Photo-cross-linked hydrogels from thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers containing multiple methacrylate groups: mechanical property, swelling, protein release, and cytotoxicity.

Photo-cross-linked hydrogels from thermoresponsive polymers can be used as advanced injectable biomaterials via a combination of physical interaction (in situ thermal gelation) and covalent cross-links (in situ photopolymerization). This can lead to gels with significantly enhanced mechanical properties compared to non-photo-cross-linked thermoresponsive hydrogels. Moreover, the thermally phase-separated gels have attractive advantages over non-thermoresponsive gels because thermal gelation upon injection allows easy handling and holds the shape of the gels prior to photopolymerization. In this study, water-soluble thermoresponsive copolymers containing multiple methacrylate groups were synthesized via one-step deactivation enhanced atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M(n) = 475), poly(propylene glycol) methacrylate (PPGMA, M(n) = 375), and ethylene glycol dimethacrylate (EGDMA) and were used to form covalent cross-linked hydrogels by photopolymerization. The cross-linking density was found to have a significant influence on the mechanical and swelling properties of the photo-cross-linked gels. Release studies using lysozyme as a model protein demonstrated a sustained release profile that varied dependent on the copolymer composition, cross-linking density, and the temperature. Mouse C2C12 myoblast cells were cultured in the presence of the copolymers at concentrations up to 1 mg/mL. It was found that the majority of the cells remained viable, as assessed by Alamar Blue, lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays. These studies demonstrate that thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers offer potential as in situ photopolymerizable materials for tissue engineering and drug delivery applications through a combination of facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible functionality for further structure modifications.

PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors.

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.

Enhanced target recognition of nanoparticles by cocktail PEGylation with chains of varying lengths.

Monodispersed gold nanoparticles (AuNPs) were simultaneously decorated with lactosylated and non-modified shorter poly(ethylene glycol)s (PEGs) to enhance their target recognition. The decoration with sufficiently shorter PEGs dramatically enhanced the multivalent binding ability of lactosylated AuNPs to the lectin-fixed surface, possibly due to the enhanced mobility of the ligands via the spacer effect generated by the shorter PEG chains.

Colloidal Au replacement assay for highly sensitive quantification of low molecular weight analytes by surface plasmon resonance.

A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.

Ligand density effect on biorecognition by PEGylated gold nanoparticles: regulated interaction of RCA120 lectin with lactose installed to the distal end of tethered PEG strands on gold surface.

PEGylated gold nanoparticles (diameter: 20 nm) possessing various functionalities of lactose ligand on the distal end of tethered PEG ranging from 0 to 65% were prepared to explore the effect of ligand density of the nanoparticles on their lectin binding property. UV-visible spectra of the aqueous solution of the nanoparticles revealed that the strong steric stabilization property of the PEG layer lends the nanoparticles high dispersion stability even under the physiological salt concentration (ionic strength, I = 0.15 M). The number of PEG strands on a single particle was determined to be 520 from thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) observation under controlled acceleration voltage revealed the thickness of the PEG layer on the nanoparticle to be approximately 7 nm. The area occupied by a single lactose molecule on the surface of PEGylated gold nanoparticles was then calculated based on TGA and SEM results and was varied in the range of 10-34 nm2 depending on the lactose functionality (65 approximately 20%). PEGylated gold nanoparticles with 40% and 65% lactose functionality showed a selective and time-dependent aggregation in phosphate buffer with the addition of Ricinus communis agglutinin (RCA120) lectin, a bivalent galactose-specific protein. The aggregates can be completely redispersed by adding an excess amount of galactose. Time-lapse monitoring of UV-visible spectra at 600-750 nm revealed that the aggregation of PEGylated gold nanoparticles was accelerated with an increase in both RCA120 concentration in the solution and the lactose density of the nanoparticles. Furthermore, the sensitivity of lectin detection could be controlled by the regulation of lactose density on the particle surface. Interestingly, there was a critical lactose density (>20%) observed to induce detectable particle aggregation, indicating that the interaction between the particles is triggered by the multimolecular bridging via lectin molecules.

Supramolecular assemblies for the cytoplasmic delivery of antisense oligodeoxynucleotide: polyion complex (PIC) micelles based on poly(ethylene glycol)-SS-oligodeoxynucleotide conjugate.

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL).