Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1−6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.

Since therapeutic agents target specific compartments inside the cells, their efficiency depends on their intracellular release from drug delivery systems (DDS). However, control over the intracellular release of therapeutic agents is a challenging issue and can only be achieved by governing their interactions with the DDS. In this work, polyglycerol amine- and polyglycerol sulfate-functionalized graphene sheets as positively and negatively charged 2D nanomaterials with 150 nm lateral size were used to deliver and control the release of doxorubicin (DOX) inside cells. A pH-sensitive dye was conjugated onto the surfaces of graphene sheets and used as an antenna to obtain specific signals from the acidic cell compartments. It was found that both positively and negatively charged graphene sheets undergo similar acidification processes after cellular uptake. Nevertheless, the intracellular drug release of these DOX-loaded nanomaterials was distinctly different. As an overall effect of the π-π stacking and electrostatic interactions, the release of DOX from the positively charged graphene sheets was much faster than that from their analogs with a negative surface charge. Therefore, therapeutic efficiency in the first case was much higher than that in the latter. Based on our findings, the intracellular release of drugs from the surfaces of graphene sheets can be finely tuned by manipulating their functionalities, which is of great importance in the designing of the future graphene-based nanomedicines.

Dyads consisting of a photochromic switch covalently linked to a fluorescent dye allow the emission from the dye to be controlled by reversible photoisomerization of the switch; one form of...

Patzelt et al. have described the size-dependent uptake of 100-1000 nm sized nanoparticles into the HF. Mediumsized nanoparticles (500-700 nm) achieved a maximum in penetration depth, [4] due to the surface structure of the hair and the hair follicle. Particles that are of the same size thickness of the keratin cells (530 nm in human hair and 320 nm in porcine hair) can be easily transported into the HF. The movements of the hair acts as a pumping system. Thus, the optimum size is also dependent on the species. [5,6] But even if the particles penetrate into the HF, they will not pass the follicular barrier. Consequently, the particles could be used as a transport system for drugs. Particles in the required nanoscale range can be produced in various ways. [7][8][9] A possibility to achieve particles in the nanometer range is the synthesis of nanogels which exist of crosslinked polymer chains and form 3D nanosized networks. [10,11] They can be prepared by mini [12][13][14][15] and microemulsion. [16,17] In this case reactive macromolecules are templated into stabilized droplets and then reacted to the desired nanoparticles. However, high energy input by ultrasonication or large amounts of surfactants are needed to stabilize the droplet reactors. To avoid these harsh In the present study, a pH responsive dendritic polyglycerol nanogel (dPG-NG) is developed to measure the pH values inside the hair follicle (HF) using an ex vivo porcine ear model. The macromolecular precursors are labeled with a pH sensitive indodicarbocyanine dye (pH-IDCC) and a control dye (indocarbocyanine dye: ICC) and crosslinked via a mild and surfactantfree Thiol-Michael reaction using an inverse nanoprecipitation method. With this method, it is possible to prepare tailor-made particles in the range of 100 nm to 1 µm with a narrow polydispersity. The dPG-NGs are characterized using dynamic light scattering, nanoparticle tracking analysis, and atomic force microscopy. Systematic analysis of confocal microscope images of histological sections of the skin enables accurate determination of the pH gradient inside the HF. The results show that these novel pH-nanosensors deeply penetrate the skin via the follicular pathway and the pH of the pig hair follicles increase from 6.5 at the surface of the skin to 7.4 in deeper areas of the HF. The pH-nanosensor shows no toxicity potentials.