Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.

In this study, we synthesized empty core-shell structured nanocapsules of Pluronic F127 and chitosan and characterized the thermal responsiveness of the nanocapsules in size and wall-permeability. Moreover, we determined the feasibility of using the nanocapsules to encapsulate small molecules for temperature-controlled release and intracellular delivery. The nanocapsules are ∼37 nm at 37 °C and expand to ∼240 nm when cooled to 4 °C in aqueous solutions, exhibiting >200 times change in volume. Moreover, the permeability of the nanocapsule wall is high at 4 °C (when the nanocapsules are swollen), allowing free diffusion of small molecules (ethidium bromide, MW = 394.3 Da) across the wall, while at 37 °C (when the nanocapsules are swollen), the wall-permeability is so low that the small molecules can be effectively withheld in the nanocapsule for hours. As a result of their thermal responsiveness in size and wall-permeability, the nanocapsules are capable of encapsulating the small molecules for temperature-controlled release and intracellular delivery into the cytosol of both cancerous (MCF-7) and noncancerous (C3H10T1/2) mammalian cells. The cancerous cells were found to take up the nanocapsules much faster than the noncancerous cells during 45 min incubation at 37 °C. Moreover, toxicity of the nanocapsules as a delivery vehicle was found to be negligible. The Pluronic F127-chitosan nanocapsules should be very useful for encapsulating small therapeutic agents to treat diseases particularly when it is combined with cryotherapy where the process of cooling and heating between 37 °C and hypothermic temperatures is naturally done.

) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble A␤ aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in ␤-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to A␤ protofibrils, the HFIP-induced aggregates seeded elongation by A␤ monomer deposition very poorly. The techniques used to distinguish these two classes of soluble A␤ aggregates may be useful in characterizing A␤ aggregates formed in vivo.