5 mg of paclitaxel were dissolved in 2 ml of dichloromethane and mixed in 10 ml of a 4% (w / v) PVA aqueous solution and sonicated for 1 minute (Model 100, Fisher Scientific Inc., Pittsburg, PA, USA). ). It was mixed with 40 ml of 1% PVA aqueous solution and stirred at 40 ° C. overnight. This was placed in a 50 ml centrifuge tube and centrifuged at 919.7 g for 10 minutes, and the supernatant was ultracentrifuged at 41137.3 g for 20 minutes to separate nanoparticles. The obtained precipitate was resuspended in distilled water (DDW), sonicated again, ultracentrifuged at 41137.3 g for 20 minutes to remove PVA, and this process was repeated once more. The product was lyophilized for one day, and the obtained nanoparticles (NP ₀ in Table 1 below) were stored at −10 ° C. for the next experiment.

PLGA nanoparticles (NP ₁ , NP ₂ , NP ₃ ) surface-modified with chitosan including paclitaxel were prepared by the following method. Chitosan was dissolved in acetic acid at 1% (w / v) and diluted with distilled water (DDW) to make 0.3, 0.5, 0.7% (w / v) solution. PLGA nanoparticles (NP ₀ ) were dispersed in 10 ml of each of the obtained solutions using ultrasonic waves, and then stirred at room temperature overnight. This was ultracentrifuged at 4 ° C. and 92,566 g for 50 minutes, and then the precipitate was resuspended in distilled water (DDW) and sonicated. This process was repeated twice and lyophilized. Nanoparticles surface-modified with 0.3, 0.5, 0.7% (w / v) chitosan solution were expressed as NP ₁ , NP ₂ , and NP ₃ , respectively. For in vitro cellular uptake test of nanoparticles, PLGA nanoparticles containing coumarin 6 instead of paclitaxel were prepared, and nanoparticles surface-modified with chitosan were prepared in the same manner as above.

(3) physical properties of nanoparticles

After dilution of the nanoparticles obtained above with distilled water (DDW), electrophoretic light scattering spectrophotometry (ELS-8000, Otsuka Electronics, Otsuka, Japan) was used to measure the size, distribution, and surface charge of nanoparticles. It was. The surface of the nanoparticles was measured by scanning electron microscopy (SEM, JSM-5600, Jeol Co., Tokyo, Japan) at a magnification of 10,000 times and 15,000 times, and platinum coating was performed for 40 seconds prior to measurement. The excitability of paclitaxel in the nanoparticles was measured using X-ray diffractometry (D-5005, Siements, Karlsruhe, Germany), and the diffraction angle (2θ) was recorded from 0 ° to 64 ° at a scanning speed of 3 ° / min. It was. The properties of nanoparticles in distilled water (DDW) may change in the rate of blood flow in vivo. Therefore, the plasma of the mouse with the particles was stirred at 120 rpm for 30 minutes at 37 ℃ and the particle size and zeta potential (zeta potential) was measured. Nanoparticles were separated by centrifugation at 15,600 g for 3 min in the culture medium and the size and zeta potential were measured.

(4) Encapsulation rate of paclitaxel

In order to determine the paclitaxel encapsulation rate of the nanoparticles, about 1.5 mg of the nanoparticles were dissolved in 0.5 ml of dichloromethane, and then mixed in a mixture of 2 ml of acetonitrile and water 1: 1. After shaking the mixture vigorously for 5 minutes, dichloromethane was blown under nitrogen gas, leaving only a clear solution. The drug was quantitatively diluted using acetonitrile and water 1: 1 mixture, and then measured by HPLC. The loading efficiency (%) was calculated according to the following equation.

Loading efficiency (%)

= (Paclitaxel content recovered from nanoparticles) X100 / (Paclitaxel content used to prepare nanoparticles)

(5) In vitro dissolution test of paclitaxel

Surface-modified nanoparticles (10 mg) with paclitaxel-encapsulated chitosan were diluted in 10 ml of PBS (pH 7.4, 0.01 M) in test tubes with lids and then kept at 37 ° C in an orbital shaker gently at 120 rpm. Remove the test tube at the sampling point between 12 hr and 719 hr, ultracentrifuge at 41137.3 g for 20 minutes, resuspend the pellet in 10 ml of PBS (p. 7.4), and place it in a thermostat so that the dissolution test can be performed again. After extracting paclitaxel with dichloromethane (1.0 ml) from the supernatant, dichloromethane was removed using nitrogen, and the remaining drug was removed with 2.0 ml of solvent (50:50 of DDW and acetonitrile). (v / v)) and quantified. The drug solution was filtered using a 0.2 μm membrane filter and then injected into the HPLC column by 20 μl. ResolveTM (Spherical C18 column: 3.9 × 150 mm, 5 μm), a reversed phase column, was used, and the HPLC system used Waters' 2487 Dual λ absorbance detector, 717 plus autosampler, and 515 HPLC dual pumps. The detection wavelength was 227 nm, and the mobile phase used a 50:50 (v / v) mixture of acetonitrile and water, and the moving speed was 1.0 ml / min.

(6) Uptake of coumarin 6 from nanoparticles into cells

In vitro cellula upkate test using PLGA nanoparticles encapsulated with coumarin 6 in an A549 cell line to determine the effect of surface-modification with chitosan on uptake into cells Was performed. Coumarin 6 was selected as a model compound because it is easy to observe in cells. Cells were supplemented with RPMI 1640 medium (10% fetal bovine serum (FBS), 100 IU / ml penicillin, 100 μg / ml streptomycin and 2 mM L-glutamine) at 37 ° C. and 5% carbon dioxide conditions. Incubated. 100 ul of fresh mouse plasma containing coumarin 6-embedded PLGA nanoparticles (0.25 mg / ml) when 80% confluence was reached after seeding cells in 96-well microplates to 1 × 10 ⁴ cells / well (fresh mouse plasma) was added and incubated at 37 ° C. and 60 rpm for 2 hours. The amount of coumarin 6 in the nanoparticles used was 3.5% (w / w). 100 μl of Ice cold PBS was added to terminate the culture, and the PBS was removed. This process was repeated twice to remove nanoparticles that were not taken up by the cells. Cell membranes were dissolved using a 0.5% triton X-100 solution in 100 μl of 0.2N NaOH and the content of coumarin 6 was measured with a microplage reader (Synergy HT, BioTek Instruments, Winooski, VT, USA, And emission wavelength: 430 and 485 nm, respectively).

(7) In vitro Cytotoxicity Test

As in (6), the A549 cell line was inoculated to 1 × 10 ⁴ cells / well in a 96-well plate and reached 1, 2, 4 μg paclitaxel or paclitaxel when the 80% confluence was reached. 100 μl of fresh mouse plasma containing the encapsulated nanoparticles was added. One row of 96 well plates was treated with media without drug or nanoparticles as a control. After incubating the plates for 24, 48, and 72 hours, the medium was removed at specific times and washed three times with 100 μl of PBS. Then 100 μl of MTT assay solution was added and incubated for 3 hours. After 3 hours, the medium was removed, 100 μl of DMSO was added to dissolve the remaining precipitate in the well, and measured at 560 nm. Cell viability was calculated according to the following equation.

% Cell viability = absorbance _{test cells} X 100 / absorbance _{control cells}

Absorbance _{test cells} and absorbance _{control cells} refers to the absorbance in cells treated with drugs or nanoparticles and the cells treated with only medium, respectively.

(8) Pharmacokinetics and Distribution of Paclitaxel

Pharmacokinetics and distribution measurements of paclitaxel were used in normal or lung cancer metastasis CDF1 mice. Lung cancer metastasis was induced by injecting mouse colon cancer cell line CT-26 through the tail vein. CT-26 cell line was cultured in RPMI 1640 medium (supplemented with 10% FBS, 100 IU / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine) and administered as 1 × 10 ⁵ cells / mouse and cancer cells It was used in the experiment two weeks after the main administration.

PLGA nanoparticles (NP ₀ ) encapsulated with Taxol ^TM (paclitaxel), nanoparticles surface-modified with chitosan (0.7%) (NP ₃ ) were injected into the mouse at 10 mg / 10 ml / kg via the tail vein. Injected. Plasma, heart, liver, spleen, lung and kidney were taken at 5, 30, 2, 4, 6, 8 and 12 hr after the injection. Plasma was taken from the eye and centrifuged using Eppendorf tubes, and other organs were washed twice with physiological saline and gently wiped. Plasma and tissue samples were stored at −50 ° C. until paclitaxel quantification.

The concentration of drug in plasma was determined by the following method. 150 μl plasma samples were mixed with 50 μl n-butyl p-hydroxybenzoate (25 μl / ml, internal standard), vigorously mixed with 2 ml of ethyl acetate for 5 minutes and extracted by centrifugation at 892.6 g for 5 minutes. . After repeating the extraction process twice, the oil phase was removed under nitrogen gas, resuspended in 150 μl of the mobile phase, and 100 μl was injected into the HPLC column.

The concentration of drug in the tissue was determined by the following method. Four times the weight of each tissue (including 4% (w / v) bovine serum albumin) was added and the tissues were ground for 5 minutes at 4 ° C. 50 μl internal standard (n-butyl p-hydroxybenzoate, 5 μl / ml) was added and extracted with 2 ml of ethyl acetate, and the procedure was repeated twice as in the case of plasma. The ethyl acetate layer was obtained to remove the liquid phase under nitrogen gas and the residue was resuspended in 150 μl of mobile phase and 100 μL was injected into an HPLC column. Analytical conditions are the same as in vitro dissolution tests.

AUC, CL and Vdss of paclitaxel were calculated using WinNolin software (Pharsight, Mountain View, CA, USA).

(9) statistical analysis

All data were expressed as mean ± SD as possible, and the comparison of mean between the preparations was done using Student's t-test. In all cases, statistical significance was determined using p <0.05.

2. Test result

(1) Physical Properties of Nanoparticles

Physical properties of nanoparticles in DDW and plasma are shown in Tables 2a and 2b.

Formulation (chitosan concentration, w / v%) Distilled Water (DDW) plasma Particle Size (nm) Polydispersity Zeta Potential (mN) Particle Size (nm) Polydispersity Zeta Potential (mN) NP ₀ (0) 202.11 ± 2.34 0.11 ± 0.02 -28.42 ± 3.04 237.82 ± 58.32 0.21 ± 0.06 -29.71 ± 4.64 NP ₁ (0.3) 240.64 ± 3.21 0.15 ± 0.04 + 8.91 ± 2.02 600.91 ± 72.14 0.38 ± 0.12 + 3.57 ± 1.51 NP ₂ (0.5) 252.12 ± 2.53 0.20 ± 0.03 + 17.31 ± 2.42 1289.53 ± 135.64 0.40 ± 0.17 + 10.83 ± 2.91 NP ₃ (0.7) 280.62 ± 3.61 0.16 ± 0.04 + 31.83 ± 1.81 2670.51 ± 319.93 0.58 ± 0.30 + 20.92 ± 5.94

Formulation (chitosan concentration, w / v%) Inclusion Rate (%) content(%) NP ₀ (0) 70.21 ± 3.53 3.51 ± 0.18 NP ₁ (0.3) 71.82 ± 3.21 3.59 ± 0.16 NP ₂ (0.5) 68.73 ± 2.61 3.44 ± 0.13 NP ₃ (0.7) 69.62 ± 2.94 3.48 ± 0.15

The loading efficiency and content of nanoparticles were 69-72% and 3.4-3.6%, and were not affected by chitosan surface-modification. However, the particle size and zeta potential of distilled water (DDW) were affected by chitosan, but the particle size increased as the concentration of chitosan increased. Its size was less than 300 nm. The zeta potential of nanoparticles is positively charged depending on the concentration of chitosan.

In vitro size measurements differ from actual size in the bloodstream. This is because nanoparticles generally increase in size by more than 50% by agglomeration or opsonization in media containing proteins. In this result, the size of the nanoparticles increased with contact with plasma. The particle size increased significantly with the concentration of chitosan (in order of NP ₀ , NP ₁ , NP ₂ , and NP ₃ ), especially for NP ₃ , at 2670.5 nm, which is more than 9.5 times higher than that of DDW. The increase in particle size is also consistent with the increasing trend of zeta potential, which appears to be due to the electrostatic interaction between the positive charge of nanoparticles and the negative charge of plasma proteins.

(2) X-ray diffraction analysis

1 shows results for paclitaxel powder, chitosan powder, PLGA nanoparticles without drugs (blank NP), NP ₀ , NP ₁ , NP ₂ , and NP ₃ . Only paclitaxel had peaks at 6 ° and 12 ° but not in other particles.

(3) surface shape

SEM images of the nanoparticles are shown in FIG. 2. Paclitaxel crystals (A) were not found in the nanoparticles, which is consistent with the (B-F) X-ray diffraction results (FIG. 1). The nanoparticles were round and had a size of less than 300 nm, which is similar to the electrophoretic light scattering spectrophotometry results in Table 2.

(4) Elution of Paclitaxel from Nanoparticles

Figure 3 is the result of measuring the drug dissolution in the nanoparticles. All nanoparticles showed a biophasic elution pattern with rapid dissolution for the first 130 hours and slow dissolution for the remaining time. Because PLGA polymers degrade over about 1-2 months, drug dissolution is not directly related to the collapse of the polymer matrix. Elution tends to be slowed down by surface-modification by chitosan, suggesting that chitosan acts as a diffusion barrier for drugs. In order for the nanoparticles to be efficiently transported to the body's target organs, the release of paclitaxel from the PLGA nanoparticles must be delayed during the blood flow residence time. As can be seen in FIG. Shows that there is.

(5) cellular uptake of nanoparticles from plasma

4 shows cellular upkates of coumarin 6 from various nanoparticles after 2 hours in A549 cell line. Elution of coumarin 6 is very minimal (less than 5%) at a given 2 hour condition, so the results of FIG. 4 can be seen as a cellular upkate of the nanoparticles. Cellular upkates increase with higher chitosan concentrations and with increasing zeta potential in plasma. Cell membranes, especially cancer cell membranes, are generally negatively charged due to negatively charged cell surface material such as phosphatidylserine, and thus the surface of cancer cells in the case of nanoparticles surface-modified with chitosan (ie NP ₁ , NP ₂ and NP ₃ ). Its electrostatic interaction with the negative charge promotes drug delivery to cancer cell lines. In addition, the adhesion of the chitosan to the cell surface increases the endocytosis of the nanoparticles, it can be seen that promotes drug delivery to cancer cell lines.

(6) in vitro cytotoxicity

Cytotoxicity of PLGA nano the paclitaxel is encapsulated particles cytotoxicity measurement of eseo han A549 cells incubated with serum of the mouse 24, 48 and 72 hours cytotoxicity measurement results Taxol ^TM (paclitaxel) in the back-containing nanoparticles results and The comparison result is shown in FIG. 5. Cytotoxicity of the nanoparticles is one increases the longer the incubation time (see Fig. 5 of A, B, C) Taxol ^TM Is weaker than It cradles parent cytotoxicity of pores EL ^TM used as a solvent of Taxol ^TM this appears to contribute to the effect of Taxol ^TM. The weak cytotoxicity of nanoparticles is due to delayed drug release from the nanoparticles. However, as the concentration of chitosan increases (delayed elution), cytotoxicity increases regardless of incubation time. This is presumably due to the increased uptake of nanoparticles into cells affected by the concentration of chitosan or zeta potential.

(7) Pharmacokinetics and Distribution of Paclitaxel

In mice with normal and metastatic lung cancer, Taxol ^TM , NP ₀ , and NP ₃ were dispersed in distilled water for injection in 10 mg / 10ml / kg mice, and the concentration and time of plasma and tissues were observed. The results are shown in FIG. In addition, drug kinetics and distribution parameters are shown in Tables 3, 4a, and 4b.

Parameter Normal mouse Metastatic lung cancer mouse Taxol ^TM NP ₀ NP ₃ Taxol ^TM NP ₀ NP ₃ CL (L / h / kg) 0.88 0.50 0.31 0.91 0.53 0.33 Vdss (L / kg) 0.92 1.93 2.55 0.96 2.01 2.68

Each value was calculated based on the mean plasma concentrations of four mice.

AUC and Tissue Distribution Indices of Paclitaxel after Taxol ^TM , NP ₀ , NP ₃ Administration at 10 mg / 10ml / kg as Paclitaxel in Normal CDF1 Mice group AUC ^a (μg · h / ml) Distribution indicator ^b Taxol ^TM NP ₀ NP ₃ Taxol ^TM NP ₀ NP ₃ plasma 11.4 18.7 24.6 One One One Heart 30.2 43.6 37.2 2.6 2.3 1.51 liver 232.4 569.8 445.6 20.4 30.5 18.1 spleen 50.7 359.8 165.6 4.4 19.2 6.7 lungs 54.6 117.2 1768.5 4.8 6.3 71.9 kidney 102.7 147.6 235.9 9.0 7.9 7.9

AUC and Tissue Distribution Indices of Paclitaxel after Taxol ^TM , NP ₀ , NP ₃ Administration at 10 mg / 10ml / kg as Paclitaxel in Metastatic Lung Cancer CDF1 Mice group AUC ^a (μg · h / ml) Distribution indicator ^b Taxol ^TM NP ₀ NP ₃ Taxol ^TM NP ₀ NP ₃ plasma 10.3 17.3 24.2 One One One Heart 31.3 42.5 34.6 3.0 2.5 1.4 liver 201.7 581.6 461.3 19.6 33.6 19.1 spleen 57.2 380.5 190.6 5.6 22.0 7.9 lungs 55.4 114.5 2416.5 5.4 6.6 99.9 kidney 95.4 131.5 246.3 9.3 7.6 10.2

In Tables 4A and 4B, a) all AUC values were calculated using the average paclitaxel concentration of four mice at each sampling time point; b) distribution index (Distribution Indexes) was calculated by the AUC _organization / AUC _{blood plasma} from each formulation.

Drug reduction in plasma of normal and metastatic lung cancer mice was slower in NP ₀ than in Taxol ^™ and the slowest in NP ₃ because of delayed release of the drug from the nanoparticles. Delayed excretion of drugs into the renal and biliary pathways is well known in nanoparticles. For the surface-modified PLGA nanoparticles (NP ₃ ) with chitosan, as can be seen in Table 2, the size increased to 2.7 μm, which appears to contribute to delayed excretion of the nanoparticles.

As can be seen in Table 3, systemic plasma clearance (CL) decreased in the order of Taxol ^™ , NP ₀ , NP ₃ in both normal and metastatic mice, which is consistent with the excretion delay in FIG. 6. Since the distribution volume (Vdss) increases in the order of Taxol ^TM , NP ₀ and NP ₃ , the distribution of drugs in different organs was observed to determine whether surface-modification by chitosan improved the distribution of drugs. 6 shows concentration-time trends of paclitaxel in various organs. The concentration-time trends varied between formulations and tissues. Taxol ^TM showed the highest concentrations in liver in both normal and diseased mice, followed by kidneys. In the case of NP ₀ , liver and spleen were in order, and NP ₃ was the highest in the order of lung and liver in pathological mice. AUC _tissues showed higher values than Taxol ^TM in NP ₀ and NP ₃ (Table 4), which is consistent with large Vdss (Table 3). In particular, the NP ₃ AUC Taxol _waste compared to the case of the ^TM than 32.4 times the normal mice, boyeoteum a value approximately 43.6 times the condition mouse NP0 was elevated about two-fold. The targeting efficiency of drugs to specific organs can be expressed using the tissue distribution index. This is the AUC ratio between tissue and plasma (AUCtissue / AUCplasma) NP0, which is higher in the liver and spleen than Taxol ^TM (Table 4), which means that the nanoparticles are present in Kupper cells (liver) in the reticuloendothelial system (RES) organ of the liver and spleen. And macrophage (splenic) to match the results. Surface by Chitosan - For the modified nanoparticles was the indicator is significantly increased in the lungs, in the case of normal mice Taxol ^TM was 15-fold higher than 4.8, NP ₀ in the case of NP ₃ to 6.3, NP ₃ is 71.9 Taxol ^TM . Increased distribution to the lungs supports an increase in Vdss of NP3, as can be seen in Table 3. Unlike the results in the lungs, the indicators in other organs of NP ₃ showed no difference or decreased, suggesting the priority of PLGA nanoparticles in the transition of paclitaxel to lungs in normal mice. Metastatic lung cancer mice showed similar results with the highest indices in the lungs of NP ₃ . Taxol ^TM is 5.4, NP ₀ is 6.5, but NP ₃ is 99.9, which is about 18.5 times higher than Taxol ^TM , which is higher than that in normal mice (71.9 vs 99.9).

In contrast, the above indicators in the heart showed that NP ₃ was lower than Taxol ^™ (ie 3.0 vs 1.4) and similar in liver, spleen and kidneys, thus having the advantage of avoiding cardiotoxicity of the drug. The preferential distribution of paclitaxel into the lung and reduced distribution to other organs in the metastatic lung cancer state represents a potential as a delivery system for surface-modified PLGA nanoparticles to lung cancer by chitosan. The accumulation mechanism of NP ₃ is presumed to be due to trapping in the pulmonary capillaries through reversible association formation (2.7 μm, Table 2) with plasma proteins in the bloodstream. When incubated surface-modified PLGA nanoparticles with chitosan in vitro with plasma, they form aggregates with components in the plasma, increasing the particle size to about 2700 nm. With agitation of the aggregates again, the particle size was observed to return to less than 300 nm, the original size, indicating that the aggregates are transient aggregates, not permanent aggregates.

The interaction of positively charged nanoparticles (eg NP ₃ ) with vascular endothelial cells of negatively charged cancers has been shown to promote further accumulation (see Table 4). The negative charge of vascular endothelial cells in cancer is believed to be due to overexpressed surface molecules (negative phospholipids, glycoproteins, proteoglycans).

As described above, the nanoparticles surface-modified by drugs such as paclitaxel-encapsulated chitosan greatly increase the average diameter of the nanoparticles in contact with plasma depending on the concentration of chitosan. In particular, in normal and metastatic lung cancer mice, the accumulation of drugs into the lung increased, indicating that the size of the nanoparticles increased in the plasma, the sustained release of the drug from the nanoparticles, and the cationicity of the nanoparticles and the anionicity of lung cancer vascular endothelial cells. Electrostatic interactions are believed to be involved in lung specific accumulation of the drug. In the case of nanoparticles, even though drug cytotoxicity is reduced, absolute ratios of nanoparticle accumulation in the lung, increased uptake of nanoparticles into cancer cells, and sustained release of the drug from cancer cells are surface-modified with chitosan. Nanoparticles are useful as a lung specific drug delivery system in lung disease patients, including lung cancer patients.

1 is surface-modified with paclitaxel (A), chitosan powder (B), surface-modified PLGA nanoparticles (blank NP, C), encapsulated PLGA nanoparticles (NP ₀ , D), 0.3% chitosan PLGA nanoparticles encapsulated with paclitaxel (NP ₁ , E), PLGA nanoparticles encapsulated with paclitaxel (NP ₂ , F) surface-modified with 0.5% chitosan, and PLGA encapsulated with paclitaxel surface-modified with 0.7% chitosan The X-ray diffraction pattern of the drug of the nanoparticles (NP ₃ , G) is shown.

Figure 2 is surface-modified with paclitaxel (A), chitosan powder (B), surface-modified PLGA nanoparticles (blank NP, C), PLGA nanoparticles with paclitaxel (NP ₀ , D), 0.3% chitosan PLGA nanoparticles encapsulated with paclitaxel (NP ₁ , E), PLGA nanoparticles encapsulated with paclitaxel (NP ₂ , F) surface-modified with 0.5% chitosan, and PLGA encapsulated with paclitaxel surface-modified with 0.7% chitosan SEM images of the nanoparticles (NP ₃ , G) are shown.

Figure 3 shows the dissolution rate of paclitaxel in phosphate-buffered saline (PBS), 0.01 M, pH 7.4 at 37 ° C. Each point represents the mean ± SD of three experiments.

4 shows cellular uptake of coumarin 6 by A549 cells for 2 hours from plasma distribution, depending on the concentration of chitosan solution used for surface-modification and the zeta potential of nanoparticles in plasma (mean ± SD, n = The result of measuring 4) is shown.

5 shows Taxol ^™ (□), NP ₀ (VII), NP ₁ (VII), NP ₂ (VII), and NP ₃ (■) for 24 hours (A), 48 hours (B) or 72 hours (C). ) Shows the result of measuring cell viability (mean ± SD, n = 8) of A549 cells. *: P <0.05, **: P <0.001.

6 shows plasma of paclitaxel after administration of Taxol ^™ (■), NP ₀ (▲), and NP ₃ (●) at a dose of 10 mg / 10ml / kg through the tail vein of normal CDF1 mice and metastatic lung cancer CDF1 mice. And the results of measuring tissue concentration-time trends.

Claims (7)

A surface-modified nanoparticle coated with chitosan on the surface of a nanoparticle consisting of polylactide or a copolymer thereof and an active ingredient, wherein the active ingredient is encapsulated inside the nanoparticle. A pharmaceutical composition for lung-targeting, comprising nanoparticles. The method of claim 1, wherein the copolymer of polylactide is a copolymer of D, L-lactide and glycolide, a copolymer of L-lactide and glycolide, D, L-lactide and L-lac A group consisting of a copolymer of tide, a copolymer of L-lactide and trimethylene carbonate, a copolymer of L-lactide with ε-caprolactone, and a copolymer of glycolide with L-lactone Pharmaceutical composition, characterized in that at least one selected from. The polylactide copolymer of claim 1, wherein the copolymer of polylactide has a copolymerization ratio of lactide and glycolide in the range of 50:50 to 90:10. -lactide-co-glycolide). The pharmaceutical composition of claim 1, wherein the active ingredient is an anticancer agent or a pulmonary tuberculosis agent. The pharmaceutical composition according to claim 1, wherein the active ingredient is paclitaxel. The pharmaceutical composition according to any one of claims 1 to 5, wherein the chitosan has a weight average molecular weight of 10 to 150 kDa. The pharmaceutical composition according to any one of claims 1 to 5, wherein the content of chitosan coated on the nanoparticles is 0.1 to 5 wt% based on the total weight of the nanoparticles.

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