Photocatalytic manipulation of Ca2+ signaling for regulating cellular and animal behaviors via MOF-enabled H2O2 generation.

The in situ generation of H2O2 in cells in response to external stimulation has exceptional advantages in modulating intracellular Ca2+ dynamics, including high controllability and biological safety, but has been rarely explored. Here, we develop photocatalyst-based metal-organic frameworks (DCSA-MOFs) to modulate Ca2+ responses in cells, multicellular spheroids, and organs. By virtue of the efficient photocatalytic oxygen reduction to H2O2 without sacrificial agents, photoexcited DCSA-MOFs can rapidly trigger Ca2+ outflow from the endoplasmic reticulum with single-cell precision in a repeatable and controllable manner, enabling the propagation of intercellular Ca2+ waves (ICW) over long distances in two-dimensional and three-dimensional cell cultures. After photoexcitation, ICWs induced by DCSA-MOFs can activate neural activities in the optical tectum of tadpoles and thighs of spinal frogs, eliciting the corresponding motor behaviors. Our study offers a versatile optical nongenetic modulation technique that enables remote, repeatable, and controlled manipulation of cellular and animal behaviors.

Molecular Motor-Driven Light-Controlled Logic-Gated K+ Channel for Cancer Cell Apoptosis.

Developing artificial ion transport systems, which process complicated information and step-wise regulate properties, is essential for deeply comprehending the subtle dynamic behaviors of natural channel proteins (NCPs). Here a photo-controlled logic-gated K+ channel based on single-chain random heteropolymers containing molecular motors, exhibiting multi-core processor-like properties to step-wise control ion transport is reported. Designed with oxygen, deoxygenation, and different wavelengths of light as input signals, complicated logical circuits comprising "YES", "AND", "OR" and "NOT" gate components are established. Implementing these logical circuits with K+ transport efficiencies as output signals, multiple state transitions including "ON", "Partially OFF" and "Totally OFF" in liposomes and cancer cells are realized, further causing step-wise anticancer treatments. Dramatic K+ efflux in the "ON" state (decrease by 50% within 7 min) significantly induces cancer cell apoptosis. This integrated logic-gated strategy will be expanded toward understanding the delicate mechanism underlying NCPs and treating cancer or other diseases is expected.

Design of disintegrable nanoassemblies to release multiple small-sized nanoparticles.

The therapeutic and diagnostic effects of nanoparticles highly depend on the efficiency of their delivery to targeted tissues, such as tumors. The size of nanoparticles, among other characteristics, plays a crucial role in determining their tissue penetration and retention. Small nanoparticles may penetrate deeper into tumor parenchyma but are poorly retained, whereas large ones are distributed around tumor blood vessels. Thus, compared to smaller individual nanoparticles, assemblies of such nanoparticles due to their larger size are favorable for prolonged blood circulation and enhanced tumor accumulation. Upon reaching the targeted tissues, nanoassemblies may dissociate at the target region and release the smaller nanoparticles, which is beneficial for their distribution at the target site and ultimate clearance. The recent emerging strategy that combines small nanoparticles into larger, biodegradable nanoassemblies has been demonstrated by several groups. This review summarizes a variety of chemical and structural designs for constructing stimuli-responsive disintegrable nanoassemblies as well as their different disassembly routes. These nanoassemblies have been applied as demonstrators in the fields of cancer therapy, antibacterial infection, ischemic stroke recovery, bioimaging, and diagnostics. Finally, we summarize stimuli-responsive mechanisms and their corresponding nanomedicine designing strategies, and discuss potential challenges and barriers towards clinical translation.

Influence of the chirality of carbon nanodots on their interaction with proteins and cells.

Carbon nanodots with opposite chirality possess the same major physicochemical properties such as optical features, hydrodynamic diameter, and colloidal stability. Here, a detailed analysis about the comparison of the concentration of both carbon nanodots is carried out, putting a threshold to when differences in biological behavior may be related to chirality and may exclude effects based merely on differences in exposure concentrations due to uncertainties in concentration determination. The present study approaches this comparative analysis evaluating two basic biological phenomena, the protein adsorption and cell internalization. We find how a meticulous concentration error estimation enables the evaluation of the differences in biological effects related to chirality.

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle.

The protein corona can significantly modulate the physicochemical properties and gene delivery of polyethylenimine (PEI)/DNA complexes (polyplexes). The effects of the protein corona on the transfection have been well studied in terms of averaged gene expression in a whole cell population. Such evaluation methods give excellent and reliable statistics, but they in general provide the final transfection efficiency without reflecting the dynamic process of gene expression. In this regard the influence of bovine serum albumin (BSA) on the gene expression of PEI polyplexes also on a single cell level via live imaging is analyzed. The results reveal that although the BSA corona causes difference in the overall gene expression and mRNA transcription, the gene expression behavior on the level of individual cell is similar, including the mitosis-dependent expression, distributions of onset time, expression pattern in two daughter cells, and expression kinetics in successfully transfected cells. Comparison of single cell and ensemble data on whole cell cultures indicate that the protein corona does not alter the transfection process after nuclear entry, including cell division, polyplex dissociation, and protein expression. Its influence on other steps of in vitro gene delivery before nuclear entry shall render the difference in the overall transfection.

Stimulation of Local Cytosolic Calcium Release by Photothermal Heating for Studying Intra- and Intercellular Calcium Waves.

A methodology is described that allows for localized Ca2+ release by photoexcitation. For this, cells are loaded with polymer capsules with integrated plasmonic nanoparticles, which reside in endo-lysosomes. The micrometer-sized capsules can be individually excited by near-infrared light from a light pointer, causing photothermal heating, upon which there is a rise in the free cytosolic Ca2+ concentration ([Ca2+ ]i ). The [Ca2+ ]i can be analyzed with a Ca2+ indicator fluorophore. In this way, it is possible to excite local lysosomal Ca2+ release in a desired target cell.

Quantitative Assessment of Endosomal Escape of Various Endocytosed Polymer-Encapsulated Molecular Cargos upon Photothermal Heating.

Encapsulated molecular cargos are efficiently endocytosed by cells. For cytosolic delivery, understanding the dynamic process of cargos release from the carrier vehicles used for encapsulation and the lysosomes where the carrier vehicles are trapped (which in general is the bottleneck), followed by diffusion in the cytosol is important for improving drug/gene delivery strategies. A methodology is reported to image this process on a millisecond scale and to quantitatively analyze the data. Polyelectrolyte capsules with embedded gold nanostars to encapsulate 43 fluorescent molecular cargos with diverse properties, ranging from small fluorophores to fluorescently labeled proteins, siRNA, etc., are used. By short laser irradiation intracellular release of the molecular cargos from endocytosed capsules into the cytosol is triggered, and their intracellular spreading is imaged. Most of the released molecular cargos evenly distribute inside the entire cell, while others are enriched in certain cell compartments. The time the different molecular cargos take to distribute within cells, i.e., the spreading time, is used as a quantifier. Quantitative analysis reveals that intracellular spread cannot be described by free diffusion, but is determined by interaction of the molecular cargo with intracellular components.

Development of Silica-Based Biodegradable Submicrometric Carriers and Investigating Their Characteristics as in Vitro Delivery Vehicles.

Nanostructured silica (SiO2)-based materials are attractive carriers for the delivery of bioactive compounds into cells. In this study, we developed hollow submicrometric particles composed of SiO2 capsules that were separately loaded with various bioactive molecules such as dextran, proteins, and nucleic acids. The structural characterization of the reported carriers was conducted using transmission and scanning electron microscopies (TEM/SEM), confocal laser scanning microscopy (CLSM), and dynamic light scattering (DLS). Moreover, the interaction of the developed carriers with cell lines was studied using standard viability, proliferation, and uptake assays. The submicrometric SiO2-based capsules loaded with DNA plasmid encoding green fluorescence proteins (GFP) were used to transfect cell lines. The obtained results were compared with studies made with similar capsules composed of polymers and show that SiO2-based capsules provide better transfection rates on the costs of higher toxicity.

Lysosomal Proton Buffering of Poly(ethylenimine) Measured In Situ by Fluorescent pH-Sensor Microcapsules.

Poly(ethylenimine) (PEI) is frequently used as transfection agent for delivery of nucleic acids to the cytosol. After endocytosis of complexes of PEI and nucleic acids, a fraction of them can escape endosomes/lysosomes and reach the cytosol. One proposed mechanism is the so-called proton sponge effect, which involves buffering of the lysosomal pH by PEI. There are however also reports that report the absence of such buffering. In this work, the buffering capacity of PEI of the lysosomal pH was investigated in situ by combining PEI and pH-sensing ratiometric fluorophores in a single carrier particle. As carrier particles, hereby capsules were used, which were composed of polyelectrolyte walls based on layer-by-layer assembly, with the pH sensors located inside the capsule cavities. In this way, the local pH around individual particles could be monitored during the whole process of endocytosis. Results demonstrate the pH-buffering capability of PEI, which prevents the strong acidification of lysosomes containing PEI. This effect was related to the presence of PEI and was not related to the overall charge of the carrier particles. In case PEI was added in molecular form, no buffering of pH could be observed by endocytosed encapsulated pH-sensing ratiometric fluorophores. Co-localization experiments demonstrated that this was due to the fact that internalized free PEI and the encapsulated pH-sensing ratiometric fluorophores were not located in the same lysosomes. Missing co-localization might explain why also in other studies no pH buffering was found; in the case of co-delivery of PEI, the pH sensors could be clearly observed.

Glutathione-Specific and Intracellularly Labile Polymeric Nanocarrier for Efficient and Safe Cancer Gene Delivery.

Cationic polymers condense nucleic acids into nanosized complexes (polyplexes) that are widely explored for nonviral gene delivery, but their strong electrostatic binding with DNA causes inefficient intracellular gene release and translation and thereby unsatisfactory gene transfection efficiencies. Facilitated intracellular dissociation of polyplexes by making the polymer undergo positive-to-negative/neutral charge reversal can effectively solve these problems, but they must be sufficiently stable during the delivery. Herein, we report the first glutathione (GSH)-specific intracellular labile polyplexes for cancer-targeted gene delivery. The polymers are made from p-(2,4-dinitrophenyloxybenzyl)-ammonium cationic moieties, whose p-2,4-dinitrophenyl ether is cleaved specifically by GSH, rather than other biological thiols, triggering the conversion of the ammonium cation into the carboxylate anion and thus the fast intracellular DNA release of the polyplexes. Furthermore, the polyplexes coated with PEG-functionalized lipids are stable in biological fluids to gain long blood circulation for tumor accumulation. Thus, the efficient tumor accumulation and cell transfection of the polyplexes loaded with the tumor suicide gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) give rise to potent antitumor activity similar to that of the first-line chemotherapy drug paclitaxel but with much less adverse effects.

Assembly and Degradation of Inorganic Nanoparticles in Biological Environments.

In solution, nanoparticles may be conceptually compartmentalized into cores and engineered surface coatings. Recent advances allow for simple and accurate characterization of nanoparticle cores and surface shells. After introduction into a complex biological environment, adsorption of biological molecules to the nanoparticle surface as well as a loss of original surface components occur. Thus, colloidal nanoparticles in the context of the biological environment are hybrid materials with complex structure, which may result in different chemical, physical, and biological outcomes as compared to the original engineered nanoparticles. In this review, we will discuss building up an engineered inorganic nanoparticle from its inside core to its outside surface and following its degradation in a biological environment from its outside to its inside. This will involve the way to synthesize selected inorganic nanoparticles. Then, we will discuss the environmental changes upon exposure of these nanoparticles to biological media and their uptake by cells. Next, the intracellular fate of nanoparticles and their degradation will be discussed. Based on these examples, the need to see nanoparticles in the context of the biological environment as dynamic hybrid materials will be highlighted.

The Future of Layer-by-Layer Assembly: A Tribute to ACS Nano Associate Editor Helmuth Möhwald.

Layer-by-layer (LbL) assembly is a widely used tool for engineering materials and coatings. In this Perspective, dedicated to the memory of ACS Nano associate editor Prof. Dr. Helmuth Möhwald, we discuss the developments and applications that are to come in LbL assembly, focusing on coatings, bulk materials, membranes, nanocomposites, and delivery vehicles.

Triple-Labeling of Polymer-Coated Quantum Dots and Adsorbed Proteins for Tracing their Fate in Cell Cultures.

Colloidal CdSe/ZnS quantum dots were water solubilized by overcoating with an amphiphilic polymer. Human serum albumin (HSA) as a model protein was either adsorbed or chemically linked to the surface of the polymer-coated quantum dots. As the quantum dots are intrinsically fluorescent, and as the polymer coating and the HSA were fluorescent labeled, the final nanoparticle had three differently fluorescent components: the quantum dot core, the polymer shell, and the human serum albumin corona. Cells were incubated with these hybrid nanoparticles, and after removal of non-internalized nanoparticles, exocytosis of the three components of the nanoparticles was observed individually by flow cytometry and confocal microscopy. The data indicate that HSA is partly transported with the underlying polymer-coated quantum dots into cells. Upon desorption of proteins, those initially adsorbed to the quantum dots remain longer inside cells compared to free proteins. Part of the polymer shell is released from the quantum dots by enzymatic degradation, which is on a slower time scale than protein desorption. Data are quantitatively analyzed, and experimental pitfalls, such as the impact of cell proliferation and fluorescence quenching, are discussed.

Remotely controlled opening of delivery vehicles and release of cargo by external triggers.

Tremendous efforts have been devoted to the development of future nanomedicines that can be specifically designed to incorporate responsive elements that undergo modification in structural properties upon external triggers. One potential use of such stimuli-responsive materials is to release encapsulated cargo upon excitation by an external trigger. Today, such stimuli-response materials allow for spatial and temporal tunability, which enables the controlled delivery of compounds in a specific and dose-dependent manner. This potentially is of great interest for medicine (e.g. allowing for remotely controlled drug delivery to cells, etc.). Among the different external exogenous and endogenous stimuli used to control the desired release, light and magnetic fields offer interesting possibilities, allowing defined, real time control of intracellular releases. In this review we highlight the use of stimuli-responsive controlled release systems that are able to respond to light and magnetic field triggers for controlling the release of encapsulated cargo inside cells. We discuss established approaches and technologies and describe prominent examples. Special attention is devoted towards polymer capsules and polymer vesicles as containers for encapsulated cargo molecules. The advantages and disadvantages of this methodology in both, in vitro and in vivo models are discussed. An overview of challenges associate with the successful translation of those stimuli-responsive materials towards future applications in the direction of potential clinical use is given.

Reactive Oxygen Species (ROS)-Responsive Charge-Switchable Nanocarriers for Gene Therapy of Metastatic Cancer.

The application of nonviral gene vectors has been limited by their insufficient transfection efficiency because of poor serum stability, high endosomal entrapment, limited intracellular release, and low accumulation in the targeted organelle. It is still challenging to design gene carriers with properties that can overcome all of the barriers. We previously developed a reactive oxygen species (ROS)-responsive cationic polymer, poly[(2-acryloyl)ethyl( p-boronic acid benzyl) diethylammonium bromide] (B-PDEAEA), which switches the charge at high concentrations of intracellular ROS to promote intracellular DNA release. However, its gene-delivery efficiency has been limited by serum instability and lysosomal trapping, and coating with an anionic PEGylated lipid only showed mild enhancement. Herein, we coated the ROS-responsive B-PDEAEA polymer with two cationic lipids to form ROS-responsive lipopolyplexes with integrated properties to overcome multiple delivery barriers. The surface cationic lipids endowed the nanocarrier with improved serum stability, effective cellular uptake, and lysosomal evasion. The interior B-PDEAEA/DNA polyplexes, which were highly stable in the extracellular environment, but quickly dissociated, released DNA, promoted nuclei localization, and achieved efficient transcription. The mechanisms of the ROS-responsive and charge-switchable properties of B-PDEAEA were quantitatively studied. The transfection efficiency and antitumor activity of lipopolyplexes were studied in vitro and in vivo. We found that the ROS-responsive lipopolyplexes effectively delivered therapeutic genes into cell nuclei and caused high tumor inhibition in mice bearing peritoneal or lung metastases.

Identifying modulation formats through 2D Stokes planes with deep neural networks.

A lightweight convolutional (deep) neural networks (CNNs) based modulation format identification (MFI) scheme in 2D Stokes planes for polarization domain multiplexing (PDM) fiber communication system is proposed and demonstrated. Influences of the learning rate of CNN is discussed. Experimental verifications are performed for the PDM system at a symbol rate of 28GBaud. Six modulation formats are identified with a trained CNN from images of received signals. They are PDM-BPSK, PDM-QPSK, PDM-8PSK, PDM-16QAM, PDM-32QAM, and PDM-64QAM. By taking advantage of computer vision, the results show that the proposed scheme can significantly improve the identification performance over the existing techniques.

Detailed investigation on how the protein corona modulates the physicochemical properties and gene delivery of polyethylenimine (PEI) polyplexes.

Given the various cationic polymers developed as non-viral gene delivery vectors, polyethylenimine (PEI) has been/is frequently used in in vitro transfection. However, the primary drawback limiting its in vivo applications is the sharp decrease in transfection efficiency in the presence of serum. Here, we investigated the influences of serum proteins or bovine serum albumin (BSA) on the physicochemical properties of PEI/DNA complexes (polyplexes), including hydrodynamic diameters and agglomeration behavior, zeta potentials, morphologies, and sensitivity to the presence of salt. Mechanism studies revealed that the protein corona determined the endocytic rates and pathways, intracellular transport, the rate of endo/lysosomal trafficking, vesicle escape efficiency, and thus the overall gene expression levels. This work offers mechanistic insights for the serum-induced suppression of transfection efficiency, which is the reduced endo/lysosomal escape of the protein-coated polyplexes, in contrast to the protein free polyplexes.

Facile synthesis of semi-library of low charge density cationic polyesters from poly(alkylene maleate)s for efficient local gene delivery.

Cationic polymers are one of the main non-viral vectors for gene therapy, but their applications are hindered by the toxicity and inefficient transfection, particularly in the presence of serum or other biological fluids. While rational design based on the current understanding of gene delivery process has produced various cationic polymers with improved overall transfection, high-throughput parallel synthesis of libraries of cationic polymers seems a more effective strategy to screen out efficacious polymers. Herein, we demonstrate a novel platform for parallel synthesis of low cationic charge-density polyesters for efficient gene delivery. Unsaturated polyester poly(alkylene maleate) (PAM) readily underwent Michael-addition reactions with various mercaptamines to produce polyester backbones with pendant amine groups, poly(alkylene maleate mercaptamine)s (PAMAs). Variations of the alkylenes in the backbone and the mercaptamines on the side chain produced PAMAs with tunable hydrophobicity and DNA-condensation ability, the key parameters dominating transfection efficiency of the resulting polymer/DNA complexes (polyplexes). A semi-library of such PAMAs was exampled from 7 alkylenes and 18 mercaptamines, from which a lead PAMA, G-1, synthesized from poly(1,4-phenylene bis(methylene) maleate) and N,N-dimethylcysteamine, showed remarkable transfection efficiency even in the presence of serum, owing to its efficient lysosome-circumventing cellular uptake. Furthermore, G-1 polyplexes efficiently delivered the suicide gene pTRAIL to intraperitoneal tumors and elicited effective anticancer activity.

Nonviral cancer gene therapy: Delivery cascade and vector nanoproperty integration.

Gene therapy represents a promising cancer treatment featuring high efficacy and limited side effects, but it is stymied by a lack of safe and efficient gene-delivery vectors. Cationic polymers and lipid-based nonviral gene vectors have many advantages and have been extensively explored for cancer gene delivery, but their low gene-expression efficiencies relative to viral vectors limit their clinical translations. Great efforts have thus been devoted to developing new carrier materials and fabricating functional vectors aimed at improving gene expression, but the overall efficiencies are still more or less at the same level. This review analyzes the cancer gene-delivery cascade and the barriers, the needed nanoproperties and the current strategies for overcoming these barriers, and outlines PEGylation, surface-charge, size, and stability dilemmas in vector nanoproperties to efficiently accomplish the cancer gene-delivery cascade. Stability, surface, and size transitions (3S Transitions) are proposed to resolve those dilemmas and strategies to realize these transitions are comprehensively summarized. The review concludes with a discussion of the future research directions to design high-performance nonviral gene vectors.

Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells.