Cell uptake and trafficking behavior of non-covalent, coiled-coil based polymer-drug conjugates.

This paper reports on the cell uptake and trafficking properties of a series of non-covalent polymer-drug conjugates. These nanomedicines are composed of a poly(N-(2-hydroxypropyl)methacrylamide) backbone functionalized with multiple copies of a drug. The drug moieties are attached to the polymer via a non-covalent, so called coiled coil motif, which is formed by heterodimerization of two complementary peptide strands, one of which is attached to the polymer carrier and the other to the drug. Cytotoxicity and FACS experiments, which were carried out with model anticancer drug or fluorophore conjugates, provided insight into the cell uptake and trafficking behavior of these conjugates.

Polymer coiled-coil conjugates: potential for development as a new class of therapeutic "molecular switch".

Polymer therapeutics, including polymeric drugs and polymer-protein conjugates, are clinically established as first-generation nanomedicines. Knowing that the coiled-coil peptide motif is fundamentally important in the regulation of many cellular and pathological processes, the aim of these studies was to examine the feasibility of designing polymer conjugates containing the coiled-coil motif as a putative therapeutic "molecular switch". To establish proof of concept, we prepared a mPEG-FosW(C) conjugate by reacting mPEG-maleimide (M(w) 5522 g mol(-1), M(w)/M(n) 1.1) with a FosW peptide synthesized to contain a terminal cysteine residue (FosW(C)). Its ability to form a stable coil-coil heterodimer with the target c-Jun sequence of the oncogenic AP-1 transcription factor was investigated using 2D (15)N-HSQC NMR together with a recombinantly prepared (15)N-labeled c-Jun peptide ([(15)N]r-c-Jun). Observation that heterodimerization was achieved and that the polymer did not sterically disadvantage hybridization suggests an important future for this new family of polymer therapeutics.

Hybrid polymer therapeutics incorporating bioresponsive, coiled coil peptide linkers.

This article reports the design, synthesis and results of first in vitro model studies of a conceptually novel class of polymer therapeutics in which the cargo is attached to a polymer backbone via a noncovalent, biologically inspired coiled coil linker, which is formed by heterodimerization of two complementary peptide sequences that are linked to the polymer carrier and the cargo, respectively. In contrast with the polymer-drug conjugates prepared so far, in which the drug is typically attached via an enzymatically or hydrolytically cleavable linker, the noncovalent polymer therapeutics proposed in this article offer several potential advantages, including facile access to combination therapeutics and rapid production of compound libraries to screen for structure-activity relationships. Furthermore, the coiled coil based peptide linkers may not only be useful to bind and release guests but may also play an active role in enhancing and directing intracellular transport and trafficking, which may make these constructs of particular interest for the cytosolic delivery of biomolecular therapeutics.

The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery.

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.