Protein-rich Environment Research Articles

Nanoetched Stainless Steel Architecture Enhances Cell Uptake of Biomacromolecules and Alters Protein Corona Abundancy.

Nanotexture on biocompatible surfaces promotes cell adhesion and proliferation. High aspect ratio nanoachitecture serves as an ideal interface between implant materials and host cells that is well-suited for localized therapeutic delivery. Despite this potential, nanotextured surfaces have not been widely applied for biomacromolecule delivery. Here, we employed a low-cost, industrially relevant nanoetching process to modify the surface of biocompatible stainless steel 316 (SS316L), creating nanotextured SS316L (NT-SS316L) as a material for intracellular biomacromolecule delivery. As biomacromolecule cargoes are adsorbed to the steel and ultimately would be used in protein-rich environments, we performed serum protein corona analysis on unmodified SS316L and NT-SS316L using tandem mass spectrometry. We observed an increase in proteins associated with cell adhesion on the surface of NT-SS316L compared to that of SS316L, supporting literature reports of enhanced adhesion on nanotextured materials. For delivery to adherent cells, a "hard corona" of model biomacromolecule cargoes including superfolder green fluorescent protein (sfGFP) charge variants, cytochrome c, and siRNA was adsorbed on NT-SS316L to assess delivery. Nanotextured surfaces enhanced cellular biomacromolecule uptake and delivered cytosolic-functional proteins and nucleic acids through energy-dependent endocytosis. Collectively, these findings indicate that NT-SS316L holds potential as a surface modification for implants to achieve localized drug delivery for a variety of biomedical applications.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme

Background and aimsBlood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. MethodsWe herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. ResultsFVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. ConclusionOur study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Optimizing encapsulation of black carrot extract using complex coacervation technique: Maximizing the bioaccessibility and release kinetics in different food matrixes

The encapsulation of bioactive compounds at the micro-scale presents an advanced approach for the food and nutraceutical industries. However, the challenge lies in determining optimal encapsulation parameters for each specific bioactive compound and encapsulation method. In addressing this, our study demonstrates the application of statistical programs to streamline the optimization of wet-lab experimental designs. Focusing on the encapsulation technique of complex coacervates for black carrot phenolic extract (BCPE), the response surface methodology was employed to optimize encapsulation parameters in terms of coating material, the core-to-coating material ratio, and the pH of the encapsulation environment. The optimum conditions predicted by RSM to produce BCPE-loaded complex coacervates were found to be a pH of 3.02, a core-to-coating ratio of 10g/100g, and a coating material composition of 59.10g/100 mL maltodextrin, and 0.90g/100 mL whey protein isolate. According to the RSM pattern with 84% desirability, encapsulation efficiency was found 86.08%. In addition, the effect of different food matrixes was examined on the in vitro bioaccessibility of spray-dried BCPE loaded complex coacervated powder (BCPE-CCp). To explore the impact of protein and carbohydrate richness, along with food temperature on capsule stability, the BCPE-CCp was incorporated into skim milk, apple juice, and chocolate beverages (1g/100 mL). Notably, heat treatment had no significant effect on the in vitro bioaccessibility of BCPE-CCp in terms of total phenolic compound and antioxidant activity (p < 0.05), indicating its suitability for hot formulations. Furthermore, the release of BCPE in a protein-rich environment was observed to be higher than in a carbohydrate-rich food matrix under both gastric and intestinal conditions. These findings provide valuable insights into the stability and release dynamics of BCPE-CCp in different food settings, supporting its adaptability for diverse formulations, including those involving elevated temperatures.

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation.

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 μg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR.

Basic protein- and peptide-induced stabilization of long-loop DNA G-guadruplexes

The human genome contains many G-quadruplex-forming sequences, including sequences containing long single-stranded loops that are believed to be unfavorable for G-quadruplex formation. The intracellular environment of biological cells is crowded with proteins with charged surfaces. Understanding the effects of protein-rich environments is important for understanding the formation of G-quadruplexes in an intracellular environment. In this study, we investigated the structural stability of DNA G-quadruplexes in the presence of several types of globular proteins (lysozyme, cytochrome c, bovine serum albumin, myoglobin, histone proteins, and serum proteins), unstructured polypeptides (protamine and poly-l-lysine), and oligopeptides (RGG/RG-domain peptides and short repeated peptides). Thermal melting studies of G-quadruplex-forming oligonucleotides derived from the human telomeric repeat sequence revealed that environments containing high concentrations of proteins and peptides differently affected the G-quadruplex stability according to their loop lengths. We found that weak electrostatic interactions of G-quadruplex loops with basic proteins and peptides improved the stability of long-loop G-quadruplexes and the interactions were strengthened under crowded conditions simulated by dextran. The comparison of the effects of different types of proteins and peptides indicated that excluded volume interactions and structural flexibility of both DNA and polypeptide chains influenced the efficiency of their interactions. This study provides insights into long-loop G-quadruplex stability in a crowded intracellular environment and the recognition of G-quadruplexes by arginine-rich domains of G-quadruplex-binding proteins.

Native Study of the Behaviour of Magnetite Nanoparticles for Hyperthermia Treatment during the Initial Moments of Intravenous Administration.

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve long-term stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle-proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration.

Recrystallization of Adenosine for Localized Drug Delivery.

Adenosine (ADO) is an endogenous metabolite with immense potential to be repurposed as an immunomodulatory therapeutic, as preclinical studies have demonstrated in models of epilepsy, acute respiratory distress syndrome, and traumatic brain injury, among others. The currently licensed products Adenocard and Adenoscan are formulated at 3 mg/mL of ADO for rapid bolus intravenous injection, but the systemic administration of the saline formulations for anti-inflammatory purposes is limited by the nucleoside's profound hemodynamic effects. Moreover, concentrations that can be attained in the airway or the brain through direct instillation or injection are limited by the volumes that can be accommodated in the anatomical space (<5 mL in humans) and the rapid elimination by enzymatic and transport mechanisms in the interstitium (half-life <5 s). As such, highly concentrated formulations of ADO are needed to attain pharmacologically relevant concentrations at sites of tissue injury. Herein, we report a previously uncharacterized crystalline form of ADO (rcADO) in which 6.7 mg/mL of the nucleoside is suspended in water. Importantly, the crystallinity is not diminished in a protein-rich environment, as evidenced by resuspending the crystals in albumin (15% w/v). To the best of our knowledge, this is the first report of crystalline ADO generated using a facile and organic solvent-free method aimed at localized drug delivery. The crystalline suspension may be suitable for developing ADO into injectable formulations for attaining high concentrations of the endogenous nucleoside in inflammatory locales.

Viability of cultured human skin cells treated with 1,6-hexamethylene diisocyanate monomer and its oligomer isocyanurate in different culture media

The isocyanate monomer 1,6-hexamethylene diisocyanate (HDI) and one of its trimers, HDI isocyanurate, are airway and skin sensitizers contained in polyurethane paint. The toxic response of cultured skin cells to these compounds was measured by evaluating the isocyanate concentrations at which 50% of the cells die (i.e., lethal concentration 50%, LC50) because the relative toxicity of each form of HDI should be considered when exposure limits of HDI-based paints are set. By using a luminescent ATP-viability assay, we compared the cytotoxic effects of HDI monomer and HDI isocyanurate on cultured human skin cells (keratinocytes, fibroblasts, and melanocytes) after 4-h isocyanate exposures using culture media with varying levels of nutrients in order to also determine the effects of media composition on isocyanate toxicity. Before analysis, experimental wells were normalized to controls containing cells that were cultured with the same vehicle and media. The measured mean LC50 values ranged from 5 to 200 µM across the experimental conditions, in which HDI isocyanurate in protein-devoid media was the most toxic to cells, producing the lowest LC50 values. For HDI monomer, keratinocytes were the most resistant to its toxicity and melanocytes were the most susceptible. However, when exposed to HDI isocyanurate, the opposite was observed, with melanocytes being the most resilient and the keratinocytes and fibroblasts were more susceptible. Depending on the type of skin cells, dose–response data indicated that HDI isocyanurate was 2–6 times more toxic than HDI monomer when using protein-devoid media whereas HDI isocyanurate was 4–13 times more toxic than HDI monomer when protein-rich media was used. Therefore, if the protein-devoid saline medium alone were used for these experiments, then a significant under-estimation of their relative toxicities in protein-rich environments would have resulted. This difference is because HDI monomer toxicity was more attenuated by the presence of protein in the culture media than HDI isocyanurate toxicity. Thus, conclusions based on comparative toxicity studies and consequent inference applied to potential human toxicity can be affected by in vitro culture media conditions. The physiochemical difference in reactivity of the two forms of HDI to biological molecules most likely explains the observed toxicity differences and may have implications for skin penetration, adverse effects like skin sensitization, and systemic responses like asthma. Future studies are warranted to investigate differences in the biological availability, cellular toxicity, and immunologic sensitization mechanisms for HDI monomer and HDI isocyanurate.

Inhibition Mechanism of α-Amylase/α-Glucosidase by Silibinin, Its Synergism with Acarbose, and the Effect of Milk Proteins.

As a natural flavonolignan, silibinin is reported to possess multiple biological activities, while the inhibitory potential of silibinin on carbohydrate-hydrolyzing enzymes is still unclear. Therefore, in this study, the inhibitory effect and underlying mechanism of silibinin against α-amylase/α-glucosidase were investigated. The results indicated that silibinin showed a strong inhibitory efficiency against α-amylase/α-glucosidase in noncompetitive manners and exhibited synergistic inhibition against α-glucosidase with acarbose. However, interestingly, the inhibitory effect of silibinin was significantly hindered in various milk protein-rich environments, but this phenomenon disappeared after simulated gastrointestinal digestion of milk proteins in vitro. Furthermore, silibinin could combine with the inactive site of α-amylase/α-glucosidase and change the microenvironment and secondary structure of the enzymes, thereby influencing the catalytic efficiency of enzymes. This research suggested that silibinin could be used as a novel carbohydrate-hydrolyzing enzyme inhibitor, and milk beverages rich in silibinin had the potential for further application in antidiabetic dietary or medicine.

Super-resolution microscopy reveals the arrangement of inner membrane protein complexes in mammalian mitochondria.

The mitochondrial inner membrane is a protein-rich environment containing large multimeric complexes, including complexes of the mitochondrial electron transport chain, mitochondrial translocases and quality control machineries. Although the inner membrane is highly proteinaceous, with 40-60% of all mitochondrial proteins localised to this compartment, little is known about the spatial distribution and organisation of complexes in this environment. We set out to survey the arrangement of inner membrane complexes using stochastic optical reconstruction microscopy (STORM). We reveal that subunits of the TIM23 complex, TIM23 and TIM44 (also known as TIMM23 and TIMM44, respectively), and the complex IV subunit COXIV, form organised clusters and show properties distinct from the outer membrane protein TOM20 (also known as TOMM20). Density based cluster analysis indicated a bimodal distribution of TIM44 that is distinct from TIM23, suggesting distinct TIM23 subcomplexes. COXIV is arranged in larger clusters that are disrupted upon disruption of complex IV assembly. Thus, STORM super-resolution microscopy is a powerful tool for examining the nanoscale distribution of mitochondrial inner membrane complexes, providing a 'visual' approach for obtaining pivotal information on how mitochondrial complexes exist in a cellular context.

In vitro and in vivo assessments of inspired Ag/80S bioactive nanocomposites against carbapenem-resistant Klebsiella pneumoniae

In 2017 the World Health Organization listed carbapenem-resistant K. pneumoniae as a critical priority for developing a novel antimicrobial agent. Here we report on our investigation of the antibacterial efficacy of silver nanoparticles (AgNPs), confined to a mesostructured material and designated as an Ag/80S bioactive nanocomposite, against carbapenem-resistant K. pneumoniae. Results from a textural analysis indicate a 7.5 nm mesopore size and 307.6 m2/g surface area for Ag/80S. UV–Vis spectrum and transmission electron microscope images of Ag/80S revealed a uniform AgNP size distribution with an approximately 3.5 nm average. ICP-MS analysis demonstrated a significantly higher silver content in TSB (a protein-rich environment) compared to ultrapure water, suggesting a controllable release of Ag/80S and thus designated as the inspired Ag/80S. Minimum inhibitory concentration (MIC) values against 16 K. pneumoniae isolates ranged from 0.25 to 0.5% (2.5 to 5.0 mg/ml). NIH 3T3 fibroblast viability at 0.25% exceeded 80% and at 0.5% just under 70%, suggesting low cytotoxicity. Mechanistic study results indicate that the inspired Ag/80S attached to and deformed bacterial cells and induced a time-dependent accumulation of reactive oxygen species, leading to bacterial death. Further, inspired Ag/80S significantly extended median survival time in a Caenorhabditis elegans animal model infected with carbapenem-resistant K. pneumoniae ATCC BAA-1705. Combined, we found a novel Ag/80S which could prevent aggregation of AgNP and control its release via a specific environment for medical use against carbapenem-resistant K. pneumoniae.

Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells.

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G0/G1 phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria

Novel classes of antibiotics and new strategies to prevent and treat infections are urgently needed because the rapid rise in drug-resistant bacterial infections in recent decades has been accompanied by a parallel decline in development of new antibiotics. Membrane permeabilizing antimicrobial peptides (AMPs) have long been considered a potentially promising, novel class of antibiotic, especially for wound protection and treatment to prevent the development of serious infections. Yet, despite thousands of known examples, AMPs have only infrequently proceeded as far as clinical trials, especially the chemically simple, linear examples. In part, this is due to impediments that often limit their applications in vivo. These can include low solubility, residual toxicity, susceptibility to proteolysis, and loss of activity due to host cell, tissue, and protein binding. Here we show how synthetic molecular evolution can be used to evolve potentially advantageous antimicrobial peptides that lack these impediments from parent peptides that have at least some of them. As an example of how the antibiotic discovery pipeline can be populated with more promising candidates, we evolved and optimized one family of linear AMPs into a new generation with high solubility, low cytotoxicity, potent broad-spectrum sterilizing activity against a panel of gram-positive and gram-negative ESKAPE pathogens, and antibiofilm activity against gram-positive and gram-negative biofilms. The evolved peptides have these activities in vitro even in the presence of concentrated host cells and also in vivo in the complex, cell- and protein-rich environment of a purulent animal wound model infected with drug-resistant bacteria.

Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

Surface functionalization of carbon-based sensors with biocompatible polymer to enable electrochemical measurement in protein-rich environment

Electrochemical biosensors have been widely applied to elucidate various information on pharmaceutically important neurotransmitters such as dopamine (DA). However, the biofouling of electrodes has been a critical issue for electrochemical measurements performed in physiological environments. In this study, highly biocompatible polymer brush made of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) was employed to modify carbon-based sensors to prevent biofouling. The PMPC brushes with thicknesses ranging from 15 to 30 nm on top of glassy carbon surfaces successfully suppressed protein adsorption in serum environments, and the polymer modification remained stable for at least 8 h in such conditions. The PMPC-modified glassy carbon electrode (GCE) achieved a limit of detection (LOD) as low as 1.8 μM for DA, as determined from cyclic voltammetry (CV), which exhibited better performance than the bovine serum albumin (BSA)-coated GCE with a LOD of 3.9 μM. The results from this study open new avenues for drug discovery and pharmacokinetics by enabling carbon-based biosensors to accurately and sensitively detect pharmaceuticals in protein-rich environments for an extensive period of time.

Analysis of complexes formed by small gold nanoparticles in low concentration in cell culture media.

New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates’ morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.

Heat Stability of Differently Stabilized Solid Lipid Nanoparticles in the Presence of Excess Bulk Phase Protein

In order to apply emulsion-based delivery systems to food, they have to be stable in a protein rich environment. This study investigated the stability of solid lipid nanoparticles (SLN) during heat treatment in the presence or absence of β-lactoglobulin (BLG). SLN were stabilized either by Tween 20 (TS) or by the protein itself (BS) and were enriched to a total BLG content of 56 mg/mL. The sizes of both types of SLN were initially in the range of 170 nm. The amount of free protein was determined before and after enrichment with BLG. As revealed by particle size and zeta potential measurements, a protein layer of BLG (hard corona) adsorbed on BS but not on TS. By contrast, a soft corona was formed around both BS and TS. SLN were heat treated in the presence and absence of protein and were characterized regarding size and zeta potential. According to transmission electron microscopy imaging, heating did not affect the shape of TS and BS: TS were platelets, whereas BS exhibited a spherical or platelet like shape. Upon heat treatment, the particle size of TS increased to about 3.5 fold of the initial size (to appr. 600 nm) in the presence and in the absence of excess protein. The cloudy protein layer (soft corona) around TS could thus not prevent coalescence of TS. By contrast, BS did not experience a change in particle size. Hence, by the choice of emulsifier, an encapsulation system that is stable against heat treatment can be obtained.

The dipeptidyl peptidase-4 inhibitory effect of flavonoids is hindered in protein rich environments.

Dipeptidyl peptidase-4 (DPP-4) inhibitors present a unique approach for the management of type 2 diabetes (T2D). In the present study, the inhibition of DPP-4 was evaluated for a large panel of flavonoids, important components of the human diet, using in vitro and ex vivo models. The activity of the isolated enzyme was assayed in vitro. Subsequently, the most active flavonoids were tested ex vivo in human whole blood and plasma. In this study, contrary to the in vitro fluorometric tests, flavonoids did not show inhibitory activity against DPP-4. Due to the discrepancy in the results between the in vitro and ex vivo approaches, plasma protein binding values were determined, presenting values from 43.9 to 100.0%. This work provides a new insight into the inhibitory activity for DPP-4, based on the flavonoid scaffold. Additionally, the obtained results showed that the inhibitory effect of flavonoids against DPP-4 was hindered in protein rich environments, like that occurring in blood, and indicated the need for experimental refinement in drug discovery for blood targets.

The Next Generation of Healthcare

Health is an issue that concerns everyone, regardless of economic status, geography, or background. As a result of pioneering research in the biomedical sciences, the quality of human health has increased markedly over recent centuries. In order to continue this progress, supporting researchers in the biomedical sciences is crucial. With this first issue of Advanced Therapeutics, we are opening a new pathway for biomedical researchers to communicate their work. Advanced Therapeutics will cover a broad range of healthcare topics. Articles from the vast interdisciplinary fields of drug discovery and delivery systems will be complemented by research on therapeutic targets, targeted delivery, and design of dosage forms and formulations, as well as sustained-release systems. Focusing on the next generation of pharmaceuticals, targeting, and delivery, the journal publishes cutting-edge developments in the emerging areas of precision, predictive and personalized medicine, and pharmacogenomics. Beyond original research and review-style content on advances in drug design, drug dispensing, and drug metabolism, Advanced Therapeutics publishes essays and editorials to inform readers of the political, societal, ethical, and industrial issues associated with research within the journal's scope. This issue marks the expansion of the Advanced family of journals further into health research, following the successful launches of Advanced Healthcare Materials in 2012 and Advanced Biosystems last year. In contrast to Advanced Healthcare Materials and Advanced Biosystems, the main focus of work published in Advanced Therapeutics will be the application of materials in pharmacology and medicine—not on the materials themselves. These three Advanced journals are published in close collaboration with the editorial teams of other established Wiley journals in the life sciences, such as Macromolecular Bioscience, Biotechnology Journal, Proteomics, BioEssays, and Molecular Nutrition & Food Research. Advanced Therapeutics is supported by an executive advisory board that is made up of internationally renowned leaders from a wide range of backgrounds: The journal is also highly indebted to the International Advisory Board members for lending their support and expertise: Cameron Alexander (University of Nottingham), Karen Christman (University of California, San Diego), Mingyuan Gao (Institute of Chemistry, Chinese Academy of Sciences), Concepción González-Bello (Universidade de Santiago de Compostela), Zhen Gu (University of North Carolina at Chapel Hill), Dean Ho (University of California, Los Angeles), Doo Sung Lee (Sungkyunkwan University), Xinjie Liang (National Center for Nanoscience and Technology, China), Samir Mitragotri (University of California, Santa Barbara), Dan Peer (Tel Aviv University), Hélder Santos (University of Helsinki), Weihong Tan (Hunan University; Florida University), Arto Urtti (University of Helsinki), Ralph Weissleder (Harvard University), Alexander Zelikin (Aarhus University), and Liangfang Zhang (University of California, San Diego). The content of this issue is intended to give an impression of the broad range of topics that will be covered by Advanced Therapeutics. Firstly, Michael Decker and co-workers have a close look at receptor agonists of the human cannabinoid receptor 2 and develop affinity switches based on a benzimidazole core through azologization and computational studies (adtp.201700032). An effective technique to treat retinoblastoma cancer cells using femtosecond laser pulses and EpCAM retinoblastoma cell targeting gold nanorods is demonstrated by Nir Katchensky and co-workers in their article (adtp.201800009). Next, Mingyuan Cao and his group propose a novel histochemical staining approach for identifying rare earth–based nanoprobes in tissues through unique chromogenic reactions (adtp.201800005). Patrick S. Doyle and his group present a bottom-up and scalable method that formulates nanoparticles of hydrophobic drugs directly embedded in composite hydrogel beads (adtp.201700020). Finally, Hélder Santos and co-workers design a smart transformable nanosystem with a photo-triggered antifouling surface to efficiently reduce protein adsorption on nanoparticles and modulate their cell interaction in protein-rich environment (adtp.201800013). These articles offer a window into the critical subject matter that Advanced Therapeutics will be devoted to highlighting. This represents our first steps toward providing an exciting new platform for researchers designing materials for therapeutic applications to showcase their work. The editorial team is looking forward to the journey. The Editorial Team of Advanced Therapeutics.