Tailored polyethylene glycol grafting on porous nanoparticles for enhanced targeting and intracellular siRNA delivery

Surface functionalization of nanoparticles with polyethylene glycol (PEG) has been widely demonstrated as an anti-opsonization strategy to reduce protein corona formation which is one of the major concerns affecting target receptor recognition. However, excessive surface passivation with PEG can lead to the strong inhibition of cellular uptake and less efficient binding to target receptors, resulting in reduced potential of targeted delivery. To improve specific cell targeting while reducing the nonspecific protein adsorption, a secondary packaging of the nanoparticles with shorter PEG chains, making the targeting ligands densely stretched out for enhanced molecular recognition is demonstrated. Particularly, we report the tailored surface functionalization of the porous nanoparticles that require the stealth shielding onto the open-pore region. This study shows that, in addition to the surface chemistry, the conformation of the PEG layers controls the cellular interaction of nanoparticles. Since the distance between neighboring PEG chains determines the structural conformation of the grafted PEG molecules, tailored PEG combinations can efficiently resist the adsorption of serum proteins onto the pores by transitioning the conformation of the PEG chains, thus significantly enhance the targeting efficiency (>5-fold). The stretched brush PEG conformation with secondary packaging of shorter PEG chains could be a promising anti-opsonization and active targeting strategy for efficient intracellular delivery of nanoparticles

Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

Induced pluripotent stem cells (iPSCs) have intrinsic properties, such as self-renewal ability and pluripotency, which are also shown in embryonic stem cells (ESCs). The challenge of improving the iPSC generation efficiency has been an important issue and there have been many attempts to develop iPSC generation methods. In this research, we added Lin28, known as one of the reprogramming factors, in the form of a soluble recombinant protein from E. coli to improve the efficiency of human iPSC (hiPSC) generation, in respect of alkaline phosphatase (AP)-positive colonies. To deliver Lin28 inside the cells, we generated a soluble Lin28-30Kc19 fusion protein, with 30Kc19 at the C-terminal domain of Lin28. 30Kc19, a silkworm hemolymph-derived protein, was fused due to its cell-penetrating and protein-stabilizing properties. The Lin28-30Kc19 was treated to human dermal fibroblasts (HDFs), in combination with four defined reprogramming factors (Oct4, Sox2, c-Myc, and Klf4). After 14 days of cell culture, we confirmed the generated hiPSCs through AP staining. According to the results, the addition of Lin28-30Kc19 increased the number and size of generated AP-positive hiPSC colonies. Through this research, we anticipate that this recombinant protein would be a valuable material for increasing the efficiency of hiPSC generation and for enhancing the possibility as a substitute of the conventional method

Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein

As the acute lymphoblastic leukaemia (ALL) develops, expression of L-asparaginase (ASNase) protein is known to decrease. Therefore, deficiency of the ASNase protein would be regarded as one of the significant indications of the ALL. For the treatment of ALL, recombinant ASNase protein derived from bacterial origin is used which causes cytotoxicity by deprivation of Asn. However, short half-life of the protein is an obstacle for medical use. In order to overcome this limit, recombinant ASNase was fused to 30Kc19 with protein-stabilizing and cell-penetrating properties. As the 30Kc19 protein may induce steric hindrance, we further added a PLGLAG linker sequence (LK) between the ASNase and 30Kc19. The treatment of ASNase-LK-30Kc19 fusion protein demonstrated enhanced stability, cell-penetrating property, and anti-cancer activity. Intracellular delivery of both the non-cleaved and cleaved forms of the protein were observed, suggesting that ASNase acted both internally and externally, performing high anti-cancer activity by effective depletion of intracellular Asn. Additionally, ASNase-LK-30Kc19 showed high selectivity towards cancer cells. In terms of the dosage, releasable ASNase from ASNase-LK-30Kc19 reached the same half-maximal inhibitory concentration at a concentration five times lower than non-releasable ASNase-30Kc19. Altogether, the findings suggest that this fusion approach has potential applications in the treatment of ALL

Optimization of ZnO Nanorod-Based Surface Enhanced Raman Scattering Substrates for Bio-Applications

Nanorods based on ZnO for surface enhanced Raman spectroscopy are promising for the non-invasive and rapid detection of biomarkers and diagnosis of disease. However, optimization of nanorod and coating parameters is essential to their practical application. With the goal of establishing a baseline for early detection in biological applications, gold-coated ZnO nanorods were grown and coated to form porous structures. Prior to gold deposition, the grown nanorods were 30-50 nm in diameter and 500-600 nm in length. Gold coatings were grown on the nanorod structure to a series of thicknesses between 100 and 300 nm. A gold coating of 200 nm was found to optimize the Rhodamine B model analyte signal, while performance for rat urine depended on the biomarkers to be detected. These results establish design guidelines for future use of Au-ZnO nanorods in the study and early diagnosis of inflammatory diseases