Enhancing Optical Forces on Fluorescent Up‐Converting Nanoparticles by Surface Charge Tailoring

3D remote control of multifunctional fluorescent up‐converting nanoparticles (UCNPs) using optical forces is being required for a great variety of applications including single‐particle spectroscopy, single‐particle intracellular sensing, controlled and selective light‐activated drug delivery and light control at the nanoscale. Most of these potential applications find a serious limitation in the reduced value of optical forces (tens of fN) acting on these nanoparticles, due to their reduced dimensions (typically around 10 nm). In this work, this limitation is faced and it is demonstrated that the magnitude of optical forces acting on UCNPs can be enhanced by more than one order of magnitude by a controlled modification of the particle/medium interface. In particular, substitution of cationic species at the surface by other species with higher mobility could lead to UCNPs trapping with constants comparable to those of spherical metallic nanoparticles.     

Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons

New techniques for single‐cell analysis enable new discoveries in gene expression and systems biology. Time‐dependent measurements on individual cells are necessary, yet the common single‐cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP‐E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP‐E is used to transfect a DNA‐based beacon that detects glyceraldehyde 3‐phosphate dehydrogenase and an RNA‐based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time‐dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.     

Plant Polyphenol‐Assisted Green Synthesis of Hollow CoPt Alloy Nanoparticles for Dual‐Modality Imaging Guided Photothermal Therapy

Theranostic nanomedicines that integrate diagnostic and therapeutic moieties into a single nanoscale platform are playing an increasingly important role in fighting cancer. Here, a facile and green synthetic strategy for hollow CoPt alloy nanoparticles (HCPA‐NPs) using plant polyphenols as assisted agents is reported for the first time. This novel strategy enables size‐controlled synthesis of HCPA‐NPs through the control of the molecular sizes of polyphenols. It is also a versatile strategy for synthesizing other hollow alloy nanoparticles with various metal compositions due to the diverse metal‐chelating ability of the polyphenols. Further studies show that HCPA‐NPs have good biocompatibility and can be successfully implemented for magnetic resonance and photoacoustic dual‐modal imaging guided photothermal therapy. This work brings new insights for the green synthesis of hollow nanoparticles and extends these biocompatible nanoparticles for theranostic applications.     

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single‐Cell Electroporation

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time‐consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub‐micron resolution and resistance‐based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor‐intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.     

Cancer‐Cell‐Phenotype‐Dependent Differential Intracellular Trafficking of Unconjugated Quantum Dots

A diverse array of nanoparticles, including quantum dots (QDs), metals, polymers, liposomes, and dendrimers, are being investigated as therapeutics and imaging agents in cancer diseases. However, the role of the cancer‐cell phenotype on the uptake and intracellular fate of nanoparticles in cancer cells remains poorly understood. Reported here is that differences in cancer‐cell phenotypes can lead to significant differences in intracellular sorting, trafficking, and localization of nanoparticles. Unconjugated anionic QDs demonstrate dramatically different intracellular profiles in three closely related human‐prostate‐cancer cells used in the investigation: PC3, PC3‐flu, and PC3‐PSMA. QDs demonstrate punctated intracellular localization throughout the cytoplasm in PC3 cells. In contrast, the nanoparticles localize mainly at a single juxtanuclear location (“dot‐of‐dots”) inside the perinuclear recycling compartment in PC3‐PSMA cells, where they co‐localize with transferrin and the prostate‐specific membrane antigen. The results indicate that nanoparticle sorting and transport is influenced by changes in cancer‐cell phenotype and can have significant implications in the design and engineering of nanoscale drug delivery and imaging systems for advanced tumors.     

Erythrocyte‐Membrane‐Enveloped Perfluorocarbon as Nanoscale Artificial Red Blood Cells to Relieve Tumor Hypoxia and Enhance Cancer Radiotherapy

Hypoxia, a common feature within many types of solid tumors, is known to be closely associated with limited efficacy for cancer therapies, including radiotherapy (RT) in which oxygen is essential to promote radiation‐induced cell damage. Here, an artificial nanoscale red‐blood‐cell system is designed by encapsulating perfluorocarbon (PFC), a commonly used artificial blood substitute, within biocompatible poly(d ,l ‐lactide‐co‐glycolide) (PLGA), obtaining PFC@PLGA nanoparticles, which are further coated with a red‐blood‐cell membrane (RBCM). The developed PFC@PLGA‐RBCM nanoparticles with the PFC core show rather efficient loading of oxygen, as well as greatly prolonged blood circulation time owing to the coating of RBCM. With significantly improved extravascular diffusion within the tumor mass, owing to their much smaller nanoscale sizes compared to native RBCs with micrometer sizes, PFC@PLGA‐RBCM nanoparticles are able to effectively deliver oxygen into tumors after intravenous injection, leading to greatly relieved tumor hypoxia and thus remarkably enhanced treatment efficacy during RT. This work thus presents a unique type of nanoscale RBC mimic for efficient oxygen delivery into solid tumors, favorable for cancer treatment by RT, and potentially other types of therapy as well.     

Photosensitizer‐Incorporated Quadruplex DNA‐Gated Nanovechicles for Light‐Triggered,Targeted Dual Drug Delivery to Cancer Cells

A novel light‐operated vehicle for targeted intracellular drug delivery is constructed using photosensitizer‐incorporated G‐quadruplex DNA‐capped mesoporous silica nanoparticles. Upon light irradiation, the photosensitizer generates ROS, causing the DNA capping to be cleaved and allowing cargo to be released. Importantly, this platform makes it possible to develop a drug‐carrier system for the synergistic combination of chemotherapy and PDT for cancer treatment with spatial/temporal control. Furthermore, the introducing of targeting ligands further improves tumor targeting efficiency. The excellent biocompatibility, cell‐specific intracellular drug delivery, and cellular uptake properties set up the basis for future biomedical application that require in vivo controlled, targeted drug delivery.     

Layer‐by‐Layer Coated Gold Nanoparticles: Size‐Dependent Delivery of DNA into Cells

Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size‐dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer‐by‐layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC₅₀ value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface.     

Multifunctional Hybrid Nanoparticles for Traceable Drug Delivery and Intracellular Microenvironment‐Controlled Multistage Drug‐Release in Neurons

Innovative nanoparticles hold promising potential for disease therapy as drug delivery systems. For brain‐disease therapy, a drug delivery system that can sustainably control drug‐release and monitor fluorescence of the drug cargos is highly desirable. In this study, a light‐traceable and intracellular microenvironment‐responsive drug delivery system was developed based on the combination of glutathione‐responsive autoflurescent nanogel, dendrimer‐like mesoporous silica nanoparticles, and gold nanoparticles. The resulting hybrid nanoparticles represent a new class of delivery system that can efficiently load, transport, and control multistage‐release of sulfydryl‐containing drugs into neurons, with light‐traceable monitoring for future brain‐disease therapy.     

Engineered Multifunctional Albumin‐Decorated Porous Silicon Nanoparticles for FcRn Translocation of Insulin

The last decade has seen remarkable advances in the development of drug delivery systems as alternative to parenteral injection‐based delivery of insulin. Neonatal Fc receptor (FcRn)‐mediated transcytosis has been recently proposed as a strategy to increase the transport of drugs across the intestinal epithelium. FcRn‐targeted nanoparticles (NPs) could hijack the FcRn transcytotic pathway and cross the epithelial cell layer. In this study, a novel nanoparticulate system for insulin delivery based on porous silicon NPs is proposed. After surface conjugation with albumin and loading with insulin, the NPs are encapsulated into a pH‐responsive polymeric particle by nanoprecipitation. The developed NP formulation shows controlled size and homogeneous size distribution. Transmission electron microscopy (TEM) images show successful encapsulation of the NPs into pH‐sensitive polymeric particles. No insulin release is detected at acidic conditions, but a controlled release profile is observed at intestinal pH. Toxicity studies show high compatibility of the NPs with intestinal cells. In vitro insulin permeation across the intestinal epithelium shows approximately fivefold increase when insulin is loaded into FcRn‐targeted NPs. Overall, these FcRn‐targeted NPs offer a toolbox in the development of targeted therapies for oral delivery of insulin.     

Magnetically Controlled Growth‐Factor‐Immobilized Multilayer Cell Sheets for Complex Tissue Regeneration

The scaffold‐free cell‐sheet technique plays a significant role in stem‐cell‐based regeneration. Furthermore, growth factors are known to direct stem cell differentiation and enhance tissue regeneration. However, the absence of an effective means to incorporate growth factors into the cell sheets hinders further optimization of the regeneration efficiency. Here, a novel design of magnetically controlled “growth‐factor‐immobilized cell sheets” is reported. A new Fe₃O₄ magnetic nanoparticle (MNP) coated with nanoscale graphene oxide (nGO@Fe₃O₄) is developed to label stem cells and deliver growth factors. First, the nGO@Fe₃O₄ MNPs can be easily swallowed by dental‐pulp stem cells (DPSCs) and have no influence on cell viability. Thus, the MNP‐labeled cells can be organized via magnetic force to form multilayered cell sheets in different patterns. Second, compared to traditional Fe₃O₄ nanoparticles, the graphene oxide coating provides plenty of carboxyl groups to bind and deliver growth factors. Therefore, with these nGO@Fe₃O₄ MNPs, bone‐morphogenetic‐protein‐2 (BMP2) is successfully incorporated into the DPSCs sheets to induce more bone formation. Furthermore, an integrated osteochondral complex is also constructed using a combination of DPSCs/TGFβ3 and DPSCs/BMP2. All these results demonstrate that the new cell‐sheet tissue‐engineering approach exhibits promising potential for future use in regenerative medicine.     

Dual‐Functional Alginic Acid Hybrid Nanospheres for Cell Imaging and Drug Delivery

An effective and facile approach to prepare gold‐nanoparticle‐encapsulated alginic acid‐poly[2‐(diethylamino)ethyl methacrylate] monodisperse hybrid nanospheres (ALG–PDEA–Au) is developed by using monodisperse ALG–PDEA nanospheres as a precursor nanoparticulate reaction system. This approach utilizes particle‐interior chemistry, which avoids additional reductant or laborious separation process and, moreover, elegantly ensures that all the gold nanoparticles are located inside the hybrid nanospheres and every nanosphere is loaded with gold nanoparticles. These obtained ALG–PDEA–Au hybrid nanospheres have not only uniform size, similar surface properties, and good biocompatibility but also unique optical properties provided by the embedded gold nanoparticles. It is demonstrated that negatively charged ALG–PDEA–Au hybrid nanospheres can be internalized by human colorectal LoVo cancer cells and hence act as novel optical‐contrast reagents in tumor‐cell imaging by optical microscopy. Moreover, these hybrid nanospheres can also serve as biocompatible carriers for the loading and delivery of an anti‐cancer drug doxorubicin. In vitro cell viability tests reveal that drug‐loaded ALG–PDEA–Au hybrid nanospheres exhibit similar tumor cell inhibition to the free drug doxorubicin. Therefore, the obtained hybrid nanospheres successfully combine two functions, that is, cell imaging and drug delivery, into one single system, and may be of great application potential in other biomedical‐related areas.     

Spray‐Dried Nanoparticle‐in‐Microparticle Delivery Systems (NiMDS) for Gene Delivery,Comprising Polyethylenimine (PEI)‐Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix

Nucleic acid‐based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid‐based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready‐to‐use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine‐based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray‐dried nanoparticle‐in‐microparticle delivery system is presented for the encapsulation of polyethylenimine‐based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy‐to‐handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine‐based nanoparticles from the nanoparticle‐in‐microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle‐in‐microparticle delivery system is demonstrated as ready‐to‐use dry powder to be an efficient device for the inhalative delivery of polyethylenimine‐based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.     

Magnetoresponsive Virus‐Mimetic Nanocapsules with Dual Heat‐Triggered Sequential‐Infected Multiple Drug‐Delivery Approach for Combinatorial Tumor Therapy

Stimuli‐responsive drug‐delivery systems constitute an appealing approach to direct and restrict drug release spatiotemporally at the specific site of interest. However, it is difficult for most systems to affect every cancer cell in a tumor tissue due to the presence of the natural tumor barrier, leading to potential tumor recurrence. Here, core–shell magnetoresponsive virus‐mimetic nanocapsules (VNs), which can infect cancer cells sequentially and double as a magnetothermal agent fabricated through anchoring iron oxide nanoparticles in a single‐component protein (lactoferrin) shell, are reported. With large payload of hydrophilic/hydrophobic anticancer cargos, doxorubicin and palictaxel, VNs can simultaneously give a rapid drug release and intense heat while applying an external high‐frequency magnetic field (HFMF). Furthermore, after being liberated from dead cells by HFMF manipulation, the constructive VNs can sequentially infect neighboring cancer cells and deliver sufficient therapeutic agents to next targeted sites. With high efficiency for sequential cell infections, VNs have successfully eliminated subcutaneous tumor after a combinatorial treatment. These results demonstrate that the VNs could be used for locally targeted, on‐demand, magnetoresponsive chemotherapy/hyperthermia, combined with repeated cell infections for tumor therapy and other therapeutic applications.     

Functionalized NIR‐II Semiconducting Polymer Nanoparticles for Single‐cell to Whole‐Organ Imaging of PSMA‐Positive Prostate Cancer

Development of molecular probes holds great promise for early diagnosis of aggressive prostate cancer. Here, 2‐[3‐(1,3‐dicarboxypropyl) ureido] pentanedioic acid (DUPA)‐conjugated ligand and bis‐isoindigo‐based polymer (BTII) are synthesized to formulate semiconducting polymer nanoparticles (BTII‐DUPA SPN) as a prostate‐specific membrane antigen (PSMA)‐targeted probe for prostate cancer imaging in the NIR‐II window. Insights into the interaction of the imaging probes with the biological targets from single cell to whole organ are obtained by transient absorption (TA) microscopy and photoacoustic (PA) tomography. At single‐cell level, TA microscopy reveals the targeting efficiency, kinetics, and specificity of BTII‐DUPA SPN to PSMA‐positive prostate cancer. At organ level, PA tomographic imaging of BTII‐DUPA SPN in the NIR‐II window demonstrates superior imaging depth and contrast. By intravenous administration, BTII‐DUPA SPN demonstrates selective accumulation and retention in the PSMA‐positive tumor, allowing noninvasive PA detection of PSMA overexpressing prostate tumors in vivo. The distribution of nanoparticles inside the tumor tissue is further analyzed through TA microscopy. These results collectively demonstrate BTII‐DUPA SPN as a promising probe for prostate cancer diagnosis by PA tomography.     

Dynamic Self‐Assembly of Homogenous Microcyclic Structures Controlled by a Silver‐Coated Nanopore

The self‐assembly of nanoparticles is a challenging process for organizing precise structures with complicated and ingenious structures. In the past decades, a simple, high‐efficiency, and reproducible self‐assembly method from nanoscale to microscale has been pursued because of the promising and extensive application prospects in bioanalysis, catalysis, photonics, and energy storage. However, microscale self‐assembly still faces big challenges including improving the stability and homogeneity as well as pursuing new assembly methods and templates for the uniform self‐assembly. To address these obstacles, here, a novel silver‐coated nanopore is developed which serves as a template for electrochemically generating microcyclic structures of gold nanoparticles at micrometers with highly homogenous size and remarkable reproducibility. Nanopore‐induced microcyclic structures are further applied to visualize the diffusion profile of ionic flux. Based on this novel strategy, a nanopore could potentially facilitate the delivery of assembled structures for many practical applications including drug delivery, cellular detection, catalysis, and plasmonic sensing.     

MOFBOTS: Metal–Organic‐Framework‐Based Biomedical Microrobots

Motile metal?organic frameworks (MOFs) are potential candidates to serve as small‐scale robotic platforms for applications in environmental remediation, targeted drug delivery, or nanosurgery. Here, magnetic helical microstructures coated with a kind of zinc‐based MOF, zeolitic imidazole framework‐8 (ZIF‐8), with biocompatibility characteristics and pH‐responsive features, are successfully fabricated. Moreover, it is shown that this highly integrated multifunctional device can swim along predesigned tracks under the control of weak rotational magnetic fields. The proposed systems can achieve single‐cell targeting in a cell culture media and a controlled delivery of cargo payloads inside a complex microfluidic channel network. This new approach toward the fabrication of integrated multifunctional systems will open new avenues in soft microrobotics beyond current applications.     

Immune Cell‐Mediated Biodegradable Theranostic Nanoparticles for Melanoma Targeting and Drug Delivery

Although tremendous efforts have been made on targeted drug delivery systems, current therapy outcomes still suffer from low circulating time and limited targeting efficiency. The integration of cell‐mediated drug delivery and theranostic nanomedicine can potentially improve cancer management in both therapeutic and diagnostic applications. By taking advantage of innate immune cell's ability to target tumor cells, the authors develop a novel drug delivery system by using macrophages as both nanoparticle (NP) carriers and navigators to achieve cancer‐specific drug delivery. Theranostic NPs are fabricated from a unique polymer, biodegradable photoluminescent poly (lactic acid) (BPLP‐PLA), which possesses strong fluorescence, biodegradability, and cytocompatibility. In order to minimize the toxicity of cancer drugs to immune cells and other healthy cells, an anti‐BRAF V600E mutant melanoma specific drug (PLX4032) is loaded into BPLP‐PLA nanoparticles. Muramyl tripeptide is also conjugated onto the nanoparticles to improve the nanoparticle loading efficiency. The resulting nanoparticles are internalized within macrophages, which are tracked via the intrinsic fluorescence of BPLP‐PLA. Macrophages carrying nanoparticles deliver drugs to melanoma cells via cell–cell binding. Pharmacological studies also indicate that the PLX4032 loaded nanoparticles effectively kill melanoma cells. The “self‐powered” immune cell‐mediated drug delivery system demonstrates a potentially significant advancement in targeted theranostic cancer nanotechnologies.     

On The Latest Three‐Stage Development of Nanomedicines based on Upconversion Nanoparticles

Following the “detect‐to‐treat” strategy, by biological engineering, the emerging upconversion nanoparticles (UCNPs) have become one of the most promising inorganic nanomedicines, and their biomedical applications have gradually shifted from multimodal tumor imaging to highly efficient cancer therapy. The past few years have witnessed a three‐stage development of UCNP‐based nanomedicines. On one hand, UCNPs can optimize each clinical treatment tool (chemotherapy, photodynamic therapy (PDT), radiotherapy (RT)) by controlled drug delivery/release, near‐infrared (NIR)‐excited deep PDT, and radiosensitization, respectively, all of which contribute greatly to the optimized treatment efficacy along with minimized side effects. On the other hand, several individual treatments can be “smartly” integrated into a single UCNP‐based nanotheranostic system for multimodal synergetic therapy, which can further improve the overall therapeutic effectiveness. Especially, UCNPs provide more‐effective strategies for overcoming tumor hypoxia, thus leading to an ideal treatment efficacy for complete eradication of solid tumors. Finally, the critical issues regarding the future development of UCNPs are discussed to promote the clinic‐translational applications of UCNP‐based nanomedicines, as well as realization of our “one drug fits all” dream.     

Light‐Responsive Hierarchically Structured Liquid Crystal Polymer Networks for Harnessing Cell Adhesion and Migration

Extracellular microenvironment is highly dynamic where spatiotemporal regulation of cell‐instructive cues such as matrix topography tightly regulates cellular behavior. Recapitulating dynamic changes in stimuli‐responsive materials has become an important strategy in regenerative medicine to generate biomaterials which closely mimic the natural microenvironment. Here, light responsive liquid crystal polymer networks are used for their adaptive and programmable nature to form hybrid surfaces presenting micrometer scale topographical cues and changes in nanoscale roughness at the same time to direct cell migration. This study shows that the cell speed and migration patterns are strongly dependent on the height of the (light‐responsive) micrometer scale topographies and differences in surface nanoroughness. Furthermore, switching cell migration patterns upon in situ temporal changes in surface nanoroughness, points out the ability to dynamically control cell behavior on these surfaces. Finally, the possibility is shown to form photoswitchable topographies, appealing for future studies where topographies can be rendered reversible on demand.