Preparation of Nanoparticle-Loaded Extracellular Vesicles Using Direct Flow Filtration.

Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20-100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells.

Synthesis and Characterization of Quercetin-Iron Complex Nanoparticles for Overcoming Drug Resistance.

Quercetin, one of the major natural flavonoids, has demonstrated great pharmacological potential as an antioxidant and in overcoming drug resistance. However, its low aqueous solubility and poor stability limit its potential applications. Previous studies suggest that the formation of quercetin-metal complexes could increase quercetin stability and biological activity. In this paper, we systematically investigated the formation of quercetin-iron complex nanoparticles by varying the ligand-to-metal ratios with the goal of increasing the aqueous solubility and stability of quercetin. It was found that quercetin-iron complex nanoparticles could be reproducibly synthesized with several ligand-to-iron ratios at room temperature. The UV-Vis spectra of the nanoparticles indicated that nanoparticle formation greatly increased the stability and solubility of quercetin. Compared to free quercetin, the quercetin-iron complex nanoparticles exhibited enhanced antioxidant activities and elongated effects. Our preliminary cellular evaluation suggests that these nanoparticles had minimal cytotoxicity and could effectively block the efflux pump of cells, indicating their potential for cancer treatment.

Fabrication of highly porous, functional cellulose-based microspheres for potential enzyme carriers.

Here, we present highly porous, cellulose-based microspheres using (2,2,6,6-tetramethylpiperidine-1-oxyl) TEMPO-oxidized cellulose fibers (TOCFs) as starting materials. The TOCFs were first dissolved in a NaOH/urea solvent and transformed into microspheres via an emulsification method. The carboxyl groups on the surface of TOCFs were successfully carried on the cellulose-based microspheres, which provides them numerous reacting or binding sites, allowing them to be easily functionalized or immobilized with biomolecules for multi-functional applications. Furthermore, the introduction of magnetic nanoparticles awards these microspheres magnetic properties, allowing them to be attracted by a magnetic field. As a proof of concept, we demonstrate the application of using these carboxylate cellulose-based microspheres for enzyme immobilization. The cellulose-based microspheres can successfully create stable covalent bonds with enzymes after the activation of carboxyl groups. The enhanced pH tolerance, thermal stability, convenient recovery, and reusability position the emulsified microspheres as promising carriers for enzyme immobilization.

Inorganic Nanoparticle-Loaded Exosomes for Biomedical Applications.

Exosomes are intrinsic cell-derived membrane vesicles in the size range of 40-100 nm, serving as great biomimetic nanocarriers for biomedical applications. These nanocarriers are known to bypass biological barriers, such as the blood-brain barrier, with great potential in treating brain diseases. Exosomes are also shown to be closely associated with cancer metastasis, making them great candidates for tumor targeting. However, the clinical translation of exosomes are facing certain critical challenges, such as reproducible production and in vivo tracking of their localization, distribution, and ultimate fate. Recently, inorganic nanoparticle-loaded exosomes have been shown great benefits in addressing these issues. In this review article, we will discuss the preparation methods of inorganic nanoparticle-loaded exosomes, and their applications in bioimaging and therapy. In addition, we will briefly discuss their potentials in exosome purification.

Sodium ion channels as potential therapeutic targets for cancer metastasis.

Is it possible to develop drugs for the treatment of a specific type of metastatic cancer by targeting sodium ion channels?

Identification of TrkB Binders from Complex Matrices Using a Magnetic Drug Screening Nanoplatform.

Brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) have been shown to play an important role in numerous neurological disorders, such as Alzheimer's disease. The identification of biologically active compounds interacting with TrkB serves as a drug discovery strategy to identify drug leads for neurological disorders. Here, we report effective immobilization of functional TrkB on magnetic iron oxide nanoclusters, where TrkB receptors behave as "smart baits" to bind compounds from mixtures and magnetic nanoclusters enable rapid isolation through magnetic separation. The presence of the immobilized TrkB was confirmed by specific antibody labeling. Subsequently, the activity of the TrkB on iron oxide nanoclusters was evaluated with ATP/ADP conversion experiments using a known TrkB agonist. The immobilized TrkB receptors can effectively identify binders from mixtures containing known binders, synthetic small molecule mixtures, and Gotu Kola (Centella asiatica) plant extracts. The identified compounds were analyzed by an ultrahigh-performance liquid chromatography system coupled with a quadrupole time-of-flight mass spectrometer. Importantly, some of the identified TrkB binders from Gotu Kola plant extracts matched with compounds previously linked to neuroprotective effects observed for a Gotu Kola extract approved for use in a clinical trial. Our studies suggest that the possible therapeutic effects of the Gotu Kola plant extract in dementia treatment, at least partially, might be associated with compounds interacting with TrkB. The unique feature of this approach is its ability to fast screen potential drug leads using less explored transmembrane targets. This platform works as a drug-screening funnel at early stages of the drug discovery pipeline. Therefore, our approach will not only greatly benefit drug discovery processes using transmembrane proteins as targets but also allow for evaluation and validation of cellular pathways targeted by drug leads.

Focused ultrasound blood brain barrier opening mediated delivery of MRI-visible albumin nanoclusters to the rat brain for localized drug delivery with temporal control.

There is an ongoing need for noninvasive tools to manipulate brain activity with molecular, spatial and temporal specificity. Here we have investigated the use of MRI-visible, albumin-based nanoclusters for noninvasive, localized and temporally specific drug delivery to the rat brain. We demonstrated that IV injected nanoclusters could be deposited into target brain regions via focused ultrasound facilitated blood brain barrier opening. We showed that nanocluster location could be confirmed in vivo with MRI. Additionally, following confirmation of nanocluster delivery, release of the nanocluster payload into brain tissue can be triggered by a second focused ultrasound treatment performed without circulating microbubbles. Release of glutamate from nanoclusters in vivo caused enhanced c-Fos expression, indicating that the loading capacity of the nanoclusters is sufficient to induce neuronal activation. This novel technique for noninvasive stereotactic drug delivery to the brain with temporal specificity could provide a new way to study brain circuits in vivo preclinically with high relevance for clinical translation.

Synthesis and characterization of iron oxide superparticles with various polymers.

Iron oxide superparticles referring to a cluster of smaller nanoparticles have recently attracted much attention because of their enhanced magnetic moments but maintaining superparamagnetic behavior. In this study, iron oxide superparticles have been synthesized using a solvothermal method in the presence of six different polymers (e.g., sodium polyacrylate, pectin sodium alginate, chitosan oligosaccharides, polyethylene glycol, and polyvinylpyrrolidine). The functional group variation in these polymers affected their interactions with precursor iron ions, and subsequently influenced crystalline grain sizes within superparticles and their magnetic properties. These superparticles were extensively characterized by transmission electron microscopy, dynamic light scattering, x-ray diffraction, Fourier transform infrared spectroscopy, and vibrating sample magnetometry.

Role of Surface Chemistry in Mediating the Uptake of Ultrasmall Iron Oxide Nanoparticles by Cancer Cells.

Ultrasmall iron oxide nanoparticles (USIONPs) (<4 nm) have recently attracted significant attention because of their potential as positive T1 magnetic resonance imaging (MRI) contrast agent contrary to larger superparamagnetic iron oxide nanoparticles (>6 nm) which act as negative T2 MRI contrast agents. However, studies on the cellular uptake behavior of these nanoparticles are very limited compared to their counterpart, larger-sized superparamagnetic iron oxide nanoparticles. In particular, the effects of specific nanoparticle parameters on the cellular uptake behavior of USIONPs by various cancer cells are not available. Here, we specifically investigated the role of USIONPs' surface functionalities [tannic acid (TA) and quinic acid (QA)] in mediating cellular uptake behavior of cancer cells pertaining to primary (U87 cells) and metastatic (MDA-MB-231Br cells) brain malignancies. Here, we chose TA and QA as representative capping molecules, wherein TA coating provides a general negatively charged nontargeting surface while QA provides a tumor-targeting surface as QA and its derivatives are known to interact with selectin receptors expressed on tumor cells and tumor endothelium. We observed differential cellular uptake in the case of TA- and QA-coated USIONPs by cancer cells. Both the cell types showed significantly higher cellular uptake of QA-coated USIONPs compared to TA-coated USIONPs at 4, 24, and 72 h. Blocking studies indicated that P-selectin cell surface receptors, in part, mediated the cellular uptake of QA-coated USIONPs. Given that P-selectin is overexpressed in cancer cells, tumor microenvironment, and at the metastatic niche, QA-coated USIONPs hold potential to be utilized as a platform for tumor-targeted drug delivery and in imaging and detection of primary and metastatic tumors.

Cell-membrane coated iron oxide nanoparticles for isolation and specific identification of drug leads from complex matrices.

The lack of suitable tools for the identification of potential drug leads from complex matrices is a bottleneck in drug discovery. Here, we report a novel method to screen complex matrices for new drug leads targeting transmembrane receptors. Using α3ß4 nicotinic receptors as a model system, we successfully demonstrated the ability of this new tool for the specific identification and effective extraction of binding compounds from complex mixtures. The formation of cell-membrane coated nanoparticles was confirmed by transmission electron microscopy. In particular, we have developed a direct tool to evaluate the presence of functional α3ß4 nicotinic receptors on the cell membrane. The specific ligand binding to α3ß4 nicotinic receptors was examined through ligand fishing experiments and confirmed by high-performance liquid chromatography coupled with diode-array detection and electrospray ionization mass spectrometry. This tool has a great potential to transform the drug discovery process focusing on identification of compounds targeting transmembrane proteins, as more than 50% of all modern pharmaceuticals use membrane proteins as prime targets.

Wet-spun nanoTiO2/chitosan nanocomposite fibers as efficient and retrievable absorbent for the removal of free fatty acids from edible oil.

Here we propose a wet-spinning assembly approach to continuously spin nanoTiO2/chitosan (CS) nanocomposite fibers, which are used directly as absorbents to remove free fatty acids (FFA) from edible oils. The morphology of nanoTiO2 and nanoTiO2/CS nanocomposite fibers was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), respectively. The structure of the fibers was studied by Fourier transform infrared spectroscopy (FTIR), and wide angel X-ray diffractometry (WAXD). Moreover, the mechanical property, thermal stability, and antibacterial activity of the fibers were evaluated. These fibers were used for the deacidification of rice bran oil and the acid value of the oil was found decreased from 4.53 ± 0.15 to 1.07 ± 0.06 mg KOH/g within 5 h with a 10 wt % load at 50 ℃. The combination of wet-spinning technology and excellent performance of nanoTiO2/CS nanocomposite fibers paves the way to eco-friendly and sustainable material for FFA removal.

Rice bran polysaccharide-metal complexes showed safe antioxidant activity in vitro.

The effect on the intracellular reactive oxygen species (ROS) generation, and the antioxidant and cytotoxicity properties of rice bran polysaccharides (RBP) and RBP-metal complexes RBP-Fe(III), RBP-Cu, RBP-Zn and RBP-Ca, were evaluated using atomic absorption spectroscopy (AAS), scavenging activity assays, cell viability assay and fluorescence microscopy. The RBP-metal complexes were prepared using the hydrothermal method. The RBP-Fe(III) complexes were found to be potent scavengers for superoxide (O2-) free radicals. The RBP alone and RBP-Ca complex showed high scavenging activity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In addition, the RBP-Fe(III) complex also showed good biocompatibility and lowered the intracellular ROS levels, while RBP alone, RBP-Zn and RBP-Ca complexes were observed to increase the intracellular ROS level. Our findings suggest that among the tested RBP-metal complexes, RBP-Fe(III) complex is a strong candidate as an antioxidant therapeutic.

Fabrication of cellulose nanowhiskers reinforced chitosan-xylan nanocomposite films with antibacterial and antioxidant activities.

Antibacterial and antioxidant chitosan-xylan/cellulose nanowhiskers (CNW) nanocomposite films were successfully prepared using CNW as nanofillers. The structure and morphology of the nanocomposite films were investigated by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), and scanning electron microscopy (SEM). The optical transmittance, thermal stability, mechanical property, and swelling property of the nanocomposite films were also evaluated. These results revealed the microstructure of the films and confirmed the good miscibility between chitosan-xylan and CNW. The improvements of tensile strength and elongation at break of the nanocomposite films confirmed the reinforcement effects of CNW. Moreover, the inhibitory effects against S. aureus and E. coli and the ABTS+ scavenging activity indicated antibacterial and antioxidant functions of the nanocomposite films. In this work, the prepared chitosan-xylan/CNW nanocomposite films, combined the antibacterial property of chitosan, the antioxidant property of xylan, and good mechanical property of CNW, could be potentially applied in food and health-related areas.

Cellulose-Based Composite Macrogels from Cellulose Fiber and Cellulose Nanofiber as Intestine Delivery Vehicles for Probiotics.

Cellulose-based composite macrogels made by cellulose fiber/cellulose nanofiber (CCNM) were used as an intestine delivery vehicle for probiotics. Cellulose nanofiber (CNF) was prepared by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation system, and the carboxyl groups in CNF acted as pore size and pH responsibility regulators in CCNMs to regulate the probiotics loading and controlled release property. The macrogel presented a porosity of 92.68% with a CNF content of 90%, and the corresponding released viable Lactobacillus plantarum (L. plantarum) was up to 2.68 × 108 cfu/mL. The porous structure and high porosity benefited L. plantarum cells to infiltrate into the core of macrogels. In addition, the macrogels made with high contents of CNF showed sustainable release of L. plantarum cells and delivered enough viable cells to the desired region of intestine tracts. The porous cellulose macrogels prepared by a green and environmental friendly method show potential in the application of fabricating targeted delivery vehicles of bioactive agents.

Development of poly (lactic acid) microspheres and their potential application in Pickering emulsions stabilization.

The aim of the present work was to study the feasibility of fabricating poly (lactic acid) (PLA) microspheres stabilized Pickering emulsions. For this purpose, the PLA microspheres were first prepared by oil-water emulsion solvent evaporation method. The effects of preparation conditions such as hydrophilic-lipophilic balance (HLB) value, emulsifier concentration, oil-water ratio and preparation temperature were evaluated by using optical microscopy. Besides, orthogonal experiments were designed to investigate the influence of preparation parameters on average diameter and uniformity, include stirring time, stirring speed, and PLA and polyvinyl alcohol (PVA) concentrations. Based on the analysis of orthogonal experimental results, an optimal level of parameters was defined for the fabrication of PLA microspheres. Furthermore, these microspheres were applied to the stabilization of Pickering emulsions, and the optimal Pickering emulsion with uniform microstructure was obtained through the adjustment of PLA microspheres concentrations. This study opens up a promising way for producing PLA microspheres stabilized Pickering emulsions.

Cellulose Anionic Hydrogels Based on Cellulose Nanofibers As Natural Stimulants for Seed Germination and Seedling Growth.

Cellulose anionic hydrogels were successfully prepared by dissolving TEMPO-oxidized cellulose nanofibers in NaOH/urea aqueous solution and being cross-linked with epichlorohydrin. The hydrogels exhibited microporous structure and high hydrophilicity, which contribute to the excellent water absorption property. The growth indexes, including the germination rate, root length, shoot length, fresh weight, and dry weight of the seedlings, were investigated. The results showed that cellulose anionic hydrogels with suitable carboxylate contents as plant growth regulators could be beneficial for seed germination and growth. Moreover, they presented preferable antifungal activity during the breeding and growth of the sesame seed breeding. Thus, the cellulose anionic hydrogels with suitable carboxylate contents could be applied as soilless culture mediums for plant growth. This research provided a simple and effective method for the fabrication of cellulose anionic hydrogel and evaluated its application in agriculture.

Citric Acid Capped Iron Oxide Nanoparticles as an Effective MALDI Matrix for Polymers.

A new matrix-assisted laser desorption ionization (MALDI) mass spectrometry matrix is proposed for molecular mass determination of polymers. This matrix contains an iron oxide nanoparticle (NP) core with citric acid (CA) molecules covalently bound to the surface. With the assistance of additives, the particulate nature of NPs allows the matrix to mix uniformly with polar or nonpolar polymer layers and promotes ionization, which may simplify matrix selection and sample preparation procedures. Several distinctively different polymer classes (polyethyleneglycol (PEG), polywax/polyethylene, perfluoropolyether, and polydimethylsiloxane) are effectively detected by the water or methanol dispersed NPCA matrix with NaCl, NaOH, LiOH, or AgNO3 as additives. Furtheremore, successful quantitative measurements of PEG1000 using polypropylene glycol 1000 as an internal standard are demonstrated. Graphical Abstract ᅟ.

Modeling the atomistic growth behavior of gold nanoparticles in solution.

The properties of gold nanoparticles strongly depend on their three-dimensional atomic structure, leading to an increased emphasis on controlling and predicting nanoparticle structural evolution during the synthesis process. In order to provide this atomistic-level insight and establish a link to the experimentally-observed growth behavior, a kinetic Monte Carlo simulation (KMC) approach is developed for capturing Au nanoparticle growth characteristics. The advantage of this approach is that, compared to traditional molecular dynamics simulations, the atomistic nanoparticle structural evolution can be tracked on time scales that approach the actual experiments. This has enabled several different comparisons against experimental benchmarks, and it has helped transition the KMC simulations from a hypothetical toy model into a more experimentally-relevant test-bed. The model is initially parameterized by performing a series of automated comparisons of Au nanoparticle growth curves versus the experimental observations, and then the refined model allows for detailed structural analysis of the nanoparticle growth behavior. Although the Au nanoparticles are roughly spherical, the maximum/minimum dimensions deviate from the average by approximately 12.5%, which is consistent with the corresponding experiments. Also, a surface texture analysis highlights the changes in the surface structure as a function of time. While the nanoparticles show similar surface structures throughout the growth process, there can be some significant differences during the initial growth at different synthesis conditions.

The responses of immune cells to iron oxide nanoparticles.

Immune cells play an important role in recognizing and removing foreign objects, such as nanoparticles. Among various parameters, surface coatings of nanoparticles are the first contact with biological system, which critically affect nanoparticle interactions. Here, surface coating effects on nanoparticle cellular uptake, toxicity and ability to trigger immune response were evaluated on a human monocyte cell line using iron oxide nanoparticles. The cells were treated with nanoparticles of three types of coatings (negatively charged polyacrylic acid, positively charged polyethylenimine and neutral polyethylene glycol). The cells were treated at various nanoparticle concentrations (5, 10, 20, 30, 50 µg ml(-1) or 2, 4, 8, 12, 20 µg cm(-2)) with 6 h incubation or treated at a nanoparticle concentration of 50 µg ml(-1) (20 µg cm(-2)) at different incubation times (6, 12, 24, 48 or 72 h). Cell viability over 80% was observed for all nanoparticle treatment experiments, regardless of surface coatings, nanoparticle concentrations and incubation times. The much lower cell viability for cells treated with free ligands (e.g. ~10% for polyethylenimine) suggested that the surface coatings were tightly attached to the nanoparticle surfaces. The immune responses of cells to nanoparticles were evaluated by quantifying the expression of toll-like receptor 2 and tumor necrosis factor-α. The expression of tumor necrosis factor-α and toll-like receptor 2 were not significant in any case of the surface coatings, nanoparticle concentrations and incubation times. These results provide useful information to select nanoparticle surface coatings for biological and biomedical applications.

Magnetic Nanoparticles: Material Engineering and Emerging Applications in Lithography and Biomedicine.

We present an interdisciplinary overview of material engineering and emerging applications of iron oxide nanoparticles. We discuss material engineering of nanoparticles in the broadest sense, emphasizing size and shape control, large-area self-assembly, composite/hybrid structures, and surface engineering. This is followed by a discussion of several non-traditional, emerging applications of iron oxide nanoparticles, including nanoparticle lithography, magnetic particle imaging, magnetic guided drug delivery, and positive contrast agents for magnetic resonance imaging. We conclude with a succinct discussion of the pharmacokinetics pathways of iron oxide nanoparticles in the human body -- an important and required practical consideration for any in vivo biomedical application, followed by a brief outlook of the field.