Protein Corona Composition of Gold Nanocatalysts.

The interaction between nanoparticles (NPs) and biological environments is profoundly influenced by a stable, strongly adsorbed "hard" protein corona. This corona significantly determines the NPs' pharmacokinetics and biological destiny. Our study delves into the mechanisms by which colloidal Au nanocrystals that are synthesized electrochemically without surface-capping organic ligands, known as CNM-Au8, traverse the blood-brain barrier (BBB) and target human brain tissue for treating neurodegenerative disorders. We discovered that upon interaction with human plasma, CNM-Au8 gold nanocrystals (AuNCs) effectively attract a variety of crucial apolipoproteins, notably apolipoproteins E, to their surfaces. This interaction likely facilitates their passage through the BBB. Furthermore, the coronas of these AuNCs exhibit a substantial presence of albumin and a notable absence of opsonin-based proteins, contributing to prolonged blood circulation. These characteristics align well with the clinical performance observed for the CNM-Au8 NCs. This study highlights that AuNCs with intentionally engineered structures and surfactant-free surfaces can create a distinct protein corona composition. This finding holds significant promise for the development of advanced therapeutic agents aimed at combating neurodegenerative diseases.

Improved Methodology for Evaluating Nanomedicine Antibacterial Properties in Biological Fluids.

Accurate assessment of nanomedicines' antibacterial properties is pivotal for their effective use in both in vitro and in vivo settings. Conventional antibacterial activity assessment methods, involving bacterial coculture with compounds on agar plates, may not fully suit nanomedicines due to their susceptibility to alterations in physicochemical properties induced by biological fluids. Furthermore, these biological fluids might even enhance the bacterial growth. This study introduces a novel, rigorous, and reproducible methodology for evaluating nanomedicine antibacterial properties using cell culture media (i.e., DMEM-FBS10%). To assess the antibacterial activity of the nanoparticles in cell culture media, superparamagnetic iron oxide nanoparticles (SPIONs) were chosen as the model nanomedicine due to their clinical significance. A comparative analysis between the traditional and our proposed methods yielded contrasting outcomes, shedding light on the significant impact of biological fluids on nanoparticle antibacterial activities. While the conventional approach suggested the antibacterial effectiveness of SPIONs against Staphylococcus aureus, our innovative method unveiled a substantial increase in bacterial growth in the presence of biological fluids. More specifically, we found a significant increase in bacterial growth when exposed to bare SPIONs at various concentrations, while the formation of a protein corona on SPION surfaces could markedly reduce the observed bacterial growth compared to the control group. These findings underscore the necessity for more refined evaluation techniques that can better replicate the in vivo environment when studying the nanomedicine's antibacterial capabilities.

Deep Plasma Proteome Profiling by Modulating Single Nanoparticle Protein Corona with Small Molecules.

The protein corona, a dynamic biomolecular layer that forms on nanoparticle (NP) surfaces upon exposure to biological fluids is emerging as a valuable diagnostic tool for improving plasma proteome coverage analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Here, we show that spiking small molecules, including metabolites, lipids, vitamins, and nutrients, into plasma can induce diverse protein corona patterns on otherwise identical NPs, significantly enhancing the depth of plasma proteome profiling. The protein coronas on polystyrene NPs when exposed to plasma treated with an array of small molecules (n=10) allowed for detection of 1793 proteins marking an 8.25-fold increase in the number of quantified proteins compared to plasma alone (218 proteins) and a 2.63-fold increase relative to the untreated protein corona (681 proteins). Furthermore, we discovered that adding 1000 µg/ml phosphatidylcholine could singularly increase the number of unique proteins within the protein corona (897 proteins). This specific concentration of phosphatidylcholine selectively depleted the four most abundant plasma proteins, including albumin, thus reducing concentration dynamic range of plasma proteome and boosting LC-MS/MS sensitivity for detection of proteins with lower abundance. By employing an optimized data-independent acquisition (DIA) approach, the inclusion of phosphatidylcholine led to the detection of 1436 proteins in plasma. This significant achievement is made utilizing only a single NP type and one small molecule to analyze a single plasma sample, setting a new standard in proteomic depth of the plasma sample. Given the critical role of plasma proteomics in biomarker discovery and disease monitoring, we anticipate widespread adoption of this methodology for identification and clinical translation of proteomic biomarkers into FDA approved diagnostics.

A uniform data processing pipeline enables harmonized nanoparticle protein corona analysis across proteomics core facilities.

Protein corona, a layer of biomolecules primarily comprising proteins, forms dynamically on nanoparticles in biological fluids and is crucial for predicting nanomedicine safety and efficacy. The protein composition of the corona layer is typically analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). Our recent study, involving identical samples analyzed by 17 proteomics facilities, highlighted significant data variability, with only 1.8% of proteins consistently identified across these centers. Here, we implement an aggregated database search unifying parameters such as variable modifications, enzyme specificity, number of allowed missed cleavages and a stringent 1% false discovery rate at the protein and peptide levels. Such uniform search dramatically harmonizes the proteomics data, increasing the reproducibility and the percentage of consistency-identified unique proteins across distinct cores. Specifically, out of the 717 quantified proteins, 253 (35.3%) are shared among the top 5 facilities (and 16.2% among top 11 facilities). Furthermore, we note that reduction and alkylation are important steps in protein corona sample processing and as expected, omitting these steps reduces the number of total quantified peptides by around 20%. These findings underscore the need for standardized procedures in protein corona analysis, which is vital for advancing clinical applications of nanoscale biotechnologies.

Magnetic Levitation System Isolates and Purifies Airborne Viruses.

Detection of viable viruses in the air is critical in order to determine the level of risk associated with the airborne diffusion of viruses. Different methods have been developed for the isolation, purification, and detection of viable airborne viruses, but they require an extensive processing time and often present limitations including low physical efficiency (i.e., the amount of collected viruses), low biological efficiency (i.e., the number of viable viruses), or a combination of all. To mitigate such limitations, we have employed an efficient technique based on the magnetic levitation (Maglev) technique with a paramagnetic solution and successfully identified distinct variations in levitation and density characteristics among bacteria (Escherichia coli), phages (MS2), and human viruses (SARS-CoV-2 and influenza H1N1). Notably, the Maglev approach enabled a significant enrichment of viable airborne viruses in air samples. Furthermore, the enriched viruses obtained through Maglev exhibited high purity, rendering them suitable for direct utilization in subsequent analyses such as reverse transcription-polymerase chain reaction (RT-PCR) or colorimetric assays. The system is portable, easy to use, and cost-efficient and can potentially provide proactive surveillance data for monitoring future outbreaks of airborne infectious diseases and allow for the induction of various preventative and mitigative measures.

Impact of Nanomedicine in Women's Metastatic Breast Cancer.

Metastatic breast cancer is responsible for 90% of mortalities among women suffering from various types of breast cancers. Traditional cancer treatments such as chemotherapy and radiation therapy can cause significant side effects and may not be effective in many cases. However, recent advances in nanomedicine have shown great promise in the treatment of metastatic breast cancer. For example, nanomedicine demonstrated robust capacity in detection of metastatic cancers at early stages (i.e., before the metastatic cells leave the initial tumor site), which gives clinicians a timely option to change their treatment process (for example, instead of endocrine therapy they may use chemotherapy). Here recent advances in nanomedicine technology in the identification and treatment of metastatic breast cancers are reviewed.

An Overview of Nanoparticle Protein Corona Literature.

The protein corona forms spontaneously on nanoparticle surfaces when nanomaterials are introduced into any biological system/fluid. Reliable characterization of the protein corona is, therefore, a vital step in the development of safe and efficient diagnostic and therapeutic nanomedicine products. 2134 published manuscripts on the protein corona are reviewed and a down-selection of 470 papers spanning 2000-2021, comprising 1702 nanoparticle (NP) systems is analyzed. This analysis reveals: i) most corona studies have been conducted on metal and metal oxide nanoparticles; ii) despite their overwhelming presence in clinical practice, lipid-based NPs are underrepresented in protein corona research, iii) studies use new methods to improve reliability and reproducibility in protein corona research; iv) studies use more specific protein sources toward personalized medicine; and v) careful characterization of nanoparticles after corona formation is imperative to minimize the role of aggregation and protein contamination on corona outcomes. As nanoparticles used in biomedicine become increasingly prevalent and biochemically complex, the field of protein corona research will need to focus on developing analytical approaches and characterization techniques appropriate for each unique nanoparticle formulation. Achieving such characterization of the nano-bio interface of nanobiotechnologies will enable more seamless development and safe implementation of nanoparticles in medicine.

Sex-Specific Silica Nanoparticle Protein Corona Compositions Exposed to Male and Female BALB/c Mice Plasmas.

As various nanoparticles (NPs) are increasingly being used in nanomedicine products for more effective and less toxic therapy and diagnosis of diseases, there is a growing need to understand their biological fate in different sexes. Herein, we report a proof-of-concept result of sex-specific protein corona compositions on the surface of silica NPs as a function of their size and porosity upon incubation with plasma proteins of female and male BALB/c mice. Our results demonstrate substantial differences between male and female protein corona profiles on the surface of silica nanoparticles. By comparing protein abundances between male and female protein coronas of mesoporous silica nanoparticles and Stöber silica nanoparticles of ∼100, 50, and 100 nm in diameter, respectively, we detected 17, 4, and 4 distinct proteins, respectively, that were found at significantly different concentrations for these constructs. These initial findings demonstrate that animal sex can influence protein corona formation on silica NPs as a function of the physicochemical properties. A more thorough consideration of the role of plasma sex would enable nanomedicine community to design and develop safer and more efficient diagnostic and therapeutic nanomedicine products for both sexes.

Multi-omics analysis of magnetically levitated plasma biomolecules.

We recently discovered that superparamagnetic iron oxide nanoparticles (SPIONs) can levitate plasma biomolecules in the magnetic levitation (MagLev) system and cause formation of ellipsoidal biomolecular bands. To better understand the composition of the levitated biomolecules in various bands, we comprehensively characterized them by multi-omics analyses. To probe whether the biomolecular composition of the levitated ellipsoidal bands correlates with the health of plasma donors, we used plasma from individuals who had various types of multiple sclerosis (MS), as a model disease with significant clinical importance. Our findings reveal that, while the composition of proteins does not show much variability, there are significant differences in the lipidome and metabolome profiles of each magnetically levitated ellipsoidal band. By comparing the lipidome and metabolome compositions of various plasma samples, we found that the levitated biomolecular ellipsoidal bands do contain information on the health status of the plasma donors. More specifically, we demonstrate that there are particular lipids and metabolites in various layers of each specific plasma pattern that significantly contribute to the discrimination of different MS subtypes, i.e., relapsing-remitting MS (RRMS), secondary-progressive MS (SPMS), and primary-progressive MS (PPMS). These findings will pave the way for utilization of MagLev of biomolecules in biomarker discovery for identification of diseases and discrimination of their subtypes.

Measurements of heterogeneity in proteomics analysis of the nanoparticle protein corona across core facilities.

Robust characterization of the protein corona-the layer of proteins that spontaneously forms on the surface of nanoparticles immersed in biological fluids-is vital for prediction of the safety, biodistribution, and diagnostic/therapeutic efficacy of nanomedicines. Protein corona identity and abundance characterization is entirely dependent on liquid chromatography coupled to mass spectroscopy (LC-MS/MS), though the variability of this technique for the purpose of protein corona characterization remains poorly understood. Here we investigate the variability of LC-MS/MS workflows in analysis of identical aliquots of protein coronas by sending them to different proteomics core-facilities and analyzing the retrieved datasets. While the shared data between the cores correlate well, there is considerable heterogeneity in the data retrieved from different cores. Specifically, out of 4022 identified unique proteins, only 73 (1.8%) are shared across the core facilities providing semiquantitative analysis. These findings suggest that protein corona datasets cannot be easily compared across independent studies and more broadly compromise the interpretation of protein corona research, with implications in biomarker discovery as well as the safety and efficacy of our nanoscale biotechnologies.

Two-Dimensional Nanomaterials beyond Graphene for Biomedical Applications.

Two-dimensional (2D) nanomaterials (e.g., graphene) have shown to have a high potential in future biomedical applications due to their unique physicochemical properties such as unusual electrical conductivity, high biocompatibility, large surface area, and extraordinary thermal and mechanical properties. Although the potential of graphene as the most common 2D nanomaterials in biomedical applications has been extensively investigated, the practical use of other nanoengineered 2D materials beyond graphene such as transition metal dichalcogenides (TMDs), topological insulators (TIs), phosphorene, antimonene, bismuthene, metal-organic frameworks (MOFs) and MXenes for biomedical applications have not been appreciated so far. This review highlights not only the unique opportunities of 2D nanomaterials beyond graphene in various biomedical research areas such as bioelectronics, imaging, drug delivery, tissue engineering, and regenerative medicine but also addresses the risk factors and challenges ahead from the medical perspective and clinical translation of nanoengineered 2D materials. In conclusion, the perspectives and future roadmap of nanoengineered 2D materials beyond graphene are outlined for biomedical applications.

In situ monitoring of photo-crosslinking reaction of water-soluble bifunctional macromers using magnetic levitation.

Crosslinking is one of the fundamental phenomena in polymer science, which happens by forming covalent bonds or relatively short sequences of chemical bonds to join two polymer chains. Crosslinking and the subsequent volume shrinkage of monomers/macromers result in changes in their corresponding density which can be measured using density-based measurement techniques (e.g., dilatometry). Here, we demonstrate a method that allows in situ monitoring of photopolymerization of water-soluble bifunctional macromers using magnetic levitation (MagLev) system. We use a hydrophobic paramagnetic solution to monitor the photopolymerization of water-soluble polyethylene glycol diacrylate (PEGDA) as a model of bifunctional macromers using a ring MagLev system. Based on changes in levitation heights (densities) after illumination of blue light, we have successfully monitored the double bond conversion of PEGDA 700 macromers at various polymerization conditions. Our findings suggest that MagLev can be used as a new and complementary analytical technique for rapid screening of the photopolymerization reactions and measurement of conversions using changes in the levitation height of the macromers.

Conformation- and phosphorylation-dependent electron tunnelling across self-assembled monolayers of tau peptides.

We report on charge transport across self-assembled monolayers (SAMs) of short tau peptides by probing the electron tunneling rates and quantum mechanical simulation. We measured the electron tunneling rates across SAMs of carboxyl-terminated linker molecules (C6H12O2S) and short cis-tau (CT) and trans-tau (TT) peptides, supported on template-stripped gold (AuTS) bottom electrode, with Eutectic Gallium-Indium (EGaIn)(EGaIn) top electrode. Measurements of the current density across thousands of AuTS/linker/tau//Ga2O3/EGaIn single-molecule junctions show that the tunneling current across CT peptide is one order of magnitude lower than that of TT peptide. Quantum mechanical simulation demonstrated a wider energy bandgap of the CT peptide, as compared to the TT peptide, which causes a reduction in its electron tunneling current. Our findings also revealed the critical role of phosphorylation in altering the charge transport characteristics of short peptides; more specifically, we found that the presence of phosphate groups can reduce the energy band gap in tau peptides and alter their electrical properties. Our results suggest that conformational and phosphorylation of short peptides (e.g., tau) can significantly change their charge transport characteristics and energy levels.

The File Drawer Problem in Nanomedicine.

The role of the 'file drawer' problem in nanomedicine, which partly drives the current limited clinical success of therapeutic nanoparticles, has been poorly investigated. We propose an integrated functioning of all stakeholders as the only effective way to address the file drawer problem in an efficient and timely manner.

Charge-driven condensation of RNA and proteins suggests broad role of phase separation in cytoplasmic environments.

Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well-established drivers of phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Phase diagrams were mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The resulting condensates were found to retain at least some degree of internal dynamics varying as a function of the molecular composition. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components.

Magnetic Levitation Systems for Disease Diagnostics.

Magnetic levitation (MagLev) is a well-documented, robust technique for density measurements and separations. Although the potential of MagLev as an emerging tool in biotechnology has been recently investigated, the practical use of MagLev in diagnosis and disease detection merits further attention. This review highlights the diagnostic capacity of a simple and portable MagLev system and the possibilities and limitations of the MagLev technique for density-based separation, classification, and manipulation of soft matter and biological systems (e.g., cells, proteins), which in turn may pave the way for the discovery of disease-specific biomarkers.

Mapping the heterogeneity of protein corona by ex vivo magnetic levitation.

In the past decade, we witnessed limited success in the clinical translation of therapeutic nanoparticles (NPs). One of the main reasons for this limited success is our poor understanding of the biological identity of NPs. Herein, we report magnetic levitation (MagLev) as a complementary analytical tool to investigate the homogeneity of the created protein corona (PC) coated NPs through an ex vivo model. Our results demonstrate that the MagLev system not only has the capacity to separate corona coated NPs, but also enables us to study the homogeneity/heterogeneity of the PC. Our findings suggest that current ex vivo isolation methods cause a heterogeneous coverage of PC profiles at the surface of NPs. The MagLev technique, therefore, would be instrumental in identifying and separating fully PC coated NPs which, in turn, enables us to achieve more accurate information on protein corona composition. Ultimately, we believe that the MagLev technique can be used for the fast screening of the homogeneity of corona coated NPs before quantitative analysis of the corona profile/composition, hence definitely improving our fundamental understanding of nano-bio interfaces.

Evolving Magnetically Levitated Plasma Proteins Detects Opioid Use Disorder as a Model Disease.

There are several methods (e.g., enzyme-linked immunosorbent assay and liquid chromatography mass spectroscopy) that already use human plasma to detect a variety of possible diseases. However, this paper introduces the capabilities of magnetic levitation (Maglev) to detect disease (Opioid Use Disorder, used here as a model disease) by using levitation of human plasma proteins. The presented proof-of-concept findings revealed that the optical images of magnetically levitated plasma proteins carry important information about the health spectrum of plasma donors. In addition, the liquid chromatography mass spectroscopy analysis of the magnetically levitated plasma proteins demonstrated remarkable differences between the plasma of healthy individuals and patients with opioid use disorders. Overall, the presented method provides diagnostic value for disease detection using optical images of evolving magnetically levitated plasma proteins and/or proteomic information.

Magnetically Levitated Plasma Proteins.

Surprisingly, the densities of proteins in solution, which are important fundamental biophysical quantities, have not been accurately measured. The lack of such data can limit meaningful interpretation of physical and chemical features of proteins and enzymes. Here, we demonstrate a new technique using superparamagnetic iron oxide nanoparticles (SPIONs) for magnetic levitation (MagLev), which promises to more precisely measure the density of proteins in solution. As a test of our new technique, we have levitated human plasma proteins using MagLev. By using standard density glass beads for calibration, MagLev showed that the levitated plasma proteins have a measured density in solution of 1.03 ± 0.02 g/cm3, which is much lower than those reported or assumed in the past literature (i.e., ∼1.35 g/cm3). Our findings suggest that MagLev may provide useful insights into the measurement of densities for better understanding the solution properties of proteins and their interactions both with other proteins in solution and with solvating water molecules.

Synergistic effect of shape-selective silver nanostructures decorating reduced graphene oxide nanoplatelets for enhanced cytotoxicity against breast cancer.

Graphene-based nanomaterials contain unique physicochemical properties and have been widely investigated due to a variety of applications particularly in cancer therapy. Furthermore, Ag has been known for its extensive historical background for biomedical applications. Therefore, conjugation of shape-selective Ag nanostructures with graphene may provide new horizons for pharmaceutical applications such as cancer treatments. Here we report on the synthesis of Ag nanoparticles (NPs)/reduced graphene oxide (AgNPs/RGO) conjugate nanomaterials containing various shapes of AgNPs by a novel and simple synthesis route using the deformation of dimethylformamide (DMF) as the reducing and coupling agent. The cytotoxicity and anticancer properties of AgNPs, AgNPs/RGO conjugate nanomaterials, RGO and graphene oxide (GO) were probed against MDA-MB-231 cancer and MCF-10A normal human breast cells in vitro. The AgNPs/RGO nanocomposites exhibited a strong anticancer effect by penetration and apoptosis in cancer cells as well as the lowest influence on the viability of normal cells. It was found that cancer cell viability not only depends on the geometry of Ag nanostructures but also on the interaction between AgNPs and RGO nanoplatelets. It is suggested that AgNPs/RGO conjugate nanomaterials with various shapes of AgNPs is a promising therapeutic platform for cancer therapy.