Hui Qian

As part of an antimicrobial resistance (AMR) strategy, we have prepared α,ε-N,N′-di-stearoyl lysine-based amide lipids to improve the chemical and biological stabilities of nanoparticles. Those amide lipids incorporated a variety of head groups, including lipid A-binding ligand (polymyxin B nonapeptide, PMBN) for bacterial targeting and sialic acid as an alternative to PEGylation for phagocytosis resistance. The study demonstrated that the PMBN-liposome specifically targeted lipid A-containing Gram-negative Acinetobacter baumannii bacteria, but not Gram-positive Staphylococcus aureus. However, such interaction was interrupted by the adsorption of serum proteins onto liposomes, demonstrating the complexity and challenge of targeted delivery. As expected, slower uptake of sialic acid-liposomes by human leukemia monocytic THP-1 cells was observed, suggesting their resistance to phagocytosis. Additionally, in a mouse model, the sialic acid-containing liposomes showed more favorable biodistribution and longer retention time than the comparable phospholipid-only liposomes. We observed that both sialic acid-incorporated and PEGylated liposomes distributed over the whole mouse bodies and remained for over 48 h. In contrast, the phospholipid-only liposomes rapidly migrated to the liver (5–15 min). In conclusion, although this study did not achieve bacteria-targeted liposome delivery, it provided evidence that the sialic acid-amide lipid can serve as an alternative to PEGylation in future nanomedicine.

The human mast cell-1 (HMC-1) cell line was established from a patient with mast cell leukemia and is the first established continuously growing HMC line which has been widely employed for in vitro studies of human mast cell biology. HMC-1 has been further subcloned into two sublines: HMC-1.1 and HMC-1.2, having one point and two-point mutations in the protooncogene c-kit, respectively. It has been hypothesized that these specific mutations in c-kit are associated with changes in granule biogenesis except for the phenotype and cellular growth rate [ 1, 2]. In our previous study, transmission electron microscopy (TEM) micrography of ultra-thin sections showed that the secretory granules in the two sublines of resting HMC-1 cells have different ultrastructures. And the structural evolution of granules in HMC-1.2 is prominent upon drug treatment with sodium butyrate [ 2]. However, individual 2D images of cell thin sections couldn’t give a full picture of differences in terms of granular distribution, content density or ultrastructures. It is therefore necessary to develop approaches that can better distinguish changes in granule using more robust techniques.

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. They have classically been prepared using a thin-film hydration method with an average particle size of 100–200 nm. In this study, we developed methods to generate SLA archaeosomes at different sizes, i.e., 30 nm and 100 nm, via microfluidic mixing technology and evaluated their physicochemical characteristics, as well as adjuvant activity and in vivo biodistribution in mice. Archaeosomes, prepared using thin-film and microfluidic mixing techniques, had similar nanostructures and physicochemical characteristics, with both appearing stable during the course of this study when stored at 4 °C or 37 °C. They also demonstrated similar adjuvant activity when admixed with ovalbumin antigen and used to immunize mice, generating equivalent antigen-specific immune responses. Archaeosomes, labeled with CellVue^TM NIR815, had an equivalent biodistribution with both sizes, namely the highest signal at the injection site at 24 h post injection, followed by liver, spleen and inguinal lymph node. The presence of SLA archaeosomes of either size helped to retain OVA antigen (OVA-Cy5.5) longer at the injection site than unadjuvanted OVA. Overall, archaeosomes of two sizes (30 nm and 100 nm) prepared using microfluidic mixing maintained similar physicochemical properties, adjuvant activity and biodistribution of antigen, in comparison to those compared by the conventional thin film hydration method. This suggests that microfluidics based approaches could be applied to generate consistently sized archaeosomes for use as a vaccine adjuvant.

Size distributions of cellulose nanocrystals (CNCs), extracted from softwood pulp via strong sulfuric acid hydrolysis, exhibit large variability when analyzed from transmission electron microscopy (TEM) images. In this article, the causes of this variability are studied and discussed. In order to obtain results comparable with those reported, a reference material of CNCs (CNCD-1) was used to evaluate size distribution. CNC TEM specimens were prepared as-stained and dried with a rapid-flushing staining method or hydrated and embedded in vitreous ice with the plunge-freezing method. Several sets of bright-field TEM (BF-TEM), annular dark-field scanning TEM (ADF-STEM) and cryogenic-TEM (cryo-TEM) images were acquired for size distribution analysis to study the contributing factors. The rapid-flushing staining method was found to be the most effective for contrast enhancement of CNCs, not only revealing the helical structure of single CNCs but also resolving the laterally jointed CNCs. During TEM specimen preparation, CNCs were fractionated on TEM grids driven by the coffee-ring effect, as observed from contrast variation of CNCs with a stain-depth gradient. From the edge to the center of the TEM grids, the width of CNCs increases, while the aspect ratio (length to width) decreases. This fractionated dispersion of CNCs suggests that images taken near the center of a droplet would give a larger mean width. In addition to particle fractionation driven by the coffee-ring effect, the arrangement and orientation of CNC particles on the substrate significantly affect the size measurement when CNC aggregation cannot be resolved in images. The coexistence of asymmetric cross-section CNC particles introduces a large variation in size measurement, as TEM images of CNCs are mixed projections of the width and height of particles. As a demonstration of how this contributes to inflated size measurement, twisted CNC particles, rectangular cross-section particles and end-to-end jointed CNCs were revealed in reconstructed three-dimensional (3D) micrographs by electron tomography (ET).

Biodegradable polymeric nanoparticles (NPs) such as poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) have been used as drug delivery systems for natural and synthetic compounds and are designed to control the loading and release of biodegradable materials to target cells, tissues, and organs. Eremophilane-type sesquiterpenes have anti-inflammatory properties but are lipophilic, cytotoxic, and not biocompatible with many cells. To determine whether biodegradable PLGA/PVA could improve the biocompatibility of sesquiterpenes, sesquiterpene-loaded NPs were synthesized and their effects on human mast cells (LAD2), the major effector cells of allergic inflammation, were determined. NPs composed of PLGA/PVA and two types of sesquiterpenes (fukinone, PLGA/PVA-21 and 10βH-8α,12-epidioxyeremophil-7(11)-en-8β-ol, PLGA/PVA-22) were produced using a microfluidic synthesis method. The NPs’ size distribution and morphology were evaluated by dynamic light scattering and cryogenic transmission electron microscopy (TEM). PLGA/PVA-21 and PLGA/PVA-22 were 60 to 70 nm and were readily internalized by LAD2 as shown by flow cytometry, fluorescence microscopy, and TEM. While unencapsulated sesquiterpenes decreased LAD2 cell viability by 20%, PLGA/PVA-21 and PLGA/PVA-22 did not alter LAD2 viability, showing that encapsulation improved the biocompatibility of the sesquiterpenes. PLGA/PVA-21 and PLGA/PVA-22 decreased the expression of genes encoding the subunits of the high affinity immunoglobulin E receptor (FcεR1α, FcεR1β, FcεR1γ) and the stem cell factor receptor (Kit,), suggesting that hybrid NPs could alter mast cell responses to antigens and shift their maturation. Similarly, PLGA/PVA-21 and PLGA/PVA-22 inhibited tryptase expression but had no effect on chymase expression, thereby promoting a shift to the tryptase-positive phenotype (MC_T). Lastly, PLGA/PVA-21 and PLGA/PVA-22 inhibited mast cell degranulation when the LAD2 cells were activated by IgE crosslinking and FcεRI. Overall, our results suggest that PLGA/PVA-21 and PLGA/PVA-22 alter human mast cell phenotype and activation without modifying viability, making them a more biocompatible approach than treating cells with sesquiterpenes alone.

Human serum albumin forms a protein corona that stabilizes silver nanoparticles in water better than commercial polymers.