Peptides for Specifically Targeting Nanoparticles to Cellular Organelles: <i>Quo Vadis</i>?

ConspectusThe interfacing of nanomaterials and especially nanoparticles within all aspects of biological research continues to grow at a nearly unabated pace with projected applications focusing on powerful new tools for cellular labeling, imaging, and sensing, theranostic materials, and drug delivery. At the most fundamental level, many of these nanoparticles are meant to target not only very specific cell-types, regardless of whether they are in a culture, tissue, an animal model, or ultimately a patient, but also in many cases a specific subcellular organelle. During this process, these materials will undergo a complex journey that must first find the target cell of interest, then be taken up by those cells across the extracellular membrane, and ultimately localize to a desired subcellular organelle, which may include the nucleus, plasma membrane, endolysosomal system, mitochondria, cytosol, or endoplasmic reticulum. To accomplish these complex tasks in the correct sequence, researchers are increasingly interested in selecting for and exploiting targeting peptides that can impart the requisite capabilities to a given nanoparticle construct. There are also a number of related criteria that need careful consideration for this undertaking centering on the nature and properties of the peptide vector itself, the peptide–nanoparticle conjugate characteristics, and the target cell.Here, we highlight some important issues and key research areas related to this burgeoning field. We begin by providing a brief overview of some criteria for optimal attachment of peptides to nanoparticles, the predominant methods by which nanoparticles enter cells, and some of the peptide sequences that have been utilized to facilitate nanoparticle delivery to cells focusing on those that engender the initial targeting and uptake. Because almost all materials delivered to cells by peptides utilize the endosomal system of vesicular transport and in many cases remain sequestered within the vesicles, we critically evaluate the issue of endosomal escape in the context of some recently reported successes in this regard. Following from this, peptides that have been reported to deliver nanoparticles to specific subcellular compartments are examined with a focus on what they delivered and the putative mechanisms by which they were able to accomplish this. The last section focuses on two areas that are critical to realizing this overall approach in the long term. The first is how to select for peptidyl sequences capable of improved or more specific cellular or subcellular targeting based upon principles commonly associated with drug discovery. The second looks at what has been done to create modular peptides that incorporate multiple desirable functionalities within a single, contiguous sequence. This provides a viable alternative to either the almost insurmountable challenge of finding one sequence capable of all functions or, alternatively, attaching different peptides with different functionalities to the same nanoparticle in different ratios when trying to orchestrate their net effects. Finally, we conclude with a brief perspective on the future evolution and broader impact of this growing area of bionanoscience

Synthesis and Characterization of PEGylated Luminescent Gold Nanoclusters Doped with Silver and Other Metals

Doping of fluorescent noble metal nanoclusters is being pursued to manipulate the structure of such materials along with improving physicochemical characteristics such as long-term stability and photoluminescence quantum yield. Here, we synthesize metal-doped and alloyed ultrasmall gold nanoclusters (AuNCs) directly in water using a facile one-step coreduction reaction with bidentate dithiolane PEGylated ligands that terminate in different functional groups including a methoxy, carboxy, amine, and azide. Two primary types of cluster materials were the focus of synthesis and characterization: first, a series of doped/alloyed Ag-doped AuNCs, where the ratio of Au:Ag was varied across a wide range including 99:1, 98:2, 90:10, 80:20, 50:50, 20:80, 10:90, and 2:98 along with pure AuNC and AgNC controls; second, doped Au:D NCs, where D included Pt, Cu, Zn, and Cd. Physical characterization of the modified AuNCs included TEM analysis of size, XPS/EDX analysis of dopant content, and a detailed analysis of photophysical properties including absorption and photoluminescence profiles, quantum yields over time, photoluminescence lifetimes, and examination of energy levels for selected materials. The addition of just a few Ag dopant atoms per AuNC yielded significant enhancement in quantum yield along with improving long-term photostability especially in comparison to materials with a very high Ag content. Preliminary cell imaging applications of the Ag-doped AuNCs were also investigated. Facilitated cellular uptake by mammalian cells via endocytosis following modification with cell penetrating peptides was confirmed by colabeling with specific cellular markers. Long-term intracellular photostability and lack of aggregation were confirmed with microinjection studies, and cytoviability assays showed the doped clusters to be minimally toxic

Purple‑, Blue‑, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing

We report the synthesis of a series of Cd_<i>x</i>Zn_1–<i>x</i>Se/Cd_<i>y</i>Zn_1–<i>y</i>S/ZnS and ZnSe/Cd_<i>y</i>Zn_1–<i>y</i>S/ZnS multishell alloyed luminescent semiconductor quantum dots (QDs) with fluorescence maxima ranging from 410 to 530 nm which cover the purple, blue, and green portion of the spectrum. Their subsequent surface modification to prepare water-soluble blue-emitting QDs, characterization, and application for ratiometric pH sensing in aqueous buffers and in an extracellular environment are further described. QDs were synthesized starting from ZnSe cores, and the fluorescence peak positions were tuned by (i) cation exchange with cadmium ions and/or (ii) overcoating with Cd_<i>y</i>Zn_1–<i>y</i>S layers. The as-prepared QDs had reasonably high fluorescence quantum yields (∼30–55%), narrow fluorescence bands (fwhm ∼25–35 nm), and monodispersed semispherical shapes. Ligand exchange with hydrophilic compact ligands was successfully carried out to prepare a series of water-soluble blue-emitting QDs. QDs coated with the hydrophilic compact ligands preserved the intrinsic photophysical properties well and showed excellent colloidal stability in aqueous buffers for over a year. The blue-emitting QDs were further conjugated with the pH-sensitive dye, fluorescein isothiocyanate (FITC), to construct a fluorescence resonance energy transfer-based ratiometric pH sensing platform, and pH monitoring with the QD-FITC conjugates was successfully demonstrated at pHs ranging between 3 and 7.5. Further assembly of the QD-FITC conjugates with membrane localization peptides allowed monitoring of the pH in extracellular environments. High quality, water-soluble blue-emitting QDs coated with compact ligands can help expand the practical fluorescence range of QDs for a variety of biological applications

Elucidating Surface Ligand-Dependent Kinetic Enhancement of Proteolytic Activity at Surface-Modified Quantum Dots

Combining biomolecules such as enzymes with nanoparticles has much to offer for creating next generation synergistically functional bionanomaterials. However, almost nothing is known about how these two disparate components interact at this critical biomolecular-materials interface to give rise to improved activity and emergent properties. Here we examine how the nanoparticle surface can influence and increase localized enzyme activity using a designer experimental system consisting of trypsin proteolysis acting on peptide-substrates displayed around semiconductor quantum dots (QDs). To minimize the complexity of analyzing this system, only the chemical nature of the QD surface functionalizing ligands were modified. This was accomplished by synthesizing a series of QD ligands that were either positively or negatively charged, zwitterionic, neutral, and with differing lengths. The QDs were then assembled with different ratios of dye-labeled peptide substrates and exposed to trypsin giving rise to progress curves that were monitored by Förster resonance energy transfer (FRET). The resulting trypsin activity profiles were analyzed in the context of detailed molecular dynamics simulations of key interactions occurring at this interface. Overall, we find that a combination of factors can give rise to a localized activity that was 35-fold higher than comparable freely diffusing enzyme–substrate interactions. Contributing factors include the peptide substrate being prominently displayed extending from the QD surface and not sterically hindered by the longer surface ligands in conjunction with the presence of electrostatic and other productive attractive forces between the enzyme and the QD surface. An intimate understanding of such critical interactions at this interface can produce a set of guidelines that will allow the rational design of next generation high-activity bionanocomposites and theranostics