Phagocytosis is a principal component of the body's innate immunity in which macrophages internalize targets in an actin-dependent manner. Targets vary widely in shape and size and include particles such as pathogens and senescent cells. Despite considerable progress in understanding this complicated process, the role of target geometry in phagocytosis has remained elusive. Previous studies on phagocytosis have been performed using spherical targets, thereby overlooking the role of particle shape. Using polystyrene particles of various sizes and shapes, we studied phagocytosis by alveolar macrophages. We report a surprising finding that particle shape, not size, plays a dominant role in phagocytosis. All shapes were capable of initiating phagocytosis in at least one orientation. However, the local particle shape, measured by tangent angles, at the point of initial contact dictates whether macrophages initiate phagocytosis or simply spread on particles. The local shape determines the complexity of the actin structure that must be created to initiate phagocytosis and allow the membrane to move over the particle. Failure to create the required actin structure results in simple spreading and not internalization. Particle size primarily impacts the completion of phagocytosis in cases where particle volume exceeds the cell volume.drug delivery ͉ macrophages ͉ membrane ͉ shape P hagocytosis is a principal component of the body's innate immunity in which macrophages and other antigenpresenting cells internalize large (Ͼ0.5 m) particulate targets (1). Examples of targets include pathogens such as rod-shaped Escherichia coli and Bacillus anthracis and spiral-shaped Campylobacter jejuni, disk-shaped senescent cells such as aged erythrocytes, and airborne particles such as dust and pollen, all of which vary widely in both shape and size. Because macrophages in the human body encounter targets with such diversity, the question has long been asked, how does target geometry impact phagocytosis? Several studies have been conducted specifically to address this question; however, a generalized answer is still lacking. The main reason behind this shortcoming is that all phagocytosis studies have been performed with spherical targets (2-7). Exclusive use of spherical particles originated partly because of a presumption that size is the principal parameter of interest and partly because of difficulties in fabricating nonspherical particles of controlled dimensions. Use of spherical particles not only concealed the role of particle shape in phagocytosis but also created an inaccurate picture of the actual role of particle size because all parameters that describe size (volume, surface area, etc.) scale with particle radius, leaving one wondering as to which parameter is of fundamental consequence in phagocytosis. Accordingly, the precise roles of target size and shape in phagocytosis, despite their high relevance, remain largely unknown.Herein, we report, using alveolar macrophages as model phagocytes and polystyrene (PS) particles o...

Encapsulation of therapeutic agents in polymer particles has been successfully used in the development of new drug carriers. A number of design parameters that govern the functional behavior of carriers, including the choice of polymer, particle size and surface chemistry, have been tuned to optimize their performance in vivo. However, particle shape, which may also have a strong impact on carrier performance, has not been thoroughly investigated. This is perhaps due to the limited availability of techniques to produce non-spherical polymer particles. In recent years, a number of reports have emerged to directly address this bottleneck and initial studies have indeed confirmed that particle shape can significantly impact the performance of polymer drug carriers. This article provides a review of this field with respect to methods of particle preparation and the role of particle shape in drug delivery.

Purpose-Polymeric microspheres are extensively researched for applications in drug and vaccine delivery. However, upon administration into the body, microspheres are primarily cleared via phagocytosis by macrophages. Although numerous studies have reported on the biochemical pathways of phagocytosis, relatively little is known about the dependence of phagocytosis on particle size. Here, we investigate the previously unexplained dependence of phagocytosis on particle size.Methods-Rat alveolar macrophages and IgG-opsonized and non-opsonized polystyrene microspheres were used as model macrophages and drug delivery particles. Phagocytosis, attachment and internalization were measured by flow cytometry and time-lapse video microscopy.Results-Particles possessing diameters of 2-3 μm exhibited maximal phagocytosis and attachment. Rate of internalization, however, was not affected significantly by particle size. Maximal attachment of 2-3 μm microspheres is hypothesized to originate from the characteristic features of membrane ruffles in macrophages. Elimination of ruffles via osmotic swelling nearly eliminated the peculiar size-dependence of phagocytosis. A simple mathematical model is presented to describe the dependence of phagocytosis on particle size.Conclusions-The dependence of phagocytosis on particle size originated primarily from the attachment step. These results reveal the importance of controlling drug delivery particle size distribution and selecting the size appropriate for avoiding or encouraging phagocytosis.

Polymeric micro-and nanoparticles play a central role in varied applications such as drug delivery, medical imaging, and advanced materials, as well as in fundamental studies in fields such as microfluidics and nanotechnology. Functional behavior of polymeric particles in these fields is strongly influenced by their shape. However, the availability of precisely shaped polymeric particles has been a major bottleneck in understanding and capitalizing on the role of shape in particle function. Here we report a method that directly addresses this need. Our method uses routine laboratory chemicals and equipment to make particles with >20 distinct shapes and characteristic features ranging in size from 60 nm to 30 m. This method offers independent control over important particle properties such as size and shape, which is crucial to the development of nonspherical particles both as tools and products for a variety of fields.morphology ͉ nanotechnology ͉ geometry ͉ drug delivery ͉ nonspherical P olymeric particles are used in a diverse array of applications including drug delivery (1), advanced materials (2), personal care (3), and medical imaging (4). They also are used in fundamental studies in fields such as microfluidics (5) and nanotechnology (6). Particle shape is a critical parameter that can significantly influence particle function. The vast potential of shape, however, has not been fully explored due to difficulties in creating polymer particles with controlled shapes. Several reports exist on fabrication of polymeric particles with nonspherical geometries. These approaches make use of self-assembly (7-9), photolithography (10), nonwetting template molding (11), microfluidics (12, 13), and stretching of spherical particles (14,15). Collectively, these methods have produced particles of several distinct shapes. Some of these methods provide advantages such as scalability, high throughput, and precise control over particle shape. However, they also suffer from drawbacks including cost, particle size limitations, low throughput, and limited ability to sculpt particles in three dimensions. Accordingly, simple, versatile, inexpensive, and high-throughput methods of fabricating nonspherical particles still remain a bottleneck of future discoveries in a diverse array of fields. Here, we report a simple method that generates particles of Ͼ20 distinct shapes in large, reproducible quantities. ResultsSpherical polystyrene (PS) particles of diameters between 60 nm and 10 m are used here as a starting material for preparing particles of complex shapes. These particles are suspended in an aqueous solution of polyvinyl alcohol (PVA) and cast into films (14), which are then manipulated to engineer particle shape. The method for engineering shape can be classified into two general approaches (Fig. 1). In the first approach, termed scheme A, PS particles are liquefied by using solvent or by heating above the glass transition temperature (T g ) of PS and then stretched in one or two dimensions. In the second approach, scheme B...

Shape-induced inhibition of phagocytosis of drug delivery particles is possible by minimizing the size-normalized curvature of particles. We have created a high aspect ratio shape that exhibits negligible uptake by macrophages.

Current influenza vaccines provide limited protection against circulating influenza A viruses. A universal influenza vaccine will eliminate the intrinsic limitations of the seasonal flu vaccines. Here we report methodology to generate double-layered protein nanoparticles as a universal influenza vaccine. Layered nanoparticles are fabricated by desolvating tetrameric M2e into protein nanoparticle cores and coating these cores by crosslinking headless HAs. Representative headless HAs of two HA phylogenetic groups are constructed and purified. Vaccinations with the resulting protein nanoparticles in mice induces robust long-lasting immunity, fully protecting the mice against challenges by divergent influenza A viruses of the same group or both groups. The results demonstrate the importance of incorporating both structure-stabilized HA stalk domains and M2e into a universal influenza vaccine to improve its protective potency and breadth. These potent disassemblable protein nanoparticles indicate a wide application in protein drug delivery and controlled release.

Abstract. Cardiopulmonary health implications resulting from exposure to secondary organic aerosols (SOA), which comprise a significant fraction of ambient particulate matter (PM), have received increasing interest in recent years. In this study, alveolar macrophages were exposed to SOA generated from the photooxidation of biogenic and anthropogenic precursors (isoprene, α-pinene, β-caryophyllene, pentadecane, m-xylene, and naphthalene) under different formation conditions (RO 2 + HO 2 vs. RO 2 + NO dominant, dry vs. humid). Various cellular responses were measured, including reactive oxygen and nitrogen species (ROS/RNS) production and secreted levels of cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). SOA precursor identity and formation condition affected all measured responses in a hydrocarbon-specific manner. With the exception of naphthalene SOA, cellular responses followed a trend where TNF-α levels reached a plateau with increasing IL-6 levels. ROS/RNS levels were consistent with relative levels of TNF-α and IL-6, due to their respective inflammatory and anti-inflammatory effects. Exposure to naphthalene SOA, whose aromatic-ring-containing products may trigger different cellular pathways, induced higher levels of TNF-α and ROS/RNS than suggested by the trend. Distinct cellular response patterns were identified for hydrocarbons whose photooxidation products shared similar chemical functionalities and structures, which suggests that the chemical structure (carbon chain length and functionalities) of photooxidation products may be important for determining cellular effects. A positive nonlinear correlation was also detected between ROS/RNS levels and previously measured DTT (dithiothreitol) activities for SOA samples. In the context of ambient samples collected during summer and winter in the greater Atlanta area, all laboratory-generated SOA produced similar or higher levels of ROS/RNS and DTT activities. These results suggest that the health effects of SOA are important considerations for understanding the health implications of ambient aerosols.

Elevated particulate matter (PM) concentrations have been associated with cardiopulmonary risks. In this study, alveolar macrophages and ventricular myocytes were exposed to PM extracts from 104 ambient filters collected in multiple rural and urban sites in the greater Atlanta area. PM-induced reactive oxygen/nitrogen species (ROS/RNS) were measured to investigate the effect of chemical composition and determine whether chemical assays are representative of cellular responses. For summer samples, the area under the ROS/RNS doseresponse curve per volume of air (AUCvolume) was significantly correlated with dithiothreitol (DTT) 20 activity, water-soluble organic carbon (WSOC), brown carbon, titanium, and iron, while a 21 relatively flat response was observed for winter samples. EC50 was also correlated with max 22 response for all filters investigated, which suggests that certain PM constituents may be involved in cellular protective pathways. Although few metal correlations were observed, exposure to laboratory-prepared metal solutions induced ROS/RNS production, indicating that a lack of correlation does not necessarily translate to a lack of response. Collectively, these results suggest that complex interactions may occur between PM species. Furthermore, the strong correlation between organic species and ROS/RNS response highlights a need to understand the contribution 28 of organic aerosols, especially photochemically driven secondary organic aerosols (SOA), to PM-29 induced health effects.