Nanomaterial-Enabled Photothermal Heating and Its Use for Cancer Therapy via Localized Hyperthermia.

Photothermal therapy (PTT), which employs nanoscale transducers delivered into a tumor to locally generate heat upon irradiation with near-infrared light, shows great potential in killing cancer cells through hyperthermia. The efficacy of such a treatment is determined by a number of factors, including the amount, distribution, and dissipation of the generated heat, as well as the type of cancer cell involved. The amount of heat generated is largely controlled by the number of transducers accumulated inside the tumor, the absorption coefficient and photothermal conversion efficiency of the transducer, and the irradiance of the light. The efficacy of treatment depends on the distribution of the transducers in the tumor and the penetration depth of the light. The vascularity and tissue thermal conduction both affect the dissipation of heat and thereby the distribution of temperature. The successful implementation of PTT in the clinic setting critically depends on techniques for real-time monitoring and management of temperature.

Microfluidic Platform for Microparticle Fabrication and Release of a Cathepsin Inhibitor.

Cathepsins are a family of cysteine proteases responsible for a variety of homeostatic functions throughout the body, including extracellular matrix remodeling, and have been implicated in a variety of degenerative diseases. However, clinical trials using systemic administration of cathepsin inhibitors have been abandoned due to side effects, so local delivery of cathepsin inhibitors may be advantageous. In these experiments, a novel microfluidic device platform was developed that can synthesize uniform, hydrolytically degradable microparticles from a combination of poly(ethylene glycol) diacrylate (PEGDA) and dithiothreitol (DTT). Of the formulations examined, the 10-polymer weight percentage 10 mM DTT formulation degraded after 77 days in vitro. A modified assay using the DQ Gelatin Fluorogenic Substrate was used to demonstrate sustained release and bioactivity of a cathepsin inhibitor (E-64) released from hydrogel microparticles over 2 weeks in vitro (up to ∼13 µg/mL released with up to ∼40% original level of inhibition remaining at day 14). Altogether, the technologies developed in this study will allow a small-molecule, broad cathepsin inhibitor E-64 to be released in a sustained manner for localized inhibition of cathepsins for a wide variety of diseases.

Quantification, Exchange, and Removal of Surface Ligands on Noble-Metal Nanocrystals.

ConspectusSurface ligands are vital to the colloidal synthesis of noble-metal nanocrystals with well-controlled sizes and shapes for various applications. The surface ligands not only dictate the formation of nanocrystals with diverse shapes but also serve as a colloidal stabilizer to prevent the suspended nanocrystals from aggregation during their synthesis or storage. By leveraging the facet selectivity of some surface ligands, one can further control the sites for growth or galvanic replacement to transform presynthesized nanocrystals into complex structures that are otherwise difficult to fabricate using conventional methods. Furthermore, the presence of surface ligands on nanocrystals also facilitates their applications in areas such as sensing, imaging, nanomedicine, and self-assembly. Despite their popular use in enhancing the properties of nanocrystals and thus optimizing their performance in a wide variety of applications, it remains a major challenge to quantitatively determine the coverage density of ligand molecules, not to mention the difficulty of substituting or removing them without compromising the surface structure and aggregation state of the nanocrystals.In this Account, we recapitulate our efforts in developing methods capable of qualitatively or quantitatively measuring, exchanging, and removing the surface ligands adsorbed on noble-metal nanocrystals. We begin with an introduction to the typical interactions between ligand molecules and surface atoms, followed by a discussion of the Langmuir model that can be used to describe the adsorption of surface ligands. It is also emphasized that the adsorption process may become very complex in the case of a polymeric ligand due to the variations in binding configuration and chain conformation. We then highlight the capabilities of various spectroscopy methods to analyze the adsorbed ligands qualitatively or quantitatively. Specifically, surface-enhanced Raman scattering, Fourier transform infrared, and X-ray photoelectron spectroscopy are three examples of qualitative methods that can be used to confirm the absence or presence of a surface ligand. On the other hand, ultraviolet-visible spectroscopy and inductively coupled plasma mass spectrometry can be used for quantitative measurements. Additionally, the coverage density of a ligand can be derived by analyzing the morphological changes during nanocrystal growth. We then discuss how the ligands present on the surface of metal nanocrystals can be exchanged directly or indirectly to meet the requirements of different applications. The former can be done using a ligand with stronger binding, whereas the latter is achieved by introducing a sacrificial shell to the surface of the nanocrystals. Furthermore, we highlight three additional strategies besides simple washing to remove the surface ligands, including calcination, heating in a solution, and UV-ozone treatment. Finally, we showcase three applications of metal nanocrystals in nanomedicine, tumor targeting, and self-assembly by taking advantage of the diversity of surface ligands bearing different functional groups. We also offer perspectives on the challenges and opportunities in realizing the full potential of surface ligands.

Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle Antibodies (SNAbs). SNAbs, with phage-display-identified cell-targeting ligands on one "face" and Fc-mimicking ligands on the opposite "face", were synthesized using a custom, multistep, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

Radiolabeling of Gold Nanocages for Potential Applications in Tracking, Diagnosis, and Image-Guided Therapy.

Gold nanocages (AuNCs) have emerged as a novel class of multifunctional nanomaterials with an array of applications in nanomedicine, including drug delivery, controlled release, as well as disease diagnosis and treatment. Labeling AuNCs with radionuclides not only offers additional therapeutic capabilities but also makes it easy to analyze their biodistribution, monitor their uptake by the tissue or organ of interest, and optimize their performance in both diagnosis and treatment. Here, an introduction to the chemical synthesis and optical properties of AuNCs is provided in the beginning. The methods developed for their radiolabeling are then showcased, followed by the use of radiolabeled AuNCs in tracking and quantifying their pharmacokinetics, including biodistribution, tumor uptake, and intratumoral distribution. Finally, their potential applications in targeted imaging and image-guided therapy are discussed.

A Mechanistic Study of the Multiple Roles of Oleic Acid in the Oil-Phase Synthesis of Pt Nanocrystals.

Oleic acid (OAc) is commonly used as a surfactant and/or solvent for the oil-phase synthesis of metal nanocrystals but its explicit roles are yet to be resolved. Here, we report a systematic study of this problem by focusing on a synthesis that simply involves heating of Pt(acac)2 in OAc for the generation of Pt nanocrystals. When heated at 80 °C, the ligand exchange between Pt(acac)2 and OAc leads to the formation of a PtII -oleate complex that serves as the actual precursor to Pt atoms. Upon increasing the temperature to 120 °C, the decarbonylation of OAc produces CO, which can act as a reducing agent for the generation of Pt atoms and thus formation of nuclei. Afterwards, several catalytic reactions can take place on the surface of the Pt nuclei to produce more CO, which also serves as a capping agent for the formation of Pt nanocrystals enclosed by {100} facets. The emergence of Pt nanocrystals further promotes the autocatalytic surface reduction of PtII precursor to enable the continuation of growth. This work not only elucidates the critical roles of OAc at different stages in a synthesis of Pt nanocrystals, but also represents a pivotal step forward toward the rational synthesis of metal nanocrystals.

Engraving the Surface of Electrospun Microfibers with Nanoscale Grooves Promotes the Outgrowth of Neurites and the Migration of Schwann Cells.

We report a simple method based upon coaxial electrospinning for the fabrication of aligned microfibers engraved with nanoscale grooves to promote neurite outgrowth and cell migration. The success of this method relies on the immiscibility between poly(ϵ-caprolactone) (PCL) and poly(vinyl pyrrolidone) (PVP) in 2,2,2-trifluoroethanol (TFE) for the generation of PVP/TFE pockets on the surface of a PCL jet. The pockets are stretched and elongated along with the jet, eventually resulting in the formation of nanoscale grooves upon the removal of PVP. The presence of nanoscale grooves greatly enhances the outgrowth of neurites from both PC12 cells and chick embryonic dorsal root ganglia (DRG) bodies, as well as the migration of Schwann cells. The enhancements can be maximized by optimizing the dimensions of the grooves for potential use in applications involving neurite extension and wound closure.

Transforming Nanofiber Mats into Hierarchical Scaffolds with Graded Changes in Porosity and/or Nanofiber Alignment.

A method for generating hierarchical scaffolds with graded changes in porosity and/or fiber alignment through solution-masked, vapor-induced welding of electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers is reported. The success of this method relies on the fact that the PLGA nanofibers are swollen and welded more slowly by ethanol when immersed in its aqueous solution relative to direct exposure to its vapor. For a mat composed of random nanofibers, the treatment generates a gradation in porosity (both surface and bulk), with the over-welded region evolving from a highly porous mat into a dense film. If uniaxially aligned nanofibers are involved, however, graded changes are observed in both surface porosity and fiber alignment. When bone marrow stem cells are cultured on such a scaffold, they exhibit highly organized and random morphologies on the regions of uniaxially aligned nanofibers and dense film, respectively, with gradual changes in between. Such a scaffold shows promise in mimicking the connective tissue, such as the tendon-to-bone insertion, that relies on a graded transition in cell morphology from uniaxially aligned to random.

Direct Visualization and Semi-Quantitative Analysis of Payload Loading in the Case of Gold Nanocages.

Upon incubation with Au nanocages, pyrrole (Py) molecules can enter the cavities by diffusing through the porous walls and then be polymerized to generate a polypyrrole (PPy) coating on the inner surface. The thicknesses of the PPy coating can serve as a direct indicator for the amount of Py molecules that diffuse into the cavity. Py molecules are able to diffuse into the cavities throughout the polymerization process, while a prolonged incubation time increases the amount of Py accumulated on both inner and outer surfaces of the nanocages. Furthermore, it is demonstrated that the dimensions of the cavity and the size of the pores in the wall are not critical parameters in determining the loading efficiency, as they do not affect the thickness of the PPy coating on the inner surface. These findings offer direct evidence to support the applications of Au nanocages as carriers for drug delivery and controlled release.

Encapsulation of a Phase-Change Material in Nanocapsules with a Well-Defined Hole in the Wall for the Controlled Release of Drugs.

As a class of biocompatible and biodegradable phase-change materials, natural fatty acids have received considerable interest in recent years for temperature-controlled release of drugs. However, the poor dispersibility and colloidal stability of their nanoparticles under physiological conditions place a major limitation on their applications in biomedicine. Herein, we report a facile method for encapsulating a mixture of two natural fatty acids (with a eutectic melting point at 39 °C) in a biocompatible, silica-based nanocapsule to achieve both stable dispersion and controllable release of drugs. The nanocapsules have a well-defined hole in the wall to ensure easy loading of fatty acids, together with multiple types of functional components such as therapeutics and near-infrared dyes. The payloads can be released through the hole when the fatty acids are melted upon photothermal heating. The release profile can be controlled by varying the size of the hole and/or the duration of laser irradiation.

Electrospun Nanofiber-Based Patches for the Delivery of Cardiac Progenitor Cells.

Congenital heart disease is the number one cause of birth defect-related death because it often leads to right ventricular heart failure (RVHF). One promising avenue to combat this RVHF is the use of cardiac patches composed of stem cells and scaffolds. Herein, we demonstrate a reparative cardiac patch by combining neonatal or child c-kit+  progenitor cells (CPCs) with a scaffold composed of electrospun polycaprolactone nanofibers. We examined different parameters of the patch, including the alignment, composition, and surface properties of the nanofibers, as well as the age of the CPCs. The patch based on uniaxially aligned nanofibers successfully aligned the CPCs. With the inclusion of gelatin in the nanofiber matrix and/or coating of fibronectin on the surface of the nanofibers, the metabolism of both neonatal and child CPCs was generally enhanced. The conditioned media collected from both patches based on aligned and random nanofibers could reduce the fibrotic gene expression in rat cardiac fibroblasts, following stimulation with transforming growth factor ß. Furthermore, the conditioned media collected from the nanofiber-based patches could lead to the formation of tubes of human umbilical vein endothelial cells, indicating the pro-angiogenic capability of the patch. Taken together, the electrospun nanofiber-based patches are a suitable delivery vehicle for CPCs and can confer reparative benefit through anti-fibrotic and pro-angiogenic paracrine signaling.

Promoting the Outgrowth of Neurites on Electrospun Microfibers by Functionalization with Electrosprayed Microparticles of Fatty Acids.

Controlling the outgrowth of neurites is important for enhancing the repair of injured nerves and understanding the development of nervous systems. Herein we report a simple strategy for enhancing the outgrowth of neurites through a unique integration of topographical guidance and a chemical cue. We use electrospray to easily functionalize the surface of a substrate with microparticles of natural fatty acids at a controllable density. Through a synergistic effect from the surface roughness arising from the microparticles and the chemical cue offered by the fatty acids, the outgrowth of neurites from PC12 cells is greatly enhanced. We also functionalize the surfaces of uniaxially aligned, electrospun microfibers with the microparticles and further demonstrate that the substrates can guide and enhance directional outgrowth of neurites from both PC12 multicellular spheroids and chick embryonic dorsal root ganglia bodies.

Near-Infrared-Triggered Release of Ca2+ Ions for Potential Application in Combination Cancer Therapy.

Calcium ion (Ca2+ ), an abundant species in the body, is a potential therapeutic ion with manageable side effects. However, the delivery of such a highly charged species represents a great challenge. Here, a nanosystem based on Au nanocages (AuNCs) and a phase-change material (PCM) for delivering calcium chloride (CaCl2 ) into cancer cells and thereby triggering cell death upon near-infrared (NIR) irradiation is demonstrated. In the absence of NIR irradiation, the nanosystem, denoted CaCl2 -PCM-AuNC, shows negligible cytotoxicity because the Ca2+ ions are fully encapsulated in a solid matrix. Upon NIR irradiation, the Ca2+ ions are swiftly released due to the melting of PCM matrix in response to photothermal heating. The sudden increase in intracellular Ca2+ causes disruption to the mitochondrial Ca2+ homeostasis and thus the loss of mitochondrial membrane potential, subsequently resulting in cell apoptosis. This nanosystem provides a new method for cancer treatment by tightly managing the intracellular concentration of a physiologically essential element.

Continuous processing of phase-change materials into uniform nanoparticles for near-infrared-triggered drug release.

We report a method based on interfacial, anti-solvent-induced precipitation in a fluidic device for the continuous and scalable processing of phase-change materials (PCMs) into uniform nanoparticles with controlled diameters in the range of 10-100 nm. A eutectic mixture of lauric acid and stearic acid, with a well-defined melting point at 39 °C, serves as an example to demonstrate the concept. In the fluidic device, a coaxial flow is created by introducing a PCM solution in ethanol and a lipid solution in water (the anti-solvent) as the focused and focusing phases, respectively. The formation of lipid-capped PCM nanoparticles is governed by diffusion-controlled mixing of ethanol and water. During the production, both doxorubicin (DOX, an anticancer drug) and indocyanine green (ICG, a near-infrared dye) can be readily loaded into the PCM nanoparticles to give a smart drug release system. Upon irradiation with near-infrared light, the photothermal heating caused by ICG can melt the PCM and thereby trigger the release of DOX. This work not only provides a new technique for the continuous processing of PCMs and other soft materials into uniform nanoparticles with controlled sizes but also demonstrates a biocompatible system for controlled release and related applications.

The effect of adipose-derived stem cell sheets and CTGF on early flexor tendon healing in a canine model.

Intrasynovial tendon injuries are among the most challenging in orthopedics. Despite significant improvements in operative and rehabilitation methods, functional outcomes continue to be limited by adhesions, gap formation, and rupture. Adhesions result from excessive inflammation, whereas tendon gapping and rupture result from inflammation-induced matrix degradation and insufficient regeneration. Therefore, this study used a combined treatment approach to modulate inflammation with adipose-derived mesenchymal stromal cells (ASCs) while stimulating tendon regeneration with connective tissue growth factor (CTGF). ASCs were applied to the repair surface via cell sheets and CTGF was delivered to the repair center via porous sutures. The effect of the combined treatment was assessed fourteen days after repair in a canine flexor tendon injury model. CTGF, either alone or with ASCs, reduced inflammatory (IL1B and IL6) and matrix degrading (MMP3 and MMP13) gene expression, while increasing anti-inflammatory gene (IL4) expression and collagen synthesis compared to control repairs. The combined treatment was more effective than CTGF treatment alone, reducing the inflammatory IFNG and scar-associated COL3A1 gene expression and increasing CD146+ tendon stem/progenitor cells at the tendon surface and interior along the core suture tracks. Therefore, the combined approach is promising in promoting early flexor tendon healing and worthy of further investigation.

Nanofiber-Based Multi-Tubular Conduits with a Honeycomb Structure for Potential Application in Peripheral Nerve Repair.

Peripheral nerve injury is a large-scale problem and it is a great challenge to repair the long lesion in a thick nerve. The design of a multi-tubular conduit with a honeycomb structure by mimicking the anatomy of a peripheral nerve for the potential repair of large defects in thick nerves has been reported. A bilayer mat of electrospun nanofibers is rolled up to form a single tube, with the inner and outer layers comprised aligned and random nanofibers, respectively. Seven such tubes are then assembled into a hexagonal array and encased within the lumen of a larger tube to form the multi-tubular conduit. By introducing an adhesive to the regions between the tubes, the conduit is robust enough for handling during surgery. The seeded bone marrow stem cells (BMSCs) are able to proliferate in all the tubes with even circumferential and longitudinal distributions. Under chemical induction, the BMSCs are transdifferentiated into Schwann-like cells in all the tubes. While the cellular version holds great promise for peripheral nerve repair, the multi-tubular conduit can also be used to investigate the fundamental aspects involved in the development of peripheral nervous system and migration of cells.

Melanocortin 1 Receptor Targeted Imaging of Melanoma With Gold Nanocages and Positron Emission Tomography.

PURPOSE: Melanoma is a lethal skin cancer with unmet clinical needs for targeted imaging and therapy. Nanoscale materials conjugated with targeting components have shown great potential to improve tumor delivery efficiency while minimizing undesirable side effects in vivo. Herein, we proposed to develop targeted nanoparticles for melanoma theranostics. METHOD: In this work, gold nanocages (AuNCs) were conjugated with α-melanocyte-stimulating hormone (α-MSH) peptide and radiolabeled with 64Cu for melanocortin 1 receptor-(MC1R) targeted positron emission tomography (PET) in a mouse B16/F10 melanoma model. RESULTS: Their controlled synthesis and surface chemistry enabled well-defined structure and radiolabeling efficiency. In vivo pharmacokinetic evaluation demonstrated comparable organ distribution between the targeted and nontargeted AuNCs. However, micro-PET/computed tomography (CT) imaging demonstrated specific and improved tumor accumulation via MC1R-mediated delivery. By increasing the coverage density of α-MSH peptide on AuNCs, the tumor delivery efficiency was improved. CONCLUSION: The controlled synthesis, sensitive PET imaging, and optimal tumor targeting suggested the potential of targeted AuNCs for melanoma theranostics.

Combination cancer treatment through photothermally controlled release of selenous acid from gold nanocages.

Selenite, one of the inorganic forms of selenium, is emerging as an attractive chemotherapeutic agent owing to its selectivity in eradicating cancer cells. Here we demonstrate a new formulation of nanomedicine based on selenous acid, which is mixed with lauric acid (a phase-change material with a melting point around 43 °C) and then loaded into the cavities of Au nanocages. The Au nanocages can serve as a carrier during cell endocytosis and then as a photothermal agent to melt the lauric acid upon the irradiation with a near-infrared laser, triggering the swift release of selenous acid. The photothermal and chemo therapies can also work synergistically, leading to enhanced destruction of cancer cells relative to normal cells. Our systematic study suggests that the impaired mitochondrial function arising from the ROS generated through combination treatment is responsible for the cell death. This study offers an appealing candidate that holds great promise for synergistic cancer treatment.

Perspective: Aligned arrays of electrospun nanofibers for directing cell migration.

Cell migration plays an important role in a wide variety of biological processes, including embryogenesis, wound healing, inflammation, cancer metastasis, and tissue repair. Electrospun nanofibers have been extensively explored as scaffolds to manipulate cell migration owing to their unique characteristics in mimicking the hierarchical architecture of extracellular matrix. In particular, aligned arrays of electrospun nanofibers are capable of guiding and promoting the directional migration of cells. The physical parameters and properties of the aligned nanofibers, including their size, modulus, and surface chemistry, can all affect the migratory behaviors of cells, while the controlled release of growth factors and drugs from the nanofibers can also be utilized to influence cell migration. By manipulating cell migration, electrospun nanofibers have been applied to promote tissue repair and help eradicate tumors in vivo. In this perspective, we highlight recent developments in collecting electrospun nanofibers as aligned arrays and then illustrate how the aligned nanofibers can be utilized to manipulate cell migration.