친수성기로 개질된 Poly(DL-lactide-co-glycolide)의 나노입자 제조

배은영, 나재운*

순천대학교 공과대학 고분자공학과

(jwnah@sunchon.ac.kr)

Preparation of Poly(DL-lactide-co-glycolide) Nanoparticles Modified

with Hydrophilic Group

Eun Yeong Bae, Jae-Woon Nah*

Dep. of Polymer Science and Engineering, Sunchon National University, Sunchon, 540-742, South Korea

(jwnah@sunchon.ac.kr)

1. Introduction

Nanoparticles or colloidal carriers have been extensively investigated in biomedical and biotechnological ares and, especially, in drug delivery systems for drug targeting1 because their particle sizes (ranged from 10 to 100 nm) are acceptable for intravenous (i.v.) injection2.

Although various polymers are possible to make nanoparticles for drug delivery system in vitro, polymeric materials used to prepare microsphers or nanoparticles for administration into the human body are significantly limited to a few kinds of acceptable polymers. Among them, poly(L-lactide) or poly(DL-lactide) (PLA), poly(glycolide) (PGA), and their copolymers such as poly(DL-lactide-co-glycolide) (PLGA) are one of the most widely used biodegradable polymers to make micro or nanoparticles for controlled drug delivery systems. The preparation method of nanoparticles is a critical problem for small sized particles3. For this study, we have prepared surfactant-free PLGA nanoparticles by dialysis method without surfactant using various solvent. Recently, dialysis method was developed for the simple preparation of drug carriers such as liposomes and polymeric micells4. Poly(DL-lactide-co-glycolide) (PLGA) has two types ; one is PLGA (RG 502) and the other PLGA (RG 502H). RG 502 is hydrophobic due to carboxyl group and RG 502H is hydrophilic due to carboxyl group. Changes of particle size, and physicochemical properties of PLGA nanoparticles are investigated against various solvents and type of PLGA.

2. Materials and methods

2.1 Materials

PLGA (RG 502) and PLGA (RG 502H) were purchased from Sigma Chemicals, USA. Molecular weight of PLGA (RG 502) 85:15, 75:25 and 50:50 was 48400, 47500 and 40100 Da from our GPC measurement as described below. PLGA (RG 502H) 50:50 was 18013 Da.  Various solvents, i.e. dimethylfomamide (DMF), dimethylsulfoxide (DMSO), dmethylacetamide (DMAc) and acetone, as a reagent grade were used without further purification.

2.2 Preparation of PLGA nanoparticles

PLGA nanoparticles procedure was carried out as followed : Twenty milligrams of PLGA was dissolved in 10 ml of various solvents. The solution was introduced into dialysis tube (molecular cutoff 12000 g/mol) and dialyzed against 1.0 L×3 of distilled water for 3 hrs and then distilled water exchange at intervals of 3∼4 hrs during 24 hrs. The resultant solution was freeze-dried.

2.3. Gel permeation chromatography (GPC) measurement

Molecular weight of PLGA was measured from Waters LC system coupled with a Waters 410 Differential Refractometer using Waters StyragelTM HR1, HR2 and HR4 Column at a flow rate of 1 ml/min. THF was used as an eluent. Average molecular weight was evaluated by polystyrene as a standard.

2.4. Scanning electron microscope (SEM) observation

The morphology of the nanoparticles was observed using a SEM (JEOL, JSM-5400, Japan). A drop of the nanoparticle suspension was placed on a graphite surface. After freeze-drying, the sample was coated with gold/palladium using an Ion Sputter (JEOL, JFC-1100). Coating was provided at 20 mA for 4min. Observation was performed at 25 kV.

2.5. Transmission electron microscope (TEM) observation

A drop of nanoparticles suspension containing 0.01% of phosphotungstic acid was placed on a carbon film coated on a copper grid for TEM. Observation was done at 80 kV in a JEOL, JEM-2000 FX-11, Japan.

2.6. Photon correlation spectroscopy (PCS) measurements

PCS was measured with a Zetasizer 3000 (Malvern instruments, England) with He-Ne laser beam at a wavelength of 633 nm at 25℃ (scattering angle of 90°).  A nanoparticle solution prepared by dialysis method was used for particle size measurement (concentration : 0.1 wt.-%) and measured without filtering.

2.7. X-ray diffractometer measurement

X-ray release experiment in vitro was obtained with a Rigaku D/Max-1200 (Rigaku) using Ni-filtered CuK □ radiation 935 kV, 15 mA).

2.8. Atomic force microcope (AFM) observation

The morphology of PLGA nanoparticles was confirmed by AFM. Nanoparticle of 0.1 mg/ml in distilled water was placed on a silicon wafer surface. AFM image was obtained by (PARK's Science, Autoprobe CP) at room temperature with cantilever oscillation frequencies between 12 and 24 kHz.

3. Result and discussion

These surfactant-free PLGA nanoparticles were characterized by analysis of photon correlation spectroscopy, scanning electron microscope, and transmission electron microscope, etc.. The particle size of PLGA (RG 502) nanoparticles from DMF, DMSO, and DMAc were about 200∼400 nm. But, when acetone as an initial solvent was used to make particles, PLGA (RG 502) particle size was increased and their size were 500∼1000 nm and also have nice spherical shapes. And the particle size of PLGA (RG 502H) nanoparticles from DMF, DMSO, and DMAc were about 300∼600 nm. But, when acetone as an initial solvent was used to make particles, PLGA (RG 502) particle size was increased and their size were 500∼1000 nm and also have nice spherical shapes.

4. Conclusion

The surfactant-free PLGA nanoparticles were prepared by dialysis method and their physicochemical properties were investigated against used an initial solvent. The size of PLGA (RG 502) nanoparticles prepared from DMAc, DMF, and DMSO as a initial used solvent was smaller than that of acetone. Selected initial solvent used to dissolve the copolymer significantly affects the size of nanoparticles. PLGA nanopanicles have spherical shapes from the observations of SEM and TEM. The analysis of X-ray powder diffraction patterns showed potential of surfactant-free nanopanicles of PLGA (RG 502) as a drug carrier system. PLGA (RG 502H) is the similar. From these results, we have studied drug contents of PLGA (RG 502H).

References

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2. Kreuter, J., J. Control-Release 16, 169(1993)

3. Gref, R., Minamitake, Y., Peracchia, M. T., Trubetskoy, V., Torchilin, V., Langer,      R., Science 263, 1600(1994)

4. Nah, J. W., Jeong, Y. I., Cho, C. S., J. Polym-Sci. B: Polym. Phys. 36, 415(1998)

5. Peracchia, M. T., Gref, R., Minamitake, Y., Domb, A., Lotan, N., Langer, R., J.         Control-Release 46, 223(1997)