Dong Woo Lim

DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline)

A series of linear poly(ethylenimine) (L-PEI) containing varying amounts of cationic charge density in its backbone was produced by controlled hydrolysis of poly(2-ethyl-2-oxazoline) (PEtOz) for using as a nonviral DNA transfection agent. The effects of cationic charge density and molecular weight of the L-PEI on the cytotoxicity and transfection efficiency were studied. The efficiency of transfection was monitored by using a luciferase reporter gene system. Gel retardation assay and dynamic light scattering (DLS) showed that the condensation capacity of L-PEI was suitable for transfection. Highly compacted L-PEI/DNA complex ( approximately 150 nm) was obtained with a surface charge value of around +28.4 mV. Cell cytotoxicity was affected to a great extent by the hydrolysis percent of L-PEI as well as by the molecular weight. Transfection efficiency of luciferase plasmid DNA against NIH 3T3 fibroblast was largely dependent upon the hydrolysis percent (charge density) in the polymer backbone and the molecular weight of the L-PEI, but independent of the total amount of cationic charges used for DNA condensation. L-PEI with a hydrolysis percent of 88.0% exhibited comparable transfection efficiency to that of commonly used branched PEI.

In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair.

Rapid cross-linking of elastin-like polypeptides (ELPs) with hydroxymethylphosphines (HMPs) in aqueous solution is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. In order to examine the independent effect of the location and number of reactive sites on the chemical cross-linking kinetics of ELPs and the mechanical properties of the resulting hydrogels, we have designed ELP block copolymers comprised of cross-linkable, hydrophobic ELP blocks with periodic Lys residues (A block) and aliphatic, hydrophilic ELP blocks with no cross-linking sites (B block); three different block architectures, A, ABA, and BABA were synthesized in this study. All ELP block copolymers were rapidly cross-linked with HMPs within several minutes under physiological conditions. The inclusion of the un-cross-linked hydrophilic block, its length relative to the cross-linkable hydrophobic block, and the block copolymer architecture all had a significant effect on swelling ratios of the cross-linked hydrogels, their microstructure, and mechanical properties. Fibroblasts embedded in the ELP hydrogels survived the cross-linking process and remained viable for at least 3 days in vitro when the gels were formed from an equimolar ratio of HMPs and Lys residues of ELPs. DNA quantification of the embedded cells indicated that the cell viability within triblock ELP hydrogels was statistically greater than that in the monoblock gels at day 3. These results suggest that the mechanical properties of ELP hydrogels and the microenvironment that they present to cells can be tuned by the design of the block copolymer architecture.

Mussel-inspired surface modification of poly(L-lactide) electrospun fibers for modulation of osteogenic differentiation of human mesenchymal stem cells.

Development of biomaterials to control the fate of stem cells is important for stem cell based regeneration of bone tissue. The objective of this study is to develop functionalized electrospun fibers using a mussel-inspired surface coating to regulate adhesion, proliferation and differentiation of human mesenchymal stem cells (hMSCs). We prepared poly(L-lactide) (PLLA) fibers coated with polydopamine (PD-PLLA). The morphology, chemical composition, and surface properties of fiber were characterized by SEM, AFM, XPS, Raman spectra and water contact angle measurements. Incubation of fibers in dopamine solution for 1h resulted in formation of polydopamine with only negligible effects on the roughness and hydrophobicity of the fibers. However, PD-PLLA fibers modulated hMSC responses in several aspects. Firstly, adhesion and proliferation of hMSCs cultured on PD-PLLA were significantly enhanced relative to those on PLLA. In addition, the ALP activity of hMSCs cultured on PD-PLLA (1.74±0.14 nmole/DNA/30 min) was significantly higher than on PLLA (0.97±0.07 nmole/DNA/30 min). hMSCs cultured on PD-PLLA showed up-regulation of genes associated with osteogenic differentiation as well as angiogenesis. Furthermore, the calcium deposition from hMSCs cultured on PD-PLLA (41.60±1.74 μg) was significantly greater than that on PLLA (30.15±1.21 μg), which was double-confirmed by alizarin red S staining. Our results suggest that the bio-inspired coating synthetic degradable polymer can be used as a simple technique to render the surface of synthetic biodegradable fibers to be active for directing the specific responses of hMSCs.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment.

Stereocomplex formation between enantiomeric PLA–PEG–PLA triblock copolymers: Characterization and use as protein‐delivery microparticulate carriers

Two enantiomeric triblock ABA copolymers composed of poly(L-lactide)–poly(ethylene glycol)–poly(L-lactide) (PLLA–PEG–PLLA) and poly(D-lactide)–poly(ethylene glycol)–poly(D-lactide) (PDLA–PEG–PDLA) were synthesized with two different middle-block PEG chain lengths by ring-opening polymerization of L-lactide and D-lactide in the presence of PEG, respectively. A pair of enantiomeric triblock copolymers were combined to form a stereocomplex by a solvent-casting method. The triblock copolymers and their stereocomplexes were characterized by 1H- and 13C-NMR spectroscopy and gel permeation chromatography. Their crystalline structures and crystalline melting behaviors were analyzed by the wide-angle X-ray diffraction method and differential scanning calorimetry. The stereocomplex formed between a pair of enantiomeric triblock copolymers exhibited a higher crystalline melting temperature with a distinctive 3/1 helical crystalline structure. PLLA–PEG–PLLA and its stereocomplex with PDLA–PEG–PDLA were used to fabricate a series of microspheres encapsulating a model protein drug, bovine serum albumin (BSA). They were prepared by a double-emulsion solvent-evaporation method. The morphological aspects of the microspheres were characterized and BSA release profiles from them were investigated.

Simultaneous immunoassay for the detection of two lung cancer markers using functionalized SERS nanoprobes.

A quick and reproducible SERS-based immunoassay, using functionalized hollow gold nanospheres and magnetic beads, has been developed. Here, a simultaneous detection of dual cancer markers in blood serum has been achieved under a single excitation wavelength. The accuracy and sensitivity for clinical sera from five patients confirms their diagnostic feasibility.

Structurally Controlled Bio‐hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured bio-hybrid materials. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.

Synthesis, characterization and protein adsorption behaviors of PLGA:PEG di-block co-polymer blend films

A series of poly(D,L-lactic-co-glycolic acid) (PLGA)/poly(ethyleneglycol) (PEG) di-block copolymers were synthesized by ring-opening polymerization of D,L-lactide and glycolide with different molecular weights of monomethoxy polyethyleneglycol (mPEG) 750, 2000 and 5000 as an initiator. The bulk properties of these co-polymers were characterized by using 1H NMR spectroscopy, gel permeation chromatography, differential scanning calorimetry (DSC). Electron spectroscopy for chemical analysis (ESCA) results, in which the blend films with the di-block copolymers showed increasing surface oxygen atomic percentage with increasing PEG chain length, indicate that PEG chain segment in the di-block copolymers is surface oriented and enriched onto the surface of the blend films. The extent of protein adsorption onto the surface of these blend films was studied, using iodine radio-labeled human serum albumin, gamma globulin and human growth hormone. The protein adsorption amount was reduced for the blend films prepared with PLGA/PEG 750 and 2000 di-block copolymers, but increased to a great extent for PLGA/PEG 5000 di-block copolymer. This is due to the increased water uptake capacity of the blend film, which absorbed more protein molecules into a swollen polymer matrix in addition to surface adsorption.

Biomedical and Biotechnological Applications of Elastin-Like Polypeptides

Elastin‐like polypeptides (ELPs) are a unique class of genetically encoded biopolymers with tunable thermosensitivity and biocompatibility. This review provides an introduction to ELPs and an overview of their design and synthesis. It also discusses applications of these polypeptides for drug delivery, tissue engineering, protein purification, and biosensing.

Highly sensitive trace analysis of paraquat using a surface-enhanced Raman scattering microdroplet sensor

We report a rapid and highly sensitive trace analysis of paraquat (PQ) in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aqueous samples of PQ, silver nanoparticles, and NaCl as the aggregation agent were introduced into a microfluidic channel and were encapsulated by a continuous oil phase to form a microdroplet. PQ molecules were adsorbed onto particle surfaces in isolated droplets by passing through the winding part of the channel. Memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, were removed because the aqueous droplets were completely isolated by a continuous oil phase. The limit of detection (LOD) of PQ in water, determined by the SERS-based microdroplet sensor, was estimated to be below 2×10(-9) M, and this low detection limit was enhanced by one to two orders of magnitude compared to conventional analytical methods.