Nanoparticle Properties Influence Transendothelial Migration of Monocytes.

Nanoparticle-based delivery of therapeutics to the brain has had limited clinical impact due to challenges crossing the blood-brain barrier (BBB). Certain cells, such as monocytes, possess the ability to migrate across the BBB, making them attractive candidates for cell-based brain delivery strategies. In this work, we explore nanoparticle design parameters that impact both monocyte association and monocyte-mediated BBB transport. We use electrohydrodynamic jetting to prepare nanoparticles of varying sizes, compositions, and elasticity to address their impact on uptake by THP-1 monocytes and permeation across the BBB. An in vitro human BBB model is developed using human cerebral microvascular endothelial cells (hCMEC/D3) for the assessment of migration. We compare monocyte uptake of both polymeric and synthetic protein nanoparticles (SPNPs) of various sizes, as well as their effect on cell migration. SPNPs (human serum albumin/HSA or human transferrin/TF) are shown to promote increased monocyte-mediated transport across the BBB over polymeric nanoparticles. TF SPNPs (200 nm) associate readily, with an average uptake of 138 particles/cell. Nanoparticle loading is shown to influence the migration of THP-1 monocytes. The migration of monocytes loaded with 200 nm TF and 200 nm HSA SPNPs was 2.3-fold and 2.1-fold higher than that of an untreated control. RNA-seq analysis after TF SPNP treatment suggests that the upregulation of several migration genes may be implicated in increased monocyte migration (ex. integrin subunits α M and α L). Integrin ß 2 chain combines with either integrin subunit α M chain or integrin subunit α L chain to form macrophage antigen 1 and lymphocyte function-associated antigen 1 integrins. Both products play a pivotal role in the transendothelial migration cascade. Our findings highlight the potential of SPNPs as drug and/or gene delivery platforms for monocyte-mediated BBB transport, especially where conventional polymer nanoparticles are ineffective or otherwise not desirable.

Multifunctional Synthetic Protein Nanoparticles via Reactive Electrojetting.

Protein nanoparticles are a promising approach for nanotherapeutics, as proteins combine versatile chemical and biological function with controlled biodegradability. In this work, the development of an adaptable synthesis method is presented for synthetic protein nanoparticles (SPNPs) based on reactive electrojetting. In contrast to past work with electrohydrodynamic cojetting using inert polymers, the jetting solutions are comprised of proteins and chemically activated macromers, designed to react with each other during the processing step, to form insoluble nanogel particles. SPNPs made from a variety of different proteins, such as transferrin, insulin, or hemoglobin, are stable and uniform under physiological conditions and maintain monodisperse sizes of around 200 nm. SPNPs comprised of transferrin and a disulfide containing macromer are stimuli-responsive, and serve as markers of oxidative stress within HeLa cells. Beyond isotropic SPNPs, bicompartmental nanoparticles containing human serum albumin and transferrin in two distinct hemispheres are prepared via reactive electrojetting. This novel platform provides access to a novel class of versatile protein particles with nanoscale architectures that i) can be made from a variety of proteins and macromers, ii) have tunable biological responses, and iii) can be multicompartmental, a prerequisite for controlled release of multiple drugs.

Size, shape, and flexibility influence nanoparticle transport across brain endothelium under flow.

Nanoparticle-based therapeutic formulations are being increasingly explored for the treatment of various ailments. Despite numerous advances, the success of nanoparticle-based technologies in treating brain diseases has been limited. Translational hurdles of nanoparticle therapies are attributed primarily to their limited ability to cross the blood-brain barrier (BBB), which is one of the body's most exclusive barriers. Several efforts have been focused on developing affinity-based agents and using them to increase nanoparticle accumulation at the brain endothelium. Very little is known about the role of fundamental physical parameters of nanoparticles such as size, shape, and flexibility in determining their interactions with and penetration across the BBB. Using a three-dimensional human BBB microfluidic model (µHuB), we investigate the impact of these physical parameters on nanoparticle penetration across the BBB. To gain insights into the dependence of transport on nanoparticle properties, two separate parameters were measured: the number of nanoparticles that fully cross the BBB and the number that remain associated with the endothelium. Association of nanoparticles with the brain endothelium was substantially impacted by their physical characteristics. Hard particles associate more with the endothelium compared to soft particles, as do small particles compared to large particles, and spherical particles compared to rod-shaped particles. Transport across the BBB also exhibited a dependence on nanoparticle properties. A nonmonotonic dependence on size was observed, where 200 nm particles exhibited higher BBB transport compared to 100 and 500 nm spheres. Rod-shaped particles exhibited higher BBB transport when normalized by endothelial association and soft particles exhibited comparable transport to hard particles when normalized by endothelial association. Tuning nanoparticles' physical parameters could potentially enhance their ability to cross the BBB for therapeutic applications.

Effect of Nanoparticle Composition, Size, Shape, and Stiffness on Penetration Across the Blood-Brain Barrier.

The delivery of therapeutics to the brain in an efficient, noninvasive manner continues to be a major unmet need in the field of drug delivery. One significant impediment to brain delivery results from the existence of the physical yet dynamic blood-brain barrier (BBB). Despite the many, often complex strategies that currently exist to breach the BBB, adequate delivery of effective therapeutics from the bloodstream continues to remain quite low. Nanotechnology has emerged as a promising tool for brain delivery, but little is known about the important particle parameters that influence delivery. Here, we synthesized and characterized a library of nanoparticles with distinct properties ranging from size, shape, stiffness, and composition to investigate and identify the key attributes influencing particle uptake and transport for brain delivery. To accomplish this task, an in vitro human BBB model was developed and validated using human cerebral microvascular endothelial cells (hCMEC/D3). Particle uptake and apparent permeability coefficients (Papp) were then determined for each particle group. To elucidate the roles of different parameters on particle uptake and transport across the BBB, the predominant mechanisms of endocytosis were also investigated. Our results show that particle composition yielded the greatest impact on penetration across the BBB model. This work lays the foundation and provides new insights into the role of particle parameters on penetration across the BBB.

A microfluidic model of human brain (µHuB) for assessment of blood brain barrier.

Microfluidic cellular models, commonly referred to as "organs-on-chips," continue to advance the field of bioengineering via the development of accurate and higher throughput models, captivating the essence of living human organs. This class of models can mimic key in vivo features, including shear stresses and cellular architectures, in ways that cannot be realized by traditional two-dimensional in vitro models. Despite such progress, current organ-on-a-chip models are often overly complex, require highly specialized setups and equipment, and lack the ability to easily ascertain temporal and spatial differences in the transport kinetics of compounds translocating across cellular barriers. To address this challenge, we report the development of a three-dimensional human blood brain barrier (BBB) microfluidic model (µHuB) using human cerebral microvascular endothelial cells (hCMEC/D3) and primary human astrocytes within a commercially available microfluidic platform. Within µHuB, hCMEC/D3 monolayers withstood physiologically relevant shear stresses (2.73 dyn/cm2) over a period of 24 hr and formed a complete inner lumen, resembling in vivo blood capillaries. Monolayers within µHuB expressed phenotypical tight junction markers (Claudin-5 and ZO-1), which increased expression after the presence of hemodynamic-like shear stress. Negligible cell injury was observed when the monolayers were cultured statically, conditioned to shear stress, and subjected to nonfluorescent dextran (70 kDa) transport studies. µHuB experienced size-selective permeability of 10 and 70 kDa dextrans similar to other BBB models. However, with the ability to probe temporal and spatial evolution of solute distribution, µHuBs possess the ability to capture the true variability in permeability across a cellular monolayer over time and allow for evaluation of the full breadth of permeabilities that would otherwise be lost using traditional end-point sampling techniques. Overall, the µHuB platform provides a simplified, easy-to-use model to further investigate the complexities of the human BBB in real-time and can be readily adapted to incorporate additional cell types of the neurovascular unit and beyond.

HIV Provider Documentation and Actions Following Patient Reports of At-risk Behaviors and Conditions When Identified by a Web-Based Point-of-Care Assessment.

We compared same-day provider medical record documentation and interventions addressing depression and risk behaviors before and after delivering point-of-care patient-reported outcomes (PROs) feedback for patients who self-reported clinically relevant levels of depression or risk behaviors. During the study period (1 January 2006-15 October 2010), 2289 PRO assessments were completed by HIV-infected patients. Comparing the 8 months before versus after feedback implementation, providers were more likely to document depression (74% before vs. 87% after feedback, p = 0.02) in patients with moderate-to-severe depression (n = 317 assessments), at-risk alcohol use (41 vs. 64%, p = 0.04, n = 155) and substance use (60 vs. 80%, p = 0.004, n = 212). Providers were less likely to incorrectly document good adherence among patients with inadequate adherence after feedback (42 vs. 24%, p = 0.02, n = 205). While PRO feedback of depression and adherence were followed by increased provider intervention, other domains were not. Further investigation of factors associated with the gap between awareness and intervention are needed in order to bridge this divide.

Functional Genomic Analysis of the Impact of Camelina (Camelina sativa) Meal on Atlantic Salmon (Salmo salar) Distal Intestine Gene Expression and Physiology.

The inclusion of plant meals in diets of farmed Atlantic salmon can elicit inflammatory responses in the distal intestine (DI). For the present work, fish were fed a standard fish meal (FM) diet or a diet with partial replacement of FM with solvent-extracted camelina meal (CM) (8, 16, or 24 % CM inclusion) during a 16-week feeding trial. A significant decrease in growth performance was seen in fish fed all CM inclusion diets (Hixson et al. in Aquacult Nutr 22:615-630, 2016). A 4x44K oligonucleotide microarray experiment was carried out and significance analysis of microarrays (SAM) and rank products (RP) methods were used to identify differentially expressed genes between the DIs of fish fed the 24 % CM diet and those fed the FM diet. Twelve features representing six known transcripts and two unknowns were identified as CM responsive by both SAM and RP. The six known transcripts (including thioredoxin and ependymin), in addition to tgfb, mmp13, and GILT, were studied using qPCR with RNA templates from all four experimental diet groups. All six microarray-identified genes were confirmed to be CM responsive, as was tgfb and mmp13. Histopathological analyses identified signs of inflammation in the DI of salmon fed CM-containing diets, including lamina propria and sub-epithelial mucosa thickening, infiltration of eosinophilic granule cells, increased goblet cells and decreased enterocyte vacuolization. All of these were significantly altered in 24 % CM compared to all other diets, with the latter two also altered in 16 % CM compared with 8 % CM and control diet groups. Significant correlation was seen between histological parameters as well as between five of the qPCR analyzed genes and histological parameters. These molecular biomarkers of inflammation arising from long-term dietary CM exposure will be useful in the development of CM-containing diets that do not have deleterious effects on salmon growth or physiology.

Antimicrobial Peptide Mimicking Primary Amine and Guanidine Containing Methacrylamide Copolymers Prepared by Raft Polymerization.

Naturally occurring antimicrobial peptides (AMPs) display the ability to eliminate a wide variety of bacteria, without toxicity to the host eukaryotic cells. Synthetic polymers containing moieties mimicking lysine and arginine components found in AMPs have been reported to show effectiveness against specific bacteria, with the mechanism of activity purported to depend on the nature of the amino acid mimic. In an attempt to incorporate the antimicrobial activity of both amino acids into a single water-soluble copolymer, a series of copolymers containing lysine mimicking aminopropyl methacrylamide (APMA) and arginine mimicking guanadinopropyl methacrylamide (GPMA) were prepared via aqueous RAFT polymerization. Copolymers were prepared with varying ratios of the comonomers, with degree of polymerization of 35-40 and narrow molecular weight distribution to simulate naturally occurring AMPs. Antimicrobial activity was determined against Gram-negative and Gram-positive bacteria under conditions with varying salt concentration. Toxicity to mammalian cells was assessed by hemolysis of red blood cells and MTT assays of MCF-7 cells. Antimicrobial activity was observed for APMA homopolymer and copolymers with low concentrations of GPMA against all bacteria tested, with low toxicity toward mammalian cells.

Antimicrobial poly(methacrylamide) derivatives prepared via aqueous RAFT polymerization exhibit biocidal efficiency dependent upon cation structure.

Antimicrobial peptides (AMPs) show great potential as alternative therapeutic agents to conventional antibiotics as they can selectively bind and eliminate pathogenic bacteria without harming eukaryotic cells. It is of interest to develop synthetic macromolecules that mimic AMPs behavior, but that can be produced more economically at commercial scale. Herein, we describe the use of aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare primary and tertiary amine-containing polymers with precise molecular weight control and narrow molecular weight distributions. Specifically, N-(3-aminopropyl)methacrylamide (APMA) was statistically copolymerized with N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) or N-[3-(diethylamino)propyl]methacrylamide (DEAPMA) to afford a range of (co)polymer compositions. Analysis of antimicrobial activity against E. coli (Gram-negative) and B. subtilis (Gram-positive) as a function of buffer type, salt concentration, pH, and time indicated that polymers containing large fractions of primary amine were most effective against both strains of bacteria. Under physiological pH and salt conditions, the polymer with the highest primary amine content caused complete inhibition of bacterial growth at low concentrations, while negligible hemolysis was observed over the full range of concentrations tested, indicating exceptional selectivity. The cytotoxicity of select polymers was evaluated against MCF-7 cells.