Young-Il Jeong

Clonazepam release from core-shell type nanoparticles in vitro

Block copolymers consisting of poly(gamma-benzyl L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene oxide) (PEO) as the hydrophilic block were synthesized and characterized. Core-shell type nanoparticles of the block copolymers (abbreviated as GE) were prepared by the diafiltration method. The particle size diameter obtained by dynamic light scattering of GE-1 (PBLG content: 60.5 mol%), GE-2 (PBLG content: 40.0 mol %), GE-3 (PBLG content: 124.4 mol %) copolymer was 309.9 +/- 160.9, 251.9 +/- 220.6 and 200.5 +/- 177.1nm, respectively. The shape of the nanoparticles by SEM or TEM was almost spherical. The critical micelle concentration of the block copolymers obtained by fluorescence spectroscopy was dependent on the chain length of hydrophobic PBLG. The micelle structure of the copolymers nanoparticle was very stable against sodium dodecyl sulfate. Clonazepam (CZ) was loaded onto the core part of the nanoparticle as the crystalline state. Release of CZ from the nanoparticles in vitro was dependent on the drug loading contents and PBLG chain length.

Adriamycin release from flower-type polymeric micelle based on star-block copolymer composed of poly(γ-benzyl l-glutamate) as the hydrophobic part and poly(ethylene oxide) as the hydrophilic part

Star-block copolymer based on PBLG as the hydrophobic part and PEO as the hydrophilic one (as abbreviated GEG) was synthesized and characterized. Polymeric micelle was prepared by the diafiltration method. From the measurement of photon correlation spectroscopy, the nanoparticle sizes of GEG-1, GEG-2 and GEG-3 were 106.5+/-59.2, 43.8+/-0.7 and 13.5+/-1.0 nm in number average, respectively, indicating of the formation of polymeric micelle. Also, the nanoparticle sizes were dependent on the PBLG chain length, i.e. the more PBLG content in the copolymer, the larger the particle size. From the observation of transmission electron microscope(TEM), GEG-2 block copolymer had almost spherical shapes with size range about 20-70 nm, that was similar to particle size measurement. Fluorescence spectroscopy measurement indicated that GEG block copolymers associated in water to form polymeric micelles and critical micelle concentration (CMC) values of the block copolymers decreased with increasing PBLG chain length in the block copolymer. Characteristic peaks of the protons of the benzyl group in the PBLG and the methylene protons adjacent to the benzyl group of the PBLG segment in the GEG-2 nanoparticles appeared in 7.2 approximately 7.4 and 5.0 approximately 5.2 ppm, respectively, and disappeared in D(2)O, indicating the restricted motions of these protons within the micellar core and the very rigid structure of the PBLG core in the GEG polymeric micelles. Release of ADR from the polymeric micelles in vitro was slower in longer PBLG chain length and higher loading contents of ADR.

Preparation of poly(dl-lactide-co-glycolide) microspheres encapsulating all-trans retinoic acid

Poly(DL-lactide-co-glycolide) (PLGA) microspheres containing all-trans retinoic acid (atRA) were prepared by o/w solvent evaporation method and various preparation parameters, such as poly(vinyl alcohol) (PVA) concentration in aqueous solution, PVA MW, drug weight, solvent, polymer MW, and polymer weight, on the characteristics of microspheres and drug release were investigated. PVA concentration in water phase was a critical factor in making microspheres consistently with smooth surface and round shape. In our study, at least 2% (w/v) of PVA in aqueous solution was necessary for making microspheres with round shape. The particle size of microspheres ranged 10-100 microm. AtRA was slowly released from PLGA microspheres over 30 days. Sterilization of microspheres by ethylene oxide (EO) gas at 37 degrees C did not significantly affect the characteristics of drug release or its morphology. Cell growth inhibition of atRA was affected by preparation process of microspheres rather than the EO-gas sterilization process. These results indicate that PLGA microspheres containing atRA are acceptable for controlled release devices for use in the treatment of brain tumor.

Characterization of hydrophobized pullulan with various hydrophobicities.

In this study, we prepared self-assembling nanospheres of hydrophobized pullulan. Pullulan acetate (PA), as hydrophobized pullulan, was synthesized by the acetylation of pullulan. PA derivatives were synthesized by changing the degree of acetylation. PA was characterized by Fourier transform infrared (FT-IR), X-ray diffractometry (XRD), and differential scanning calorimetry (DSC). The particle size distribution of the PA was determined using photon correlation spectroscopy (PCS) and the number-average particle size was found to depend upon the degree of acetylation of PA. Morphology by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed the PA nanospheres were spherical in shape. The fluorescence probe technique was used to study the self-association behavior of hydrophobized pullulans in water using pyrene as a hydrophobic probe. The critical association concentration (CAC) values were determined from the fluorescence excitation spectra, CAC values were dependent upon the degree of acetylation. Drug release studies using clonazepam (CNZ) as a hydrophobic model drug showed that the increased drug contents and increased degree of acetylation resulted in a slower release rate of drug from the nanospheres.

Effects of temperature on diblock copolymer micelle composed of poly(γ-benzyl L-glutamate) and poly(N-isopropylacrylamide)

Abstract Diblock copolymers composed of poly( γ -benzyl L -glutamate) and poly( N -isopropylacrylamide) (PNIPAAm) (abbreviated as GN) were prepared by the ring-opening polymerization of γ -benzyl L -glutamate N -carboxy anhydride (BLG-NCA) using amine-terminated PNIPAAm as a polymeric initiator. Polymeric micelles consisting of PBLG as the hydrophobic inner core and PNIPAAm as the hydrophilic outer shell were prepared by diafiltration method. Their critical micelle concentrations (cmc) were determined by fluorescent probe techniques with pyrene as a hydrophobic probe. The cmc of the polymeric micelle with the thermosensitive outer shell is less influenced by temperature than the ordinary polymeric micelle consisting of PBLG as the same innercore and poly(ethylene oxide) (PEO) as the hydrophilic outer shell (abbreviated as GE). Fluorescence results were used to estimate the thermodynamic data of micelle formation, such as the standard Gibbs energies (Δ G 0 ), the standard enthalpies (Δ H 0 ), and the standard entropies (Δ S 0 ) of micellization. For the GN/water system, Δ S 0 was found to be positive and so was favorable to micelle formation whereas Δ G 0 , Δ H 0 , and Δ S 0 for the GE/water system were found to be negative. The partition constant of pyrene in these micelles has a value of the order of 5 and inflection of the change of that for GN with temperature was found around the LCST of PNIPAAm.

Doxorubicin-incorporated nanoparticles composed of poly(ethylene glycol)-grafted carboxymethyl chitosan and antitumor activity against glioma cells in vitro.

In this study, methoxy poly(ethylene glycol)-grafted carboxymethyl chitosan (CMCPEG) was synthesized to make nanoparticles with doxorubicin (DOX) by ion complex formation. Since DOX has positive amine groups, it can interact with the carboxymethyl group of CMCPEG. The particle size of DOX-incorporated nanoparticles of CMCPEG was < 300 nm and nanoparticles had spherical shapes at morphological observation, indicating that DOX/CMCPEG mixtures can form spherical nanoparticles. In a drug release study, higher drug content induced an extended release of drug. Drug release was significantly changed by the release media pH. DOX release was faster at an acidic pH than a neutral or basic pH. The antitumor activity of DOX-incorporated nanoparticles in vitro was tested with DOX-resistant C6 glioma cells. Nanoparticles showed increased cytotoxicity compared to DOX alone. These results suggest that DOX was unable to penetrate into cells and did not effectively inhibit cell proliferation. In contrast, nanoparticles can penetrate into cells and effectively inhibit cell proliferation. Observation of cells under red fluorescence confirmed these results, i.e., nanoparticle-treated C6 cells, unlike DOX-treated cells, had strong red fluorescence. Since DOX has strong red fluorescence, DOX-incorporated nanoparticles entered into the tumor cells more than DOX alone. As a result, we suggest that DOX-incorporated nanoparticles of CMCPEG are superior candidates for antitumor drug delivery.

Clonazepam release from core-shell type nanoparticles composed of poly(γ-benzyl L-glutamate) as the hydrophobic part and poly(ethylene oxide) as the hydrophilic part

Block copolymers consisting of poly(γ-benzyl L-glutamate) (PBLG) as the hydrophobic part and poly(ethylene oxide) (PEO) as the hydrophilic part were synthesized and characterized. Core shell type nanoparticles of the block copolymers (abbreviated GEG) were prepared by the dialysis method. Under fluorescence spectroscopy measurement, the GEG block copolymers were associated in water to form core shell type nanoparticles as polymeric micelles and the critical micelle concentrations (CMC) values of the block copolymers decreased with increasing PBLG chain length in the block copolymers. Transmission electron microscopy (TEM) observations revealed nanoparticles of spherical shapes. From dynamic light scattering (DLS) study, sizes of nanoparticles of GEG-1 and GEG-2 copolymer were 64.3 ± 28.7 nm and 28.9 ± 7.0 nm. The drug-loading contents of GEG-1 and GEG-2 nanoparticles were 15.2 and 8.3 wt %, respectively. These results indicated that the drug-loading contents were dependent on PBLG chain length in the copolymer. Then, the longer the PBLG chain length, the more the drug-loading contents. Release of clonazepam (CNZ) from the nanoparticles was slower in higher loading contents of CNZ than lower loading contents due to the hydrophobic interaction between PBLG core and CNZ.

Conformational transition of nanoparticles composed of poly(γ-benzyl l-glutamate) as the core and poly(ethylene oxide) as the shell

Abstract Diblock copolymers composed of poly(γ-benzyl l -glutamate) (PBLG) as the hydrophobic component and poly(ethylene oxide) (PEO) as the hydrophilic component were obtained by the polymerization of γ-benzyl l -glutamate N -carboxyanhydride, initiated by the primary amino end group of the α-methoxy-ω-amino PEO. Nanoparticles are formed from an organic solution of the block copolymers by the diafiltration method. From the NMR measurement of the nanoparticle in water, the PEO segments in the copolymer extend out as the shell from the nanoparticle core into the aqueous environment. From circular dichroism measurements in ethylene dichloride solution, it was found that the polypeptide block exists in the α-helical conformation and adopts right-handed helix sense, as in PBLG homopolymer. However, the helix sense of PBLG in the copolymer nanoparticles prepared from organic solvent depends on the PEO content in the copolymer and the nature of solvent.

Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model

Abstract Purpose. The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma. Methods. This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4-µm pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope. Results. We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed. Conclusion. This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.

Effect of cryoprotectants on the reconstitution of surfactant-free nanoparticles of poly(DL-lactide-co-glycolide)

Various cryoprotectants were tested to reconstitute the surfactant-free nanoparticles of poly(DL-lactide-co-glycolide) (PLGA). When 2.0% (w/v) of sucrose, trehalose and lactose were used, nanoparticles were completely reconstituted into aqueous solution and particle size was not significantly changed. Above 1.0% (w/v) of sucrose, trehalose and lactose, nanoparticles are well reconstituted whereas it was precipitated with 1.0% (w/v) of mannitol. Drug-encapsulated surfactant-free nanoparticles were quite reconstituted when 2.0% (w/v) of sucrose, trehalose and lactose. Drug release kinetics of nanoparticles was not significantly changed by cryoprotectants.