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.
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.
Pharmacophore models for nicotinic agonists have been proposed for four decades. Central to these models is the presence of a cationic nitrogen and a hydrogen bond acceptor. It is now ...well-established that the cationic center makes an important cation-π interaction to a conserved tryptophan, but the donor to the proposed hydrogen bond acceptor has been more challenging to identify. A structure of nicotine bound to the acetylcholine binding protein predicted that the binding partner of the pharmacophore's second component was a water molecule, which also hydrogen bonds to the backbone of the complementary subunit of the receptors. Here we use unnatural amino acid mutagenesis coupled with agonist analogs to examine whether such a hydrogen bond is functionally significant in the α4β2 neuronal nAChR, the receptor most associated with nicotine addiction. We find evidence for the hydrogen bond with the agonists nicotine, acetylcholine, carbamylcholine, and epibatidine. These data represent a completed nicotinic pharmacophore and offer insight into the design of new therapeutic agents that selectively target these receptors.
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-GX
PLX
, 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 (IMesH
)C
H
N)
(Cl)
Ru=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.
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.
The scarcity of effective means to deliver functional proteins to living cells is a central problem in biotechnology and medicine. Herein, we report the efficient delivery of an active DNA‐modifying ...enzyme to human stem cells through high‐density cell penetrating peptide brush polymers. Cre recombinase is mixed with a fluorophore‐tagged polymer carrier and then applied directly to induced pluripotent stem cells or HEK293T cells. This results in efficient delivery of Cre protein as measured by activation of a genomically integrated Cre‐mediated recombination reporter. We observed that brush polymer formulations utilizing cell penetrating peptides promoted Cre delivery but oligopeptides alone or oligopeptides displayed on nanoparticles did not. Overall, we report the efficient delivery of a genome‐modifying enzyme to stem cells that may be generalizable to other, difficult‐to‐transduce cell types.
(B)rush delivery: The efficient delivery of an active DNA‐modifying enzyme to human stem cells through high‐density cell penetrating peptide brush polymers is reported. Brush polymer formulations utilizing cell penetrating peptides could promote delivery of Cre recombinase, whereas oligopeptides alone or oligopeptides displayed on nanoparticles did not.
N-Heterocyclic carbene (NHC)-containing metal complexes are prepared through a simple, base-free method involving the decomposition of 2-(pentafluorophenyl)imidazolidines under mild thermolytic ...conditions. Ruthenium, iridium, and rhodium complexes containing NHC ligands with different electronic and steric parameters are reported.
We present an untemplated, single-component antisense oligonucleotide delivery system capable of regulating mRNA abundance in live human cells. While most approaches to nucleic acid delivery rely on ...secondary carriers and complex multicomponent charge-neutralizing formulations, we demonstrate efficient delivery using a simple locked nucleic acid (LNA)-polymer conjugate that assembles into spherical micellar nanoparticles displaying a dense shell of nucleic acid at the surface. Cellular uptake of soft LNA nanoparticles occurs rapidly within minutes as evidenced by flow cytometry and fluorescence microscopy. Importantly, these LNA nanoparticles knockdown survivin mRNA, an established target for cancer therapy, in a sequence-specific fashion as analyzed by RT-PCR.
We report enhancement in the fluorescent signal of the carbocyanine dye Cy5 by using an engineered virus as a scaffold to attach >40 Cy5 reporter molecules at fixed locations on the viral capsid. ...Although cyanine dye loading is often accompanied by fluorescence quenching, our results demonstrate that organized spatial distribution of Cy5 reporter molecules on the capsid obviates this commonly encountered problem. In addition, we observe energy transfer from the virus to adducted dye molecules, resulting in a highly fluorescent viral nanoparticle. We have used this enhanced fluorescence for the detection of DNA−DNA hybridization. When compared with the most often used detection methods in a microarray-based genotyping assay for Vibrio cholerae O139, these viral nanoparticles markedly increased assay sensitivity, thus demonstrating their applicability for existing DNA microarray protocols.
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