The ubiquitous expression of the mannose-6-phosphate receptor on the majority of human cells makes it a valid target in the quest to deliver therapeutics selectively to the lysosome. In this work end-functionalized polyvalent mannose-6-phosphate glycopolypeptides (M6P-GPs) with high molecular weights (up to 22 kDa) have been synthesized via NCA polymerization. These synthetic M6P-GPs were found to display minimal toxicity to cells in vitro and show exceptional selectivity for trafficking into lysosomes in various cell lines. Comparison of the cellular uptake behavior of M6P-GP and the corresponding mannose-GP polymer reveals that incorporation of the phosphate moiety at the 6-position of mannose completely alters its trafficking behavior and becomes exclusively lysosome specific. We also demonstrate that trafficking of M6P-GPs in mammalian cells is likely associated with the CI-MPR receptor pathway. I n mammalian cells, mannose-6-phosphate receptors play a critical role in the sorting of lysosomal enzymes from secretory proteins and their subsequent delivery into lysosomes. 1 These receptors, which include the ∼300 kDa insulin-like growth factor-II (IGF-II)/cation-independent (CI) multifunctional transmembrane glycoprotein mannose-6-phosphate (M6P) receptor (CI-MPR), bind lysosomal enzymes containing phosphomannosyl residues in the trans-golgi network (TGN) and transport them to endosomes where the low pH leads to the dissociation of the ligand−receptor complex. 2 The receptors then return to the TGN to repeat another round of this process. As a result, the receptor CI-MPR is largely localized in the intracellular compartments and shuttles between TGN and endosomes with ∼10% of the receptor being present on the cell surface. 3 Additionally, the CI-MPR receptor is overexpressed in the early stage of several cancers, particularly in breast and prostate cancer, and hence can serve as an early marker for these cancers. 4,5 The ability of CI-MPR to deliver cargo specifically to the lysosome can thus be selectively targeted using M6P-labeled carriers to dispatch cytotoxic drugs inside lysosomes for destroying cancer cells. 6−8 In addition, CI-MPR can be targeted to deliver M6P-labeled enzymes into lysosomes for enzyme replacement therapies (ERTs) in non-neural lysosomal storage disorders (LSDs). 9,10 LSDs are a group of more than 40 metabolic disorders that result from the deficient activity of specific lysosomal enzymes inside the lysosomes leading to progressive accumulation of its substrate inside the cell. Nowadays several M6P-modified enzymes are used clinically for the treatment of LSD. However, the low levels of M6P modification dramatically reduce its binding efficiency to the receptor, and hence relatively high doses of these enzymes need to be administered to achieve significant therapeutic efficacy. 11 Modification of lysosomal enzymes with higher amounts of M6P residues for enhanced targeted delivery remains a critical challenge. 12 Previous studies demonstrate that CI-MPR exists as a dimer in the membrane ...

Receptors of the carbohydrate mannose-6-phosphate (M6P) are overexpressed in specific cancer cells (such as breast cancer) and are also involved in the trafficking of mannose-6-phosphate labeled proteins exclusively onto the...

pH-responsive "supra-amphiphiles" based on double hydrophilic, positively charged block copolypeptides such as PEG-b-poly-L-lysine together with low molecular weight stimuli-sensitive partners that contain phosphate and carboxylate groups have been widely studied. In contrast, the other widely used cationic polypeptide poly-L-arginine whose cellpenetrating properties are well-known has been much less explored for the synthesis of supra-amphiphile-based nanomaterials. It is also known that the guanidine side chain of arginine binds to carboxylate anions with binding constants that are 2.5 times higher than the corresponding amines of poly-L-lysine. Here, we demonstrate the fabrication of pH-sensitive supraamphiphilic nanoparticles by simple mixing of PEG 2k -b-poly-(homoarginine) block copolymer and carboxylic acid containing functional low molecular weight organic compounds. A high yielding three-step methodology was developed for the synthesis of ε-N,N′-di-Boc-Lhomoarginine-α-N-carboxyanhydride which was polymerized using amine-terminated PEG (2000 MW) to yield 100% guanine-functionalized polypeptide (PEG 2k -b-PHR 30 ) with controlled molecular weights and low PDIs. Incubation of PEG 2k -b-PHR 30 with four different carboxylic acids (including dexamethasone a glucocorticoid receptor used in cancer therapy) in water leads to the formation of "supra-amphiphilic" nanoparticles (<200 nm size) due to the charge neutralization resulting from the strong interaction between the guanidine group and the carboxylate group. All these nanoparticles were able to encapsulate the hydrophobic dye Nile red with varying efficiency. Although these assemblies were stable at neutral pH, upon lowering the pH of the solution between 4 and 5, the protonation of the carboxylic acids leads to disassembly of these nanoparticles. The cytotoxicity of all four "supra-amphiphilic" nanoparticles varied depending on the carboxylic acid used for their fabrication. While the nanoparticle formed using dioctylbenzoic acid displayed 80% cell viability at concentration of 60 μg/mL, those formed with the steroid deoxycholic acid or dexamethasone showed only 40% cell viability at similar concentrations. Colocalization studies performed using epifluorescence microscopy demonstrate the successful uptake of intact dyeencapsulated nanoparticle inside the cell.

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Dual enzyme responsive stable biomimetic vesicles composed of mannose-6-phosphate lipid can encapsulate and deliver dual dye/drug and protein/enzyme exclusively to the lysosome in HEK-293 cells. The release of the cargo...