Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy

Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ∼34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time (⟨τ_s⟩) in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus (⟨τ_s⟩ = 750 ± 50 ps) and cytoplasm (⟨τ_s⟩ = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ∼ 11.2 cP, ε ∼ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ∼ 21.7 cP, ε ∼ 7.11)

Solvation Dynamics of Biological Water in a Single Live Cell under a Confocal Microscope

Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CHO) cell. To select the cytoplasm and the nucleus region, two different fluorescent probes are used. A hydrophobic fluorescent dye, DCM, localizes preferentially in the cytoplasm region of a CHO cell. A DNA binding dye, DAPI, is found to be inside the nucleus of the cell. The locations of the probes are clearly seen in the image. Emission maxima of the dyes (DCM in cytoplasm and DAPI in the nucleus) are compared to those of the same dyes in different solvents. From this, it is concluded that the polarity (dielectric constant, ε) of the microenvironment of DCM in the cytoplasm is ∼15. The nucleus is found to be much more polar with ε ≈ 60 (as reported by DAPI). The diffusion coefficient (and hence viscosity) in the cytoplasm and the nucleus was determined using fluorescence correlation spectroscopy (FCS). The diffusion coefficient (Dt) of the dye (DCM) in the cytoplasm is 2 μm2 s–1 and is ∼150 times slower than that in bulk water (buffer). Dt of DAPI in the nucleus (15 μm2 s–1) is 30 times slower than that in bulk water (buffer). The extremely slow diffusion inside the cell has been ascribed to higher viscosity and also to the binding of the probes (DCM and DAPI) to large biological macromolecules. The solvation dynamics of water in the cytoplasm (monitored by DCM) exhibits an average relaxation time ⟨τsol⟩ of 1250 ± 50 ps, which is about 1000 times slower than in bulk water (1 ps). The solvation dynamics inside the nucleus (studied using DAPI) is about 2-fold faster, ⟨τsol⟩ ≈ 775 ps. The higher polarity, faster diffusion, and faster solvation dynamics in the nucleus indicates that it is less crowded and less restricted than the cytoplasm

Solvation Dynamics and Rotational Relaxation Study Inside Niosome, A Nonionic Innocuous Poly(ethylene Glycol)-Based Surfactant Assembly: An Excitation Wavelength Dependent Experiment

Excitation wavelength dependence of solvation and rotational relaxation dynamics has been investigated inside niosome, a biologically stable, nontoxic to our body, multilamellar vesicle system, by using steady state and time-resolved fluorescence spectroscopy to explore the heterogeneity of such a system. Red edge excitation shifts (REES) of 7 nm for Coumarin-153 (C-153) and 11 nm for C-480 were observed with change in λ_ex. Average solvation dynamics is composed of two types of slow components and one fast component. There are two distinct restricted regions, one at the bilayer headgroup region and the other on the two extreme surfaces, which are responsible for the slow components. An unaltered fast component is reported for the segmental chain dynamics of poly(ethylene glycol) (PEG) located at the headgroup region of niosome. The trend in λ_ex dependence obtained for C-153 is found to be similar to that obtained for C-480. Such hindered solvation is attributed to the presence of a strong H-bonding environment of water molecules in the headgroup region, and movement of these highly bound water molecules along with a hydrated oxyethyelene moiety control the observed slow relaxation

Unraveling the Interaction of Diflunisal with Cyclodextrin and Lysozyme by Fluorescence Spectroscopy

Understanding the interaction between the drug:carrier complex and protein is essential for the development of a new drug-delivery system. However, the majority of reports are based on an understanding of interactions between the drug and protein. Here, we present our findings on the interaction of the anti-inflammatory drug diflunisal with the drug carrier cyclodextrin (CD) and the protein lysozyme, utilizing steady-state and time-resolved fluorescence spectroscopy. Our findings reveal a different pattern of molecular interaction between the inclusion complex of β-CD (β-CD) or hydroxypropyl-β-CD (HP-β-CD) (as the host) and diflunisal (as the guest) in the presence of protein lysozyme. The quantum yield for the 1:2 guest:host complex is twice that of the 1:1 guest:host complex, indicating a more stable hydrophobic microenvironment created in the 1:2 complex. Consequently, the nonradiative decay pathway is significantly reduced. The interaction is characterized by ultrafast solvation dynamics and time-resolved fluorescence resonance energy transfer. The solvation dynamics of the lysozyme becomes 10% faster under the condition of binding with the drug, indicating a negligible change in the polar environment after binding. In addition, the fluorescence lifetime of diflunisal (acceptor) is increased by 50% in the presence of the lysozyme (donor), which indicates that the drug molecule is bound to the binding pocket on the surface of the protein, and the average distance between active tryptophan in the hydrophobic region and diflunisal is calculated to be approximately 50 Å. Excitation and emission matrix spectroscopy reveals that the tryptophan emission increases 3–5 times in the presence of both diflunisal and CD. This indicates that the tryptophan of lysozyme may be present in a more hydrophobic environment in the presence of both diflunisal and CD. Our observations on the interaction of diflunisal with β-CD and lysozyme are well supported by molecular dynamics simulation. Results from this study may have an impact on the development of a better drug-delivery system in the future. It also reveals a fundamental molecular mechanism of interaction of the drug–carrier complex with the protein