Protein transport is an important phenomenon in biological systems. the cellular
Protein transport is an important phenomenon in biological systems. the cellular transport mechanism in stem cells is highly desirable for diagnostics, targeting and therapeutic applications, opening up new avenues in the area of drug delivery. In BV-6 manufacture the mammalian cell, the cell membrane acts as a barrier to cargoes (ions, small molecules or macromolecules). In higher order biological systems, a nuclear localization signal (NLS) is essential for targeting macromolecular cargoes to the nucleus. Studies on stem cells hold much promise for human regenerative medicine. Recent advances in the field of nanoparticles (NPs) generated novel applications in biomedicine including regenerative medicine. Applications such as imaging, diagnostics and drug delivery (the so-called theranostics) require precise targeting approach for their successes1,2,3,4. NPs have been engineered to induce membrane receptor internalization, followed by downstream signaling and subsequent cellular responses. NPCmediated cellular response is size-dependent5. Gold NPs have been shown to be a powerful vehicle for drug delivery6. The presence of HerceptinCgold NP complexes within endosomes showed that receptor-mediated uptake is the most probable mechanism. There are several biological barriers at the cellular level that the engineered NPs must overcome, starting from cell membrane to sub-cellular compartment (cytosol, mitochondria or nucleus). Several endocytotic mechanisms can be engaged to facilitate the internalization of a carrier. In clathrin-mediated endocytosis, endosomal escape must occur before fusion with a lysosome to prevent degradation of the cargo by the harsh lysosomal conditions. More importantly, the endosomal escape is usually necessary to allow access of the carrier to the desired BV-6 manufacture sub-cellular compartment7. For example, the uptake of TAT-functionalized Au NPs was enhanced in HeLa cells and the particles initially found in the cytosol, nucleus, mitochondria and later within densely filled vesicles were released, negotiating intracellular membrane barriers quite freely, including the possibility of direct membrane transfer8. The intracellular delivery of quantum dots (QDs) is BV-6 manufacture mainly governed by size, surface charge and coatings9,10,11. BV-6 manufacture Different types of peptides have been used as the promising candidates for intracellular delivery of QDs9,10,11,12,13,14,15. Recent advances include the enhancement of intracellular delivery of streptavidin-conjugated QDs into mouse fibroblast cells using biotinylated L-arginine peptides10, TAT-functionalized QDs for selective intracellular transport, vesicle shedding and delivery9,13,14. ArginineCglycineCaspartic acid (RGD) peptides have been conjugated to target QDs specifically to tumor angiogenesis for theranostics12,16. Despite these advances, the effective nuclear targeting (since many of the anti-cancer targets are located in the nucleus) and therapeutic applications of QDs are still limited by their poor intracellular delivery and aggregation within the endosomes. We have chosen CdSe QDs as a model system owing to their superior optical properties and stability. Here we show a simple approach for the effective nuclear targeting via microtubules using a combined peptide approach involving both NLS and microtubule-associated sequence (MTAS) peptideCQD conjugates. Results Importance of endosomal escape and designing of peptide sequences In the area of NP-mediated drug delivery, it is assumed that only <10% of active drugs would escape the endosomes and reach the nucleus. Therefore, an endosomal escape is highly indispensable. To achieve this, we have initially designed a peptide sequence based on NLS and a transportan protein (TP); the latter has been used for the delivery of siRNA/proteins/PNA17,18,19. First, we demonstrate the design of a novel peptide sequence containing TP and SV40-NLS peptides to elucidate the nuclear targeting via endosomal escape using CdSe/ZnS QDCpeptide conjugates. In another interesting design, we could mimic the cellular protein transport pathway using MTAS and NLS peptideCQD conjugates. We believe this combined strategy would increase the endosomal escape of cargoes, and be useful F2RL2 for optimal drug delivery to the nucleus. We have systematically designed and studied different peptides (SV40-NLS, TP, short MTAS (s-MTAS) and long MTAS (l-MTAS)) to determine which one of them is more efficient in delivering the QDs to cell organelles, e.g. the nucleus for effective targeting (Fig. 1). We will show the efficacy of peptide coating method for labeling stem cells and transporting QDs to the nucleus via microtubules using mixed NLS and l-MTAS peptides. We believe that this is the first report of nuclear targeting of QDs through microtubules using a biomimetic approach. Figure 1 Design of different peptide sequences for making water-soluble core/shell CdSe/ZnS quantum dots (QDs). QD-peptide conjugates Our initial study suggested that a short chain peptide, e.g. glutathione (GSH) can be used in transferring the CdSe/ZnS QDs into water. The peptide coating of QDs.