A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins.

Using a chemical approach to crosslink functionally versatile bioeffectors (such as peptides) to native proteins of interest (POI) directly inside a living cell is a useful toolbox for chemical biologists. However, this goal has not been reached due to unsatisfactory chemoselectivity, regioselectivity, and protein selectivity in protein labeling within living cells. Herein, we report the proof of concept of a cytocompatible and highly selective photolabeling strategy using a tryptophan-specific Ru-TAP complex as a photocrosslinker. Aside from the high selectivity, the photolabeling is blue light-driven by a photoinduced electron transfer (PeT) and allows the bioeffector to bear an additional UV-responsive unit. The two different photosensitivities are demonstrated by blue light-photocrosslinking a UV-sensitive peptide to POI. Our visible light photolabeling can generate photocaged proteins for subsequent activity manipulation by UV light. Cytoskeletal dynamics regulation is demonstrated in living cells via the unprecedented POI photomanipulation and proves that our methodology opens a new avenue to endogenous protein modification.

A Facile NMR Method for Pre-MRI Evaluation of Trigger-Responsive T1 Contrast Enhancement.

There is a growing interest in developing paramagnetic nanoparticles as responsive magnetic resonance imaging (MRI) contrast agents, which feature switchable T1 image contrast of water protons upon biochemical cues for better discerning diseases. However, performing an MRI is pragmatically limited by its cost and availability. Hence, a facile, routine method for measuring the T1 contrast is highly desired in early-stage development. This work presents a single-point inversion recovery (IR) nuclear magnetic resonance (NMR) method that can rapidly evaluate T1 contrast change by employing a single, optimized IR pulse sequence that minimizes water signal for "off-state" nanoparticles and allows for sensitively measuring the signal change with "switch-on" T1 contrast. Using peptide-induced liposomal gadopentetic acid (Gd3+ -DTPA) release and redox-sensitive manganese oxide (MnO2 ) nanoparticles as a demonstration of generality, this method successfully evaluates the T1 shortening of water protons caused by liposomal Gd3+ -DTPA release and Mn2+ formation from MnO2 reduction. Furthermore, the NMR measurement is highly correlated to T1 -weighted MRI scans, suggesting its feasibility to predict the MRI results at the same field strength. This NMR method can be a low-cost, time-saving alternative for pre-MRI evaluation for a diversity of responsive T1 contrast systems.

Nano-On-Nano: Responsive Nanosubstrate-Mediated Liposome Delivery with High Cellular Uptake Efficiency.

Efficiently delivering liposomal content to cells in a relatively uniform dose and patterned fashion, especially bypassing the degradative endocytosis pathway, is an important technology in cell culture and potentially to tissue engineering that still remains challenging. We developed a "nano-on-nano" platform technology that consists of the following three material features: (1) high density silicon nanopillars to create a pseudo-3-dimensional nanoenvironment for cell culturing, (2) thermoresponsive polymer grafted onto silicon nanopillars to form a responsive nanosubstrate, and (3) immobilized liposomes using a biotin-streptavidin-biotin conjugation. The working principle is that the liposomes are detached for cellular uptake upon thermal stimulation and high local liposome concentration between the cells and substrates drives the cellular uptake with nonendocytic pathways. Cryo-EM images confirms that liposomes are attached to form liposome-warped nanopillars. Upon thermal stimulation, an 8 times higher increase in the liposomal fluorescence intensity is observed compared to the conventional solution-phase liposome delivery, indicating that high local concentration drives liposome uptake with greater efficiency. Moreover, preliminary mechanistic studies reveal that these liposomes are taken up by nonendocytic pathways. The ability of our nano-on-nano delivery system that achieves efficient dose-uniform cellular delivery can open a unique era in cell and tissue engineering by controlling cell behaviors with the delivery of bioactive ingredient-loaded liposomes.

Liposomal PHD2 Inhibitors and the Enhanced Efficacy in Stabilizing HIF-1α.

Prolyl hydroxylase domain-containing protein 2 (PHD2) inhibition, which stabilizes hypoxia-inducible factor (HIF)-1α and thus triggers adaptation responses to hypoxia in cells, has become an important therapeutic target. Despite the proven high potency, small-molecule PHD2 inhibitors such as IOX2 may require a nanoformulation for favorable biodistribution to reduce off-target toxicity. A liposome formulation for improving the pharmacokinetics of an encapsulated drug while allowing a targeted delivery is a viable option. This study aimed to develop an efficient loading method that can encapsulate IOX2 and other PHD2 inhibitors with similar pharmacophore features in nanosized liposomes. Driven by a transmembrane calcium acetate gradient, a nearly 100% remote loading efficiency of IOX2 into liposomes was achieved with an optimized extraliposomal solution. The electron microscopy imaging revealed that IOX2 formed nanoprecipitates inside the liposome's interior compartments after loading. For drug efficacy, liposomal IOX2 outperformed the free drug in inducing the HIF-1α levels in cell experiments, especially when using a targeting ligand. This method also enabled two clinically used inhibitors-vadadustat and roxadustat-to be loaded into liposomes with a high encapsulation efficiency, indicating its generality to load other heterocyclic glycinamide PHD2 inhibitors. We believe that the liposome formulation of PHD2 inhibitors, particularly in conjunction with active targeting, would have therapeutic potential for treating more specifically localized disease lesions.

Lipid-Wrapped Upconversion Nanoconstruct/Photosensitizer Complex for Near-Infrared Light-Mediated Photodynamic Therapy.

Photodynamic therapy (PDT) is a noninvasive medical technology that has been applied in cancer treatment where it is accessible by direct or endoscope-assisted light irradiation. To lower phototoxicity and increase tissue penetration depth of light, great effort has been focused on developing new sensitizers that can utilize red or near-infrared (NIR) light for the past decades. Lanthanide-doped upconversion nanoparticles (UCNPs) have a unique property to transduce NIR excitation light to UV-vis emission efficiently. This property allows some low-cost, low-toxicity, commercially available visible light sensitizers, which originally are not suitable for deep tissue PDT, to be activated by NIR light and have been reported extensively in the past few years. However, some issues still remain in the UCNP-assisted PDT platform such as colloidal stability, photosensitizer loading efficiency, and accessibility for targeting ligand installation, despite some advances in this direction. In this study, we designed a facile phospholipid-coated UCNP method to generate a highly colloidally stable nanoplatform that can effectively load a series of visible light sensitizers in the lipid layers. The loading stability and singlet oxygen generation efficiency of this sensitizer-loaded lipid-coated UCNP platform were investigated. We also have demonstrated the enhanced cellular uptake efficiency and tumor cell selectivity of this lipid-coated UCNP platform by changing the lipid dopant. On the basis of the evidence of our results, the lipid-complexed UCNP nanoparticles could serve as an effective photosensitizer carrier for NIR light-mediated PDT.

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

Targeted and efficient activation of channelrhodopsins expressed in living cells via specifically-bound upconversion nanoparticles.

Optogenetics is an innovative technology now widely adopted by researchers in different fields of biological sciences. However, most light-sensitive proteins adopted in optogenetics are excited by ultraviolet or visible light which has a weak tissue penetration capability. Upconversion nanoparticles (UCNPs), which absorb near-infrared (NIR) light to emit shorter wavelength light, can help address this issue. In this report, we demonstrated the target selectivity by specifically conjugating the UCNPs with channelrhodopsin-2 (ChR2). We tagged the V5 epitope to the extracellular N-terminal of ChR2 (V5-ChR2m) and functionalized the surface of UCNPs with NeutrAvidin (NAv-UCNPs). After the binding of the biotinylated antibody against V5 onto the V5-ChR2m expressed in the plasma membrane of live HEK293T cells, our results showed that the NAv-UCNPs were specifically bound to the membrane of cells expressing V5-ChR2m. Without the V5 epitope or NAv modification, no binding of UCNPs onto the cell membrane was observed. For the cells expressing V5-ChR2m and bound with NAv-UCNPs, both 488 nm illumination and the upconverted blue emission from UCNPs by 980 nm excitation induced an inward current and elevated the intracellular Ca2+ concentration. Our design reduces the distance between UCNPs and light-sensitive proteins to the molecular level, which not only minimizes the NIR energy required but also provides a way to guide the specific binding for optogenetics applications.

Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking.

The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a "switch" in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photoactivation. The photoinduced in vitro fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides in vitro and in vivo spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases.

Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.

Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme-nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector.

Characterization and real-time imaging of the FTLD-related protein aggregation induced by amyloidogenic peptides.

We identify a new amyloidogenic peptide from the glutamine/asparagine-rich region of the FTLD-related protein (TDP-43), which can seed both the full-length and N-terminus-truncated TDP-43. Through the microinjection and real-time fluorescence imaging, we also found that this novel peptide could trigger cell apoptosis and initiate TDP-43 aggregation in the cytosol.