Stimuli Induced Uptake of Protein-Like Peptide Brush Polymers.

Recently, we presented a strategy for packaging peptides as side-chains in high-density brush polymers. For this globular protein-like polymer (PLP) formulation, therapeutic peptides were shown to resist proteolytic degradation, enter cells efficiently and maintain biological function. In this paper, we establish the role charge plays in dictating the cellular uptake of these peptide formulations, finding that peptides with a net positive charge will enter cells when polymerized, while those formed from anionic or neutral peptides remain outside of cells. Given these findings, we explored whether cellular uptake could be selectively induced by a stimulus. In our design, a cationic peptide is appended to a sequence of charge-neutralizing anionic amino acids through stimuli-responsive cleavable linkers. As a proof-of-concept study, we tested this strategy with two different classes of stimuli, exogenous UV light and an enzyme (a matrix metalloproteinase) associated with the inflammatory response. The key finding is that these materials enter cells only when acted upon by the stimulus. This approach makes it possible to achieve delivery of the polymers, therapeutic peptides or an appended cargo into cells in response to an appropriate stimulus.

Directly Observing Micelle Fusion and Growth in Solution by Liquid-Cell Transmission Electron Microscopy.

Amphiphilic small molecules and polymers form commonplace nanoscale macromolecular compartments and bilayers, and as such are truly essential components in all cells and in many cellular processes. The nature of these architectures, including their formation, phase changes, and stimuli-response behaviors, is necessary for the most basic functions of life, and over the past half-century, these natural micellar structures have inspired a vast diversity of industrial products, from biomedicines to detergents, lubricants, and coatings. The importance of these materials and their ubiquity have made them the subject of intense investigation regarding their nanoscale dynamics with increasing interest in obtaining sufficient temporal and spatial resolution to directly observe nanoscale processes. However, the vast majority of experimental methods involve either bulk-averaging techniques including light, neutron, and X-ray scattering, or are static in nature including even the most advanced cryogenic transmission electron microscopy techniques. Here, we employ in situ liquid-cell transmission electron microscopy (LCTEM) to directly observe the evolution of individual amphiphilic block copolymer micellar nanoparticles in solution, in real time with nanometer spatial resolution. These observations, made on a proof-of-concept bioconjugate polymer amphiphile, revealed growth and evolution occurring by unimer addition processes and by particle-particle collision-and-fusion events. The experimental approach, combining direct LCTEM observation, quantitative analysis of LCTEM data, and correlated in silico simulations, provides a unique view of solvated soft matter nanoassemblies as they morph and evolve in time and space, enabling us to capture these phenomena in solution.

Activating Peptides for Cellular Uptake via Polymerization into High Density Brushes.

The utility of peptide therapeutics is thwarted by an inability to enter cells, preventing access to crucial intracellular targets. Herein, we describe a simple and potentially widely applicable solution involving the polymerization of a minimally modified amino acid sequence into a high density brush polymer. Specifically, non-cell penetrating peptides can be rendered competent for cell entry by first including a single Arg or Lys in their amino acid sequence, if one is not already present, along with a norbornenyl unit. This modified monomer is then polymerized by ring opening metathesis polymerization (ROMP). To demonstrate the utility of this strategy, a known therapeutic peptide, which does not penetrate cells on its own, was polymerized. The resulting polymer proficiently entered cells while maintaining its intracellular function. We anticipate that this methodology will find broad use in medicine, increasing or enabling the in vivo efficacy of promising peptide therapeutics.

Cellular Delivery of Nanoparticles Revealed with Combined Optical and Isotopic Nanoscopy.

Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.

Stimuli-responsive nanomaterials for biomedical applications.

Nature employs a variety of tactics to precisely time and execute the processes and mechanics of life, relying on sequential sense and response cascades to transduce signaling events over multiple length and time scales. Many of these tactics, such as the activation of a zymogen, involve the direct manipulation of a material by a stimulus. Similarly, effective therapeutics and diagnostics require the selective and efficient homing of material to specific tissues and biomolecular targets with appropriate temporal resolution. These systems must also avoid undesirable or toxic side effects and evade unwanted removal by endogenous clearing mechanisms. Nanoscale delivery vehicles have been developed to package materials with the hope of delivering them to select locations with rates of accumulation and clearance governed by an interplay between the carrier and its cargo. Many modern approaches to drug delivery have taken inspiration from natural activatable materials like zymogens, membrane proteins, and metabolites, whereby stimuli initiate transformations that are required for cargo release, prodrug activation, or selective transport. This Perspective describes key advances in the field of stimuli-responsive nanomaterials while highlighting some of the many challenges faced and opportunities for development. Major hurdles include the increasing need for powerful new tools and strategies for characterizing the dynamics, morphology, and behavior of advanced delivery systems in situ and the perennial problem of identifying truly specific and useful physical or molecular biomarkers that allow a material to autonomously distinguish diseased from normal tissue.

Peptides displayed as high density brush polymers resist proteolysis and retain bioactivity.

We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility.

Polymerization of Protecting-Group-Free Peptides via ROMP.

A study was conducted to survey the tolerance of ring-opening metathesis polymerization (ROMP) with respect to amino acid (a.a) identity of pentapeptide-modified norbornene-based monomers. A library of norbornyl-pentapeptides were prepared with the general structure, norbornyl-GX2PLX5, where residue 'X' was changed at each of the two positions (2 or 5) alternately to consist of the natural amino acids F, A, V, R, S, K, N, T, M, Q, H, W, C, Y, E, Q, and D. Each peptide monomer, free of protecting groups, was mixed in turn under a standard set of polymerization conditions with the ROMP initiator (IMesH2)C5H5N)2(Cl)2Ru=CHPh. Two sets of polymerization reactions were performed, one with Monomer:Initiator (M:I) ratio of 20:1, and another with M:I of 200:1. For the nucleophilic amino acids cysteine and lysine, polymerization reactions were quantitatively compared to those of their protected analogues. Furthermore, we describe polymerization of macromonomers containing up to 30 a.a. to test for tolerance of ROMP to peptide molecular weight. These reactions were studied via SEC-MALS and NMR. Finally, with knowledge of sequence scope in hand, we prepared a set of enzyme-substrate containing brush polymers and studied them with respect to their bioactivity.