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.
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.
Glucagon is a peptide hormone used for the treatment of hypoglycemia; however, its clinical potential is limited by its insolubility and instability in solution. Herein, the encapsulation, ...stabilization, and release of glucagon by trehalose glycopolymer nanogels are reported. Methacrylate‐functionalized trehalose is copolymerized with pyridyl disulfide ethyl methacrylate using free radical polymerization conditions to form trehalose glycopolymers with thiol‐reactive handles. Glucagon is chemically modified to contain two thiol groups and is subsequently utilized as the cross‐linker to form redox‐responsive trehalose nanogels with greater than 80% conjugation yield. Nanogel formation and subsequent glucagon stabilization are characterized using polyacrylamide gel electrophoresis, dynamic light scattering, and transmission electron microscopy. It is determined that the solution stability of the glucagon increased from less than 24 h to at least three weeks in the nanogel form. Additionally, in vitro activity of the synthesized glucagon analog and released glucagon is investigated, demonstrating that the glucagon remains active after modification. It is anticipated that these glucagon–nanogel conjugates will be useful as a stabilizing glucagon formulation, allowing for cargo release under mild reducing conditions.
Trehalose glycopolymer nanogels encapsulate and stabilize glucagon, an unstable therapeutic peptide hormone, for significantly improved solubility and long‐term stability in solution. Glucagon is incorporated into the nanogels via redox‐responsive cross‐links, allowing for high peptide loading and release under mild reducing conditions. The released glucagon is bioactive by cellular assays, suggesting this system may be useful as a stabilizing glucagon formulation.
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.
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.
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.
Cellular uptake of high‐density brush polymers with peptide side chains is governed by the net charge of the polymers. Stimuli responsive cleavable linkers are employed to control the charge of the polymers and to trigger cellular uptake of brush polymers possessing a net positive charge.
An undergraduate organic chemistry laboratory that provides an introduction to various spectroscopic techniques is reported. Whereas organic spectroscopy is most often learned and practiced in the ...context of reaction analyses, this laboratory experiment allows students to become comfortable with 1H NMR, 13C NMR, and IR spectroscopy, in addition to mass spectrometry. The laboratory experiment also teaches students how to perform thin-layer chromatography. The specific compound analyzed is isoamyl acetate, commonly known as “banana oil”. With the skills acquired from completing this laboratory experiment early in a given course, students are well-prepared to perform spectroscopic analyses in subsequent experiments encountered in their organic chemistry laboratory course.
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.
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.
The utility of peptide therapeutics is thwarted by an inability to enter cells, preventing access to crucial intracellular targets.
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