High Spatiotemporal Control Research Articles

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Stimuli‐Responsive DNA Circuits for High‐Performance Bioimaging Application

AbstractProbing endogenous molecules in living entities is significant to help decipher biological functions and exploit novel theranostics. DNA circuits that can recognize molecular inputs of interest and transduce them into readable signal outputs in an isothermal and autonomous manner have been actively pursued as versatile toolkits for intracellular biosensing research. Tremendous efforts are being devoted to developing integrated DNA circuits with high sensitivity, while spatiotemporal selectivity is often overlooked in the construction of functional DNA circuitry systems. This requires the development of stimuli‐responsive DNA circuits that can be activated on‐demand from the initial sensing‐blunt state to the sensing‐ready state under a programmable manner, for achieving precise bioimaging with high spatiotemporal control. In this review, an overview of recent advances in the construction of stimuli‐responsive DNA circuits that respond to particular triggers, including external physical stimuli and endogenous biological cues, and their spatiotemporally controllable molecular bioimaging applications is provided. The current challenges and potential solutions of these stimuli‐responsive DNA circuits for their future developments in this emerging field are also discussed.

Acceptor Engineering Produces Ultrafast Nonradiative Decay in NIR-II Aza-BODIPY Nanoparticles for Efficient Osteosarcoma Photothermal Therapy via Concurrent Apoptosis and Pyroptosis.

Small-molecule photothermal agents (PTAs) with intense second near-infrared (NIR-II, 1,000 to 1,700 nm) absorption and high photothermal conversion efficiencies (PCEs) are promising candidates for treating deep-seated tumors such as osteosarcoma. To date, the development of small-molecule NIR-II PTAs has largely relied on fabricating donor-acceptor-donor (D-A-D/D') structures and limited success has been achieved. Herein, through acceptor engineering, a donor-acceptor-acceptor (D-A-A')-structured NIR-II aza-boron-dipyrromethene (aza-BODIPY) PTA (SW8) was readily developed for the 1,064-nm laser-mediated phototheranostic treatment of osteosarcoma. Changing the donor groups to acceptor groups produced remarkable red-shifts of absorption maximums from first near-infrared (NIR-I) regions (~808 nm) to NIR-II ones (~1,064 nm) for aza-BODIPYs (SW1 to SW8). Furthermore, SW8 self-assembled into nanoparticles (SW8@NPs) with intense NIR-II absorption and an ultrahigh PCE (75%, 1,064 nm). This ultrahigh PCE primarily originated from an additional nonradiative decay pathway, which showed a 100-fold enhanced decay rate compared to that shown by conventional pathways such as internal conversion and vibrational relaxation. Eventually, SW8@NPs performed highly efficient 1,064-nm laser-mediated NIR-II photothermal therapy of osteosarcoma via concurrent apoptosis and pyroptosis. This work not only illustrates a remote approach for treating deep-seated tumors with high spatiotemporal control but also provides a new strategy for building high-performance small-molecule NIR-II PTAs.

Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CB1R) "Cis-On" Agonist.

Activation of the human cannabinoid receptor type 1 (hCB1R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for hCB1R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB1R (Ki (cis-form) = 0.18 μM), with a marked difference in affinity with respect to its inactive "trans-off" form (CB1R Ki trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT βarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of hCB1R in disorders associated with the endocannabinoid system.

Melding Synthetic Molecules and Genetically Encoded Proteins to Forge New Tools for Neuroscience.

Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems.

Photocaged amplified FRET nanoflares: spatiotemporal controllable of mRNA-powered nanomachines for precise and sensitive microRNA imaging in live cells.

There is considerable interest in creating a precise and sensitive strategy for in situ visualizing and profiling intracellular miRNA. Present here is a novel photocaged amplified FRET nanoflare (PAFN), which spatiotemporal controls of mRNA-powered nanomachine for precise and sensitive miRNA imaging in live cells. The PAFN could be activated remotely by light, be triggered by specific low-abundance miRNA and fueled by high-abundance mRNA. It offers high spatiotemporal control over the initial activity of nanomachine at desirable time and site, and a ‘one-to-more’ ratiometric signal amplification model. The PAFN, an unprecedented design, is quiescent during the delivery process. However, upon reaching the interest tumor site, it can be selectively activated by light, and then be triggered by specific miRNA, avoiding undesirable early activation and reducing nonspecific signals, allowing precise and sensitive detection of specific miRNA in live cells. This strategy may open new avenues for creating spatiotemporally controllable and endogenous molecule-powered nanomachine, facilitating application at biological and medical imaging.

Reactive Oxygen Species in Anticancer Immunity: A Double-Edged Sword.

Reactive oxygen species (ROS) are critical mediators in many physiological processes including innate and adaptive immunity, making the modulation of ROS level a powerful strategy to augment anticancer immunity. However, current evidences suggest the necessity of a deeper understanding of their multiple roles, which may vary with their concentration, location and the immune microenvironment they are in. Here, we have reviewed the reported effects of ROS on macrophage polarization, immune checkpoint blocking (ICB) therapy, T cell activation and expansion, as well as the induction of immunogenic cell death. A majority of reports are indicating detrimental effects of ROS, but it is unadvisable to simply scavenge them because of their pleiotropic effects in most occasions (except in T cell activation and expansion where ROS are generally undesirable). Therefore, clinical success will need a clearer illustration of their multi-faced functions, as well as more advanced technologies to tune ROS level with high spatiotemporal control and species-specificity. With such progresses, the efficacy of current immunotherapies will be greatly improved by combining with ROS-targeted therapies.

Selective Bond Cleavage in RAFT Agents Promoted by Low-Energy Electron Attachment.

Radical polymerization with reversible addition‐fragmentation chain transfer (RAFT polymerization) has been successfully applied to generate polymers of well‐defined architecture. For RAFT polymerization a source of radicals is required. Recent work has demonstrated that for minimal side‐reactions and high spatio‐temporal control these should be formed directly from the RAFT agent or macroRAFT agent (usually carbonothiosulfanyl compounds) thermally, photochemically or by electrochemical reduction. In this work, we investigated low‐energy electron attachment to a common RAFT agent (cyanomethyl benzodithioate), and, for comparison, a simple carbonothioylsulfanyl compound (dimethyl trithiocarbonate, DMTTC) in the gas phase by means of mass spectrometry as well as quantum chemical calculations. We observe for both compounds that specific cleavage of the C−S bond is induced upon low‐energy electron attachment at electron energies close to zero eV. This applies even in the case of a poor homolytic leaving group (.CH3 in DMTTC). All other dissociation reactions found at higher electron energies are much less abundant. The present results show a high control of the chemical reactions induced by electron attachment.

Azobenzene‐Based Photomechanical Biomaterials

Biomaterials with stimuli sensitivity and good mechanical properties are garnering interest as an important branch of stimuli‐responsive materials. Among them, photomechanical biomaterials are an emerging class of eco‐friendly materials for the development of various biomedical devices because they offer high spatiotemporal control, on‐demand response, and noninvasive manipulation. In particular, azobenzene can be reversibly converted from the trans to the cis isomer under irradiation by different wavelengths of light. The significant changes in structural geometry and excellent fatigue resistance of azobenzene upon isomerization allow its use in the fabrication of materials with photoresponsive properties, such as bending, twisting, coiling, buckling, expansion, or even jumping. However, studies on the photomodulated mechanical performance of azobenzene‐based bulk biomaterials have rarely been reported. This review focuses on the photomechanical effects that occur in various systems incorporated with azobenzene moieties. Within this framework, the advantages of azobenzene in different photomechanical materials, including liquid crystals, bulk films, gels, and bulk fibers, are discussed. In each section, the light‐induced modulation of mechanical properties, including tensile strength, modulus, and toughness, is highlighted. Finally, a summary and outlook for the development of azobenzene‐based photomechanical materials is presented.

A Photohormone for Light-Dependent Control of PPARα in Live Cells.

Photopharmacology enables the optical control of several biochemical processes using small-molecule photoswitches that exhibit different bioactivities in their cis- and trans-conformations. Such tool compounds allow for high spatiotemporal control of biological signaling, and the approach also holds promise for the development of drug molecules that can be locally activated to reduce target-mediated adverse effects. Herein, we present the expansion of the photopharmacological arsenal to two new members of the peroxisome proliferator-activated receptor (PPAR) family, PPARα and PPARδ. We have developed a set of highly potent PPARα and PPARδ targeting photohormones derived from the weak pan-PPAR agonist GL479 that can be deactivated by light. The photohormone 6 selectively activated PPARα in its trans-conformation with high selectivity over the related PPAR subtypes and was used in live cells to switch PPARα activity on and off in a light- and time-dependent fashion.

Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell–Cell Interactions

The regulation of cell-cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell-cell interactions with high precision are of great interest to a better understanding of their roles and building tissue-like structures. Herein, the green light-responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell-cell interactions form and lead to cell clustering in a concentration-dependent manner. Upon green light illumination, the CarH based cell-cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell-cell interactions impact cell migration, as observed in a wound-healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell-cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell-cell interactions in biological processes.

Photoactivatable 1,2-dioxetane chemiluminophores

Photochemical control of molecules has provided chemists with the ability to establish high spatiotemporal control of chemical configuration and activity. Herein, we report UVC-454, UVA-454, and Spiro-CL as photoactivatable chemiluminescence compounds. UVC-454 and UVA-454 are protected with ortho-nitrobenzyl protecting groups that provide irreversible photochemical uncaging of the chemiluminophore species through UV light irradiation. UVC-454 and UVA-454 can be selectively activated based on uncaging wavelength, and demonstrate ability to be photoactivated in water. Spiro-CL is a novel chemiluminescent spiropyran that can reversibly interconvert from its stable spiropyran form to a metastable merocyanine form through UV or visible light irradiation, respectively. Further, this compound exhibits chemiluminescence in its open form upon irradiation with UV light in DMSO.

Reversibly Photo-Modulating Mechanical Stiffness and Toughness of Bioengineered Protein Fibers.

Light‐responsive materials have been extensively studied due to the attractive possibility of manipulating their properties with high spatiotemporal control in a non‐invasive fashion. This stimulated the development of a series of photo‐deformable smart devices. However, it remained a challenge to reversibly modulate the stiffness and toughness of bulk materials. Here, we present bioengineered protein fibers and their optomechanical manipulation by employing electrostatic interactions between supercharged polypeptides (SUPs) and an azobenzene (Azo)‐based surfactant. Photo‐isomerization of the Azo moiety from the E‐ to Z‐form reversibly triggered the modulation of tensile strength, stiffness, and toughness of the bulk protein fiber. Specifically, the photo‐induced rearrangement into the Z‐form of Azo possibly strengthened cation–π interactions within the fiber material, resulting in an around twofold increase in the fiber's mechanical performance. The outstanding mechanical and responsive properties open a path towards the development of SUP‐Azo fibers as smart stimuli‐responsive mechano‐biomaterials.

Reversibly Photo‐Modulating Mechanical Stiffness and Toughness of Bioengineered Protein Fibers

AbstractLight‐responsive materials have been extensively studied due to the attractive possibility of manipulating their properties with high spatiotemporal control in a non‐invasive fashion. This stimulated the development of a series of photo‐deformable smart devices. However, it remained a challenge to reversibly modulate the stiffness and toughness of bulk materials. Here, we present bioengineered protein fibers and their optomechanical manipulation by employing electrostatic interactions between supercharged polypeptides (SUPs) and an azobenzene (Azo)‐based surfactant. Photo‐isomerization of the Azo moiety from the E‐ to Z‐form reversibly triggered the modulation of tensile strength, stiffness, and toughness of the bulk protein fiber. Specifically, the photo‐induced rearrangement into the Z‐form of Azo possibly strengthened cation–π interactions within the fiber material, resulting in an around twofold increase in the fiber's mechanical performance. The outstanding mechanical and responsive properties open a path towards the development of SUP‐Azo fibers as smart stimuli‐responsive mechano‐biomaterials.

Modular operation of microfluidic chips for highly parallelized cell culture and liquid dosing via a fluidic circuit board

Microfluidic systems enable automated and highly parallelized cell culture with low volumes and defined liquid dosing. To achieve this, systems typically integrate all functions into a single, monolithic device as a “one size fits all” solution. However, this approach limits the end users’ (re)design flexibility and complicates the addition of new functions to the system. To address this challenge, we propose and demonstrate a modular and standardized plug-and-play fluidic circuit board (FCB) for operating microfluidic building blocks (MFBBs), whereby both the FCB and the MFBBs contain integrated valves. A single FCB can parallelize up to three MFBBs of the same design or operate MFBBs with entirely different architectures. The operation of the MFBBs through the FCB is fully automated and does not incur the cost of an extra external footprint. We use this modular platform to control three microfluidic large-scale integration (mLSI) MFBBs, each of which features 64 microchambers suitable for cell culturing with high spatiotemporal control. We show as a proof of principle that we can culture human umbilical vein endothelial cells (HUVECs) for multiple days in the chambers of this MFBB. Moreover, we also use the same FCB to control an MFBB for liquid dosing with a high dynamic range. Our results demonstrate that MFBBs with different designs can be controlled and combined on a single FCB. Our novel modular approach to operating an automated microfluidic system for parallelized cell culture will enable greater experimental flexibility and facilitate the cooperation of different chips from different labs.

Immunogenicity of Externally Activated Nanoparticles for Cancer Therapy.

Simple SummaryRecent advances in treating cancer via stimulating an anti-tumor immune system response have resulted in extraordinary results for lymphomas and leukemias; however these therapies have not performed well in solid tumors. External beam therapies, such as radiotherapy, hyperthermia, and photodynamic therapy, that are clinically used for solid tumors are now being explored in combination with nanoparticle systems to stimulate a long-term anti-tumor immune system response. In this review, we detail the novel nanoparticle complexes that are being researched to activate an anti-tumor immune response in combination with external beam therapy in both the preclinical and clinical settings.Nanoparticles activated by external beams, such as ionizing radiation, laser light, or magnetic fields, have attracted significant research interest as a possible modality for treating solid tumors. From producing hyperthermic conditions to generating reactive oxygen species, a wide range of externally activated mechanisms have been explored for producing cytotoxicity within tumors with high spatiotemporal control. To further improve tumoricidal effects, recent trends in the literature have focused on stimulating the immune system through externally activated treatment strategies that result in immunogenic cell death. By releasing inflammatory compounds known to initiate an immune response, treatment methods can take advantage of immune system pathways for a durable and robust systemic anti-tumor response. In this review, we discuss recent advancements in radiosensitizing and hyperthermic nanoparticles that have been tuned for promoting immunogenic cell death. Our review covers both preclinical and clinical results, as well as an overview of possible future work.

Effect of Photocaged Isopropyl β-d-1-thiogalactopyranoside Solubility on the Light Responsiveness of LacI-controlled Expression Systems in Different Bacteria.

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non‐invasive as well as easily tuneable. In the recent past, photo‐responsive inducer molecules such as 6‐nitropiperonyl‐caged IPTG (NP‐cIPTG) have been used as optochemical tools for Lac repressor‐controlled microbial expression systems. To further expand the applicability of the versatile optochemical on‐switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria. To this end, we developed two new cIPTG derivatives with different hydrophobicity and demonstrated both an easy applicability for the light‐mediated control of gene expression and a simple transferability of this optochemical toolbox to the biotechnologically relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably, the more water‐soluble cIPTG derivative proved to be particularly suitable for light‐mediated gene expression in these alternative expression hosts.

Light-Driven Proton Transfer for Cyclic and Temporal Switching of Enzymatic Nanoreactors.

Temporal activation of biological processes by visible light and subsequent return to an inactive state in the absence of light is an essential characteristic of photoreceptor cells. Inspired by these phenomena, light-responsive materials are very attractive due to the high spatiotemporal control of light irradiation, with light being able to precisely orchestrate processes repeatedly over many cycles. Herein, it is reported that light-driven proton transfer triggered by a merocyanine-based photoacid can be used to modulate the permeability of pH-responsive polymersomes through cyclic, temporally controlled protonation and deprotonation of the polymersome membrane. The membranes can undergo repeated light-driven swelling-contraction cycles without losing functional effectiveness. When applied to enzyme loaded-nanoreactors, this membrane responsiveness is used for the reversible control of enzymatic reactions. This combination of the merocyanine-based photoacid and pH-switchable nanoreactors results in rapidly responding and versatile supramolecular systems successfully used to switch enzymatic reactions ON and OFF on demand.

Blue-Light-Switchable Bacterial Cell–Cell Adhesions Enable the Control of Multicellular Bacterial Communities

Although the fundamental importance and biotechnological potential of multibacterial communities, also called biofilms, are well-known, our ability to control them is limited. We present a new way of dynamically controlling bacteria-bacteria adhesions by using blue light and how these photoswitchable adhesions can be used to regulate multicellularity and associated bacterial behavior. To achieve this, the photoswitchable proteins nMagHigh and pMagHigh were expressed on bacterial surfaces as adhesins to allow multicellular clusters to assemble under blue light and reversibly disassemble in the dark. Regulation of the bacterial cell-cell adhesions with visible light provides unique advantages including high spatiotemporal control, tunability, and noninvasive remote regulation. Moreover, these photoswitchable adhesions make it possible to regulate collective bacterial functions including aggregation, quorum sensing, biofilm formation, and metabolic cross-feeding between auxotrophic bacteria with light. Overall, the photoregulation of bacteria-bacteria adhesions provides a new way of studying bacterial cell biology and will enable the design of biofilms for biotechnological applications.

Gap-size dependence of optical near fields in a variable nanoscale two-tip junction

Nanometer-sharp objects coupled with femtosecond laser pulses find much current interest because they allow us to reach high spatiotemporal control -- like a needle tip enables the scanning tunneling microscope (STM). With two needles facing each other, a special nanometric gap is formed, facilitating large optical near fields. Here, the authors experimentally retrieve the optical near fields of femtosecond laser pulses present in a nanogap as a function of the gap size, bearing direct ramifications for plasmonics and strong-field physics.