ABSTRACT
Protein crystals composed of protein molecules are expected as a novel porous material. They have high porosity, and the knowledge of the diffusion of intracrystalline water is important. In this study, the diffusion coefficient of intracrystalline water in intrinsic hen egg-white lysozyme (HEWL) crystals was determined by a method that combines confocal Raman spectroscopy and air convection with controlled relative humidity. Similar to common porous materials, the drying process of the protein crystals includes three periods: constant-rate drying, falling-rate drying, and equilibrium state. During the falling-rate drying period, the drying rate depends on the diffusion of intracrystalline water in the protein crystal. The gradient of the water content was measured using confocal Raman spectroscopy. The diffusion coefficient of the intrinsic HEWL crystals was determined as 3.1 × 10-7 cm2/s with a water content of 36.3 vol %. The estimated diffusion coefficients of the intrinsic HEWL crystals without cross-linking were in close agreement with those of the cross-linked protein crystals. This study is timely as the knowledge of the intrinsic diffusion coefficient is useful not only for understanding the mechanism of hydration of proteins but also in practical applications such as porous materials, drug binding, and cryoprotectant soaks.
Subject(s)
Muramidase , Water , Animals , Muramidase/chemistry , Water/chemistry , Crystallization , Spectrum Analysis, Raman , Chickens/metabolismABSTRACT
Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.
Subject(s)
Ethylenediamines/chemistry , Liposomes/chemistry , Organophosphates/chemistry , RNA, Small Interfering/metabolism , Animals , Base Sequence , Cell Line, Tumor , Cholesterol/metabolism , Fibrosarcoma/metabolism , Fibrosarcoma/pathology , Gene Silencing , Humans , Injections, Intravenous , Liposomes/administration & dosage , Liposomes/chemical synthesis , Liposomes/pharmacokinetics , Male , Mice , Mice, Inbred BALB C , Molecular Imaging , Polyethylene Glycols/chemistry , RNA, Small Interfering/genetics , Spectrophotometry, InfraredABSTRACT
Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.
