WO2012138013A1

WO2012138013A1 - Paclitaxel-loaded polymeric nanoparticle and preparation thereof

Info

Publication number: WO2012138013A1
Application number: PCT/KR2011/005062
Authority: WO
Inventors: E. Geckeler KURT; Yeonju LEE
Original assignee: Gwangju Institute Of Science And Technology
Priority date: 2011-04-07
Filing date: 2011-07-11
Publication date: 2012-10-11
Also published as: KR101314579B1; KR20120115441A

Abstract

Provided are paclitaxel-loaded polymeric nanoparticles, including paclitaxel and a water-soluble polymer with which the surface of paclitaxel is surrounded and coated, wherein the polymer is poly[2-(dimethylamino)ethyl methacrylate-co-methacrylic acid (PDM). The paclitaxel-loaded polymeric nanoparticles are those having paclitaxel surrounded with a copolymer, have water solubility and biocompatibility, and release and transport the drug, paclitaxel, to cancer cells having multi-drug resistance (MDR), thereby providing an anti-cancer effect several thousand times higher than the effect of original paclitaxel.

Description

PACLIT AXEL-LOADED POLYMERIC NANOPARTICLE AND PREPARATION

THEREOF

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to paclitaxel-loaded polymeric nanoparticle and preparation thereof.

DESCRIPTION OF THE RELATED ART

Cancer has complicated symptoms and is diagnosed in a variety of cases of ten millions or more, and thus is regarded as one of the main global diseases. Currently, anti-cancer agents are used to treat cancer but they frequently cause many problems due to their side effects, despite their therapeutic effects.

In particular, occurrence of multi-drug resistance (MDR) is a great obstacle in treating cancer with anti-cancer agents. The most general mechanism related to MDR is that a drug is not bonded to cancer cells but is released owing to ATP binding- ABC transporter protein. It is reported that one of ABC transporter proteins, i.e., P- glycoprotein (P-glycoprotein encoded by MDR-1) is a main cause of MDR. P- glycoprotein is one of the proteins serving to transport a material that acts specifically to cancer cells in various structural and functional modes. Over-expression of P- glycoprotein on cancer cells reduces drug accumulation in the cells, resulting in degradation of therapeutic effects of anti-cancer agents.

Therefore, in order to overcome the drug resistance caused by a P- glycoprotein inhibitor, some anti-cancer agents encapsulated with polymeric nanoparticles capable of interacting with P-glycoprotein are reported.

Recently, nanoparticles have received increasing attention in the field of cancer treatment. Particularly, nanoparticles have been spotlighted because they have permeability through a specific biological barrier, immunological or non-specific interaction causing cytotoxicity, and functional binding ability to a specific polymer, such as polyethylene glycol, thereby preventing interaction with blood plasma protein.

More particularly, nanoparticles improve intracellular accumulation of anticancer agents and drug delivery into cells so as to overcome drug resistance with ease. For example, some anti-tumor antibiotics, such as doxorubicin or paclitaxel, show an IC₅₀ value six to nine times lower than the effect of inhibiting cellular activity provided by doxorubicin or paclitaxel itself due to the effect of P-glycoprotein over- expressed in the human body. In addition, paclitaxel-loaded polycaprolactam/ Poloxamer 188 polymeric nanoparticles show an IC₅₀ value to drug-resistant cells up to ten times lower than taxol, an anti-cancer agent collected from Taxus cuspidate.

Paclitaxel extracted from Taxus brevifolia is one of the main anti-cancer agents. Paclitaxel is a leader of new anti-tumor drugs, is effective for treating some main cancers, such as ovarian cancer or breast cancer, and has a unique acting mechanism of inhibiting growth and isolation of cancer cells.

However, paclitaxel is problematic in that it requires addition of adjuvants, such as Cremophor EL (polyethoxylated castor oil derivative), to a drug formulation due to its insolubility in water, and it causes side effects, including allergic hypersensitivity, hyperlipidemia and abnormal protein pattern synthesis, due to ethanol toxicity in chemotherapeutic treatment.

Therefore, there is an imminent need for a paclitaxel/polymeric nanoparticle complex having water solubility and biocompatibility in order to increase the anticancer effect of paclitaxel and to overcome drug resistance.

SUMMARY OF THE INVENTION

The present inventors have made intensive studies to provide paclitaxel- loaded polymeric nanoparticles including paclitaxel and a polyampholyte, to which paclitaxel loaded, having both acidic activity and basic activity. The paclitaxel-loaded polymeric nanoparticles are capable of delivering paclitaxel effectively to cancer cells having multi-drug resistance (MDR), thereby providing an increased anti-cancer effect.

Accordingly, it is an object of this invention to provide a method for preparing the paclitaxel-loaded polymeric nanoparticles from paclitaxel and the polymeric material.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the present disclosure will become apparent from the following description of certain exemplary embodiments given in conjunction with the accompanying drawings, in which:

Fig. 1 is a schematic view of a solid state reaction for preparing the paclitaxel- loaded polymeric nanoparticles according to an embodiment of the present disclosure, wherein A represents a structural formula of paclitaxel and B represents a structural formula of copolymer, poly[2-(dimethylamino)ethyl methacrylate-co-methacrylic acid (PDM);

Fig. 2a is a transmission electron microscopy (TEM) image of paclitaxel;

Fig. 2b is an scanning electron microscopy (SEM) image of paclitaxel loaded on PDM by pretreatment according to an embodiment of the present disclosure;

Fig. 2c is a graph showing the size of the nanoparticles according to an embodiment of the present disclosure;

Fig. 3 is a graph showing the extent of drug release from the paclitaxel- loaded nanoparticles under different pH conditions of: (1) pH 5.2 and (2) pH 7.4;

Fig. 4 is a graph showing the effective values IC₅₀ against cellular activities when using paditaxel solution (PAX), paclitaxel-loaded polymeric nanopartides (PP), or mixed solution of paditaxel with PDM (PDM+PAX);

Fig. 5a is a fluorescence image showing the intracellular distribution of fluorescein isothiocyanate (FITC) conjugated with the paclitaxel-loaded nanopartides according to an embodiment of the present disclosure, in MCF7 cells;

Fig. 5b is a graph showing the results of flow cytometric analysis carried out in MCF7 cells at 37°C under 10% carbon dioxide using different conditions of: (1) non-labeled cells, (2) F1TC-PP at a temperature of 4°C or lower, (3) FITC-PP at 37°C under 10% carbon dioxide, (4) FITC-PP with depleted ATP;

Fig. 5c is a fluorescence image showing the intracellular distribution of FUC conjugated with the paclitaxel-loaded nanopartides according to an embodiment of the present disclosure, in MCF7/ADR cells; and

Fig. 5d is a graph showing the results of flow cytometric analysis carried out in MCF7/ADR cells at 37°C under 10% carbon dioxide using different conditions of: (1) non-labeled cells, (2) FITC-PP at a temperature of 4°C or lower, (3) FITC-PP at 37°C under 10% carbon dioxide, (4) FITC-PP with depleted ATP.

DETAILED DESCRIPTION OF THIS INVETNION

In an aspect of this invention, there are provided paclitaxel-loaded polymeric nanopartides, comprising paditaxel and a water-soluble polymer with which the surface of paditaxel is surrounded and coated, wherein the polymer is poly[2- (dimethylamino)ethyl methacrylate-co-methacrylic acid (PDM).

Accordingly, it is an object of this invention to provide a method for preparing the paclitaxel-loaded polymeric nanopartides from paditaxel and the polymeric material.

In addition, the present invention provides paclitaxel-loaded polymeric nanoparticles, obtained by mixing PDM with paclitaxel, followed by milling, to form a complex, dissolving the complex into water, and carrying out centrifugal separation.

According to an embodiment, PDM may be obtained by subjecting methacrylic acid (MAA) monomer and 2-(dimethylamino)ethyl methacrylate (DMAEMA) monomer to radical polymerization under nitrogen atmosphere.

According to another embodiment, MAA may be present in the polyampholyte, PDM, in a molar ratio of 5-30%.

According to still another embodiment, the paclitaxel-loaded polymeric nanoparticles may have a particle size of 120-600 nm.

In another general aspect, the present invention provides an anti-cancer agent composition including the paclitaxel-loaded polymeric nanoparticles as an active ingredient.

In still another aspect, there is provided a method for treating cancer, comprising administering to a mammalian subject a pharmaceutical composition comprising the paclitaxel-loaded polymeric nanoparticles described above as an active ingredient.

The paclitaxel-loaded polymeric nanoparticles according to the present invention include paclitaxel represented by the following Structural Formula 1, and a water soluble polymer with which the surface of paclitaxel is surrounded and coated:

[Structural Formula 1]

The polymer is a polyampholyte that may be ionized as both cation and anion and has both acidic activity and basic activity. Particularly, the polymer is poly[2- (dimethylamino)ethylmethaci late-co-methacrylic acid (PDM).

PDM is represented by the following Structural Formula 2 and includes, as repeating units, a weak base portion (2-(dimethylamino)ethyl methacrylate, D) and a weak acid portion (methacrylic acid, M):

[Structural Formula 2]

PDM is a material liable and decomposable under an acidic condition, and thus may be decomposed under an acidic pH of tissues in vivo to release paclitaxel. In other words, since tumors generally have acidity, PDM allows paclitaxel to be released and transported effectively to tumor tissues.

In addition, PDM may be obtained by radical polymerization of methacrylic acid (MM) monomer and 2-dimethylaminoethyl methacrylate (DMAEMA) monomer under nitrogen atmosphere. MM may be present in the polyampholyte, PDM, in a molar ratio of 5-30%.

In another aspect, the present disclosure provides a pharmaceutical composition for treating cancer, which includes the paclitaxel -loaded polymeric nanoparticles as an active ingredient. The cancer may be any one selected from the group consisting of leukemia, encephaloma, kidney cancer, stomach cancer, skin cancer, bladder cancer, breast cancer, uterine cancer, lung cancer, colon carcinoma, prostatic carcinoma, ovarian cancer, liver cancer, large intestine cancer, peritoneum cancer, peritoreal metastases and pancreatic cancer.

As used herein, the 'pharmaceutical composition' may include the nanoparticles according to the present disclosure in combination with a pharmaceutically acceptable carrier, diluent, excipient or a combination thereof, as desired. The term 'carrier' means a material that facilitates addition of a compound into cells or tissues. In addition, the term 'diluent' means a material that stabilizes the biologically active form of a target compound and is diluted with water into which the compound is dissolved.

The pharmaceutically acceptable carrier is one used generally in the field of pharmaceutical formulation. Particular examples of the pharmaceutically acceptable carrier include, but are not limited to: lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talcum, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition may be administered via a parenteral route. In the case of parenteral administration, any parenteral route, such as intravenous injection, intramuscular injection, intra-articular injection, intra- synovial injection, intrathecal injection, intrahepatic injection, intralesional injection or intracranial injection, may be used.

Adequate dose of the pharmaceutical composition according to the present disclosure may be determined by various factors, including formulation methods, administration modes, age, body weight, sex and physical state of a patient, diet, administration periods, administration routes, excretion rates and reaction sensitivity. For example, the pharmaceutical composition of the present invention may be administered with a daily dose of 0.001-1000 mg/kg (body weight).

Other terms and abbreviations used herein may be understood as their meanings recognized generally by those skilled in the art, unless otherwise defined.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

EXAMPLE 1: Preparation of Paclitaxel-Loaded Polymeric Nanoparticles

(1) As the monomers used to obtain the nanoparticles according to the present disclosure, methacrylic acid (MM) is purchased from Jassen Chimica Co. and 2-(dimethylamino)ethyl methacrylate (DMAEMA) is purchased from Aldrich Co.

As the anticancer agents, paclitaxel is obtained from Bolak (Lot No.

200805001, South Korea) and doxorubicin hydrochloride is obtained from AK

Scientific, Inc. (USA).

The MTT reagent used in MTT assay for a cytotoxicity test (thiazoyl blue tetrazolium bromide, 97.5% TLC), bisbenzimide H 33258, fluorescein isothiocyanate (FITC), sodium azide, rhodamine 123 and 2-deoxy-D-glucose are purchased from

Sigma-Aldrich Co. In addition, acetonitrile (HPLC Gradient grade) and dimethyl sulfoxide (DMSO) are obtained from LAB-SCAN Co. and Roth Co., respectively.

Further, DMEM (GlutaMAX) and penicillin-stereptomycin used for cell culture are purchased from Gibco Co., and fetal bovine serum (FBS) is obtained from PAA Laboratories GmbH.

(2) Synthesis of Polyampholyte

Poly[2-(dimethylamino)ethylmethacrylate-co-methacrylic acid (PDM) is prepared by radical polymerization of MAA and DMAEMA under nitrogen atmosphere.

Molar fractions of PDM are analyzed by elemental analysis (Thermo Quest Analyzer, EA-1110). Mass spectrometry is carried out by using a light scattering system (SLS, DLS-8000, Otsuka Electronics Co., Ltd., cylindrical optical cell (diameter: 1.9 cm)). The specific refractive index of the copolymer is analyzed by DRM-3000 (Otsuka Electronics Co., Ltd.)- In addition, pH measurement is carried out by a pH meter (ThermoOrion, Model 550A). Further, the surface charge of the copolymer, PDM, is determined by ELS-Z (Otsuka Electronics Co., Ltd.).

(3) Preparation of Paclitaxel-Loaded Polymeric Nanoparticles

Paclitaxel and PDM are introduced into a stainless steel capsule together with mixing balls and are mixed at room temperature under 20 Hz. Next, the resultant mixture is dissolved into water at a concentration of 1 mg/mL and subjected to centrifugal separation for 10 minutes to remove non-complexed paclitaxel. Then, water is added thereto, the resultant mixture is further subjected to centrifugal separation and the supernatant is collected to recover paclitaxel-loaded polymeric nanoparticles. Finally, the supernatant is freeze dried.

The particle shape and surface charge characteristics are determined by SEM and ELS, respectively. Then, in order to determine the particle size, the freeze dried powder is dissolved into water, followed by sonication for 1 minute. The particle size is determined by using a light scattering system and the paclitaxel loading efficiency is measured by UV-Vis spectroscopy.

It can be seen that after the paclitaxel present in the form of sticks as shown in Fig. 2a undergoes a solid state reaction with PDM, the paclitaxel is transformed into a nano-sized spherical shape as shown in Fig. 2b. As the paclitaxel is transformed into a nano-sized spherical shape, drug release and drug dispersion mechanisms may be affected thereby. Particularly, nanoparticles smaller than 250 nm are accumulated in tumor cells efficiently, thereby providing an anticancer effect.

In the case of the paclitaxel-loaded polymeric nanoparticles obtained according to an embodiment of the present disclosure, it can be seen that the average particle size is 250 nm when determined by dynamic light scattering (DLS), as shown in Fig. 2c.

EXAMPLE 2: Drug Release Test First, 10 mg of the paclitaxel-loaded polymeric nanoparticles are introduced into a dialysis bag. To carry out a drug release test, acetate solution (pH 5.2) and phosphate buffer solution (pH 7.4) containing 2% fetal bovine serum (FBS) and 0.02% sodium azide are used as releasing media.

The nanoparticles contained in the dialysis bag are introduced into 20 ml. of the releasing media, followed by incubation at 37°C. The paclitaxel content is analyzed by HPLC (Zorbax C18 column (5 pm, 4.5X250 mm), watenacetonitrile = 45:55, UV 230 nm). EXAMPLE 3: Cell Culture

Cell culture is carried out in a DMEM medium containing 10% FBS and 1% penicillin stereptomycin under the conditions of 10% carbon dioxide and 37°C. Doxorubicin-resistant cells, MCF7/ADR and MT3/ADR are cultured in a medium containing 0.1 g/mL of doxorubicin.

EXAMPLE 4: Evaluation of Cytotoxicity

To determine anti-cancer effects against MCF7, MCF7/ADR, MT3 and MT3/ADR, MTT colorimetric assay is used. To a 96-well tray, MCF7 and MCF7/ADR are seeded at a concentration of 1.5X10⁴ cells/well, and MT3 and MT3/ADR are seeded at a concentration of 1.5X10⁴ cells/well. Then, the cells are cultured in a 10% C0₂ incubator at 37°C for 24 hours. The pristine paclitaxel, PDM, and PP are treated for 72 hours.

EXAMPLE 5: Analysis of Cellular Uptake

To analyze cellular uptake, FlTC-labeled paclitaxel-loaded polymeric nanoparticles are subjected to flow cytometric analysis (FACS).

In the FITC-labeled paclitaxel-loaded polymeric nanoparticles (FITC-PP), isothiocyanate groups of FITC interact with amine groups of PDM. FITC-PP is provided as a solution of 1 mg/mL of FITC dissolved in DMSO, and 10 μΙ_ of the FITC solution is added to 10 mL of a dispersion of nanoparticles, followed by incubation for 2 hours. FUC-PP is subjected to dialysis for 2 days using a cellulose dialysis membrane in the dark with water exchanged every 12 hours.

To perform FACS, MCF7 and MCF7/ADR cells are cultured at 37°C and 4°C for

30 minutes, or pretreated with 10 mM NaN₃ and 50 mM 2-deoxy-D-glucose for 30 minutes. Then, each pretreated cell is further treated with 0.1 μΜ FUC-PP.

EXAMPLE 6: Paclitaxel Loading Efficiency

The loading efficiency is measured by UV-Vis spectroscopy. The paclitaxel loading efficiency may be measured through the interrelation between the absorbances of paclitaxel and PDM. Paclitaxel and PDM are determined at 280 nm and 230 nm, respectively. After the determination at 280 nm and 230 nm, it is shown that e_PAx,28o = 1225 L/mol cm, e_PAx₍23o = 28316 L/mol cm e_PDM,28o = 58529 L/mol cm, and e_PDM,23o = 649850 L/mol cm. As a result, the paclitaxel loading efficiency according to the present disclosure is shown to be 57.62%.

EXAMPLE 7: Paclitaxel Release

The paclitaxel-loaded polymeric nanoparticles are surrounded with a water soluble material, PDM, and thus have water solubility. Therefore, controlling release of paclitaxel from the nanoparticles is determined by pH on which PDM depends.

PDM has acid-liable and chemically decomposable functional groups on its backbone or side chain. Thus, the surfaces of paclitaxel-loaded nanoparticles are decomposed to release the drug, paclitaxel, as a function of pH. In this manner, the system capable of delivering a drug to cells as a function of pH allows effective drug release, thereby transporting the drug to a specific tissue.

In general, tissues and blood have a pH of 7.4. However, most tumor tissues have a pH of 5.7-7.8. Therefore, the paclitaxel-loaded nanoparticles according to the present disclosure respond to pH so that they release and transport the drug directly to cancer cells, thereby providing an excellent anti-cancer effect.

Fig. 3 is a graph showing the extent of drug release from the paclitaxel- loaded nanoparticles under different pH conditions of: (1) pH 5.2 and (2) pH 7.4. More particularly, it can be seen that paclitaxel is released more rapidly at pH 5.2 than pH 7.4. After carrying out incubation for 24 hours, drug release is increased by 30% under an acidic condition as compared to pH 7.4 (total drug release is about 60%).

(1) Effect of Paclitaxel-Loaded Nanoparticles upon Inhibition of Cancer Cells To determine the resistance caused by over-expression of P-glycoprotein (P- gp) on MCF7, MCF7/ADR, MT3 and MT3/ADR, western blot and rhodamine 123 assay are carried out.

Fig. 4 and the following Table 1 show the effective values IC₅₀ against cellular activities when using the paclitaxel-loaded polymeric nanoparticles according to the present disclosure.

Table 1

PAX: paclitaxel in solution

PP: paclitaxel-loaded polymer nanoparticles

PDM + PAX: poly[2-(dimethylamino)ethyl methacrylate-co-methacrylic acid]) and paclitaxel in solution

Referring to Table 1, PP of the present disclosure provides an effect of inhibiting cancer cells several ten times to several thousand times higher than the effect provided by original PAX.

EXAMPLE 8: Evaluation of Intracellular Accumulation and Internalization of Nanoparticles

To determine the intracellular accumulation and internalization of PP according to the present disclosure, FACS and a confocal microscope are used to observe the intracellular distribution of FTIC-PP. Nuclear labeling is carried out by using bisbenzimide.

The drug-loaded nanoparticles are incorporated by cellular membranes through endocytosis to increase the intracellular accumulation, thereby providing an improved therapeutic effect. Particularly, in the case of cancer cells having multidrug resistance, the drug loaded on the polymeric nanoparticles is not transported to the outside of the cells by P-gp but is accumulated in the cells, thereby providing an improved therapeutic effect.

Endocytosis is inhibited under a low temperature (4°C) or an ATP-depleted condition. To confirm this, several tests are carried out under an ATP-depleted condition (pretreatment with NaN₃ and 2-deoxy-D-glucose) at 4°C and 37°C, and under a general condition of 37°C. The test results are shown in Figs. 5a to 5d.

As shown in Figs. 5a to 5d, it can be seen that cellular uptake of PP occurs in MCF7 and MCF7/ADR. Thus, it can be seen through the fluorescence image that FITC-PP is accumulated in the cells through endocytosis and FITC-PP is internalized in the cells. As a result, paclitaxel is transported effectively to cancer cells, while providing an increased concentration in the cells. This suggests that PP according to the present disclosure provides an excellent therapeutic effect.

As mentioned above, over-expression of P-gp encoded by multi-drug resistant genes leads to drug resistance in various types of anti-cancer chemotherapy, and thus it is regarded as a main obstacle in cancer treatment.

The paclitaxel-loaded polymeric nanoparticles obtained according to the present disclosure include paclitaxel surrounded with a polymer having a hydrophilic surface, and have a nano-scaled size. Therefore, it is possible to transport and release paclitaxel effectively to a specific cancer cell, and thus provide an excellent anti-cancer effect against cells having multi-drug resistance. Particularly, the paclitaxel-loaded nanoparticles of the present disclosure have an effect 100-400 times higher than the effect of original paclitaxel.

The present application contains subject matter related to Korean Patent Application No. 10-2011-0032273, filed in the Korean Intellectual Property Office on Apr. 7. 2011, the entire contents of which are incorporated herein by reference.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present disclosure. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the disclosure as set forth in the appended claims.

Claims

What is claimed is:

1. Paclitaxel-loaded polymeric nanoparticles, comprising paclitaxel and a water- soluble polymer with which the surface of paclitaxel is surrounded and coated, wherein the polymer is poly[2-(dimethylamino)ethyl methacrylate-co-methacrylic acid (PDM).

2. The paclitaxel-loaded polymeric nanoparticles according to claim 1, which are obtained by mixing PDM with paclitaxel, followed by milling, to form a complex; and dissolving the complex into water and carrying out centrifugal separation.

3. The paclitaxel-loaded polymeric nanoparticles according to claim 2, wherein PDM is obtained by subjecting methacrylic acid (MAA) monomer and 2- (dimethylamino)ethyl methacrylate (DMAEMA) monomer to radical polymerization under nitrogen atmosphere.

4. The paclitaxel-loaded polymeric nanoparticles according to claim 1, wherein MAA is present in PDM in a molar ratio of 5-30%.

5. The paclitaxel-loaded polymeric nanoparticles according to claim 1, which have a particle size of 120-600 nm.

6. A pharmaceutical composition for treating cancer, which comprises the paclitaxel- loaded polymeric nanoparticles according to any one of claims 1 to 5 as an active ingredient.

7. A method for treating cancer, comprising administering to a mammalian subject a pharmaceutical composition comprising the paclitaxel-loaded polymeric nanoparticles according to any one of claims 1 to 5 as an active ingredient.