pH-Dependent Cellular Internalization of Paramagnetic Nanoparticle

A hallmark of the tumor microenvironment in malignant tumor is extracellular acidosis, which can be exploited for targeted delivery of drugs and imaging agents. A pH sensitive paramagnetic nanoaparticle (NP) is developed by incorporating GdDOTA-4AmP MRI contrast agent and pHLIP (pH Low Insertion Peptide) into the surface of a G5–PAMAM dendrimer. pHLIP showed pH-selective insertion and folding into cell membranes, but only in acidic conditions. We demonstrated that pHLIP-conjugated Gd₄₄-G5 paramagnetic nanoparticle binds and fuses with cellular membrane at low pH, but not at normal physiological pH, and that it promotes cellular uptake. Intracellular trafficking of NPs showed endosomal/lysosomal path ways

Concentration-independent MRI of pH with a dendrimer-based pH-responsive nanoprobe

The measurement of extracellular pH (pHe ) has significant clinical value for pathological diagnoses and for monitoring the effects of pH-altering therapies. One of the major problems of measuring pHe with a relaxation-based MRI contrast agent is that the longitudinal relaxivity depends on both pH and the concentration of the agent, requiring the use of a second pH-unresponsive agent to measure the concentration. Here we tested the feasibility of measuring pH with a relaxation-based dendritic MRI contrast agent in a concentration-independent manner at clinically relevant field strengths. The transverse and longitudinal relaxation times in solutions of the contrast agent (GdDOTA-4AmP)44 -G5, a G5-PAMAM dendrimer-based MRI contrast agent in water, were measured at 3 T and 7 T magnetic field strengths as a function of pH. At 3 T, longitudinal relaxivity (r1 ) increased from 7.91 to 9.65 mM(-1) s(-1) (on a per Gd(3+) basis) on changing pH from 8.84 to 6.35. At 7 T, r1 relaxivity showed pH response, albeit at lower mean values; transverse relaxivity (r2 ) remained independent of pH and magnetic field strengths. The longitudinal relaxivity of (GdDOTA-4AmP)44 -G5 exhibited a strong and reversible pH dependence. The ratio of relaxation rates R2 /R1 also showed a linear relationship in a pH-responsive manner, and this pH response was independent of the absolute concentration of (GdDOTA-4AmP)44 -G5 agent. Importantly, the nanoprobe (GdDOTA-4AmP)44 -G5 shows pH response in the range commonly found in the microenvironment of solid tumors

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field