Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay.

All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.

Engineering the Orientation, Density, and Flexibility of Single-Domain Antibodies on Nanoparticles To Improve Cell Targeting.

Nanoparticles targeted to specific cells have the potential to improve the delivery of therapeutics. The effectiveness of cell targeting can be significantly improved by optimizing how the targeting ligands are displayed on the nanoparticle surface. Crucial to optimizing the cell binding are the orientation, density, and flexibility of the targeting ligand on the nanoparticle surface. In this paper, we used an anti-EGFR single-domain antibody (sdAb or nanobody) to target fluorescent nanocrystals (Qdots) to epidermal growth factor receptor (EGFR)-positive cells. The sdAbs were expressed with a synthetic amino acid (azPhe), enabling site-specific conjugation to Qdots in an improved orientation. To optimize the targeting efficiency, we engineered the point of attachment (orientation), controlled the density of targeting groups on the surface of the Qdot, and optimized the length of the poly(ethylene glycol) linker used to couple the sdAb to the Qdot surface. By optimizing orientation, density, and flexibility, we improved cell targeting by more than an order of magnitude. This work highlights the importance of understanding the structure of the nanoparticle surface to achieve the optimal interactions with the intended receptors and how engineering the nanoparticle surface can significantly improve cell targeting.

Pointing in the Right Direction: Controlling the Orientation of Proteins on Nanoparticles Improves Targeting Efficiency.

Protein-conjugated nanoparticles have the potential to precisely deliver therapeutics to target sites in the body by specifically binding to cell surface receptors. To maximize targeting efficiency, the three-dimensional presentation of ligands toward these receptors is crucial. Herein, we demonstrate significantly enhanced targeting of nanoparticles to cancer cells by controlling the protein orientation on the nanoparticle surface. To engineer the point of attachment, we used amber codon reassignment to incorporate a synthetic amino acid, p-azidophenylalanine (azPhe), at specific locations within a single domain antibody (sdAb or nanobody) that recognizes the human epidermal growth factor receptor (EGFR). The azPhe modified sdAb can be tethered to the nanoparticle in a specific orientation using a bioorthogonal click reaction with a strained cyclooctyne. The crystal structure of the sdAb bound to EGFR was used to rationally select sites likely to optimally display the sdAb upon conjugation to a fluorescent nanocrystal (Qdot). Qdots with sdAb attached at the azPhe13 position showed 6 times greater binding affinity to EGFR expressing A549 cells, compared to Qdots with conventionally (succinimidyl ester) conjugated sdAb. As ligand-targeted delivery systems move toward clinical application, this work shows that nanoparticle targeting can be optimized by engineering the site of protein conjugation.

Secondary Self-Assembly of Supramolecular Nanotubes into Tubisomes and Their Activity on Cells.

The properties and structures of viruses are directly related to the three-dimensional structure of their capsid proteins, which arises from a combination of hydrophobic and supramolecular interactions, such as hydrogen bonds. The design of synthetic materials demonstrating similar synergistic interactions still remains a challenge. Herein, we report the synthesis of a polymer/cyclic peptide conjugate that combines the capability to form supramolecular nanotubes via hydrogen bonds with the properties of an amphiphilic block copolymer. The analysis of aqueous solutions by scattering and imaging techniques revealed a barrel-shaped alignment of single peptide nanotubes into a large tubisome (length: 260 nm (from SANS)) with a hydrophobic core (diameter: 16 nm) and a hydrophilic shell. These systems, which have a structure that is similar to those of viruses, were tested in vitro to elucidate their activity on cells. Remarkably, the rigid tubisomes are able to perforate the lysosomal membrane in cells and release a small molecule into the cytosol.

A thiol probe for measuring unfolded protein load and proteostasis in cells.

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

N-Terminal Fragments of Huntingtin Longer than Residue 170 form Visible Aggregates Independently to Polyglutamine Expansion.

BACKGROUND: A hallmark of Huntington's disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. OBJECTIVE: It remains unclear how the length of the N-terminal fragments relates to polyQ - mediated aggregation. We investigated the fundamental intracellular aggregation process of eight different-length N-terminal fragments of Htt in both short (25Q) and long polyQ (97Q). METHODS: N-terminal fragments were fused to fluorescent proteins and transiently expressed in mammalian cell culture models. These included the classic exon 1 fragment (90 amino acids) and longer forms of 105, 117, 171, 513, 536, 552, and 586 amino acids based on wild-type Htt (of 23Q) sequence length nomenclature. RESULTS: N-terminal fragments of less than 171 amino acids only formed inclusions in polyQ-expanded form. By contrast the longer fragments formed inclusions irrespective of Q-length, with Q-length playing a negligible role in extent of aggregation. The inclusions could be classified into 3 distinct morphological categories. One type (Type A) was universally associated with polyQ expansions whereas the other two types (Types B and C) formed independently of polyQ length expansion. CONCLUSIONS: PolyQ-expansion was only required for fragments of less than 171 amino acids to aggregate. Longer fragments aggregated predominately through a non-polyQ mechanism, involving at least one, and probably more distinct clustering mechanisms.

Measuring macromolecular crowding in cells through fluorescence anisotropy imaging with an AIE fluorogen.

We report a new strategy that allows spatiotemporal visualization of the macromolecular crowding effect in cells. An amine-reactive aggregation-induced emission fluorogen is used to label proteins in the cytoplasm and the change in the protein mobility as well as local viscosity can be monitored by using fluorescence anisotropy imaging and fluorescence lifetime imaging, respectively.

pH-Responsive Transferrin-pHlexi Particles Capable of Targeting Cells in Vitro.

Targeting nanoparticles to specific cellular receptors has the potential to deliver therapeutic compounds to target sites while minimizing side effects. To this end, we have conjugated a targeting protein, holo-transferrin (holo-Tf), to pH-responsive polymers, poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) and poly(2-(diethylamino)ethyl methacrylate)-ran-poly(2-(diisopropylamino)ethyl methacrylate (PDEAEMA-r-PDPAEMA). These protein-polymer hybrid materials were observed to self-assemble when the pH is increased above the pKa of the polymer. We demonstrate that their response to pH could be tuned depending on the polymer constituent attached to holo-Tf. Importantly, the targeting behavior of these nanoparticles could be maximized by tuning the density of holo-Tf on the nanoparticle surface by the introduction of a (PDEAEMA-r-PDPAEMA)-b-poly(ethylene glycol) (PEG) copolymer.