Cationic inhalable particles for enhanced drug delivery to M. tuberculosis infected macrophages

Inhalable microparticle-based drug delivery platforms are being investigated extensively for Tuberculosis (TB) treatment as they offer efficient deposition in lungs and improved pharmacokinetics of the encapsulated cargo. However, the effect of physical parameters of microcarriers on interaction with Mycobacterium tuberculosis (Mtb) infected mammalian cells is underexplored. In this study, we report that Mtb-infected macrophages are highly phagocytic and microparticle surface charge plays a major role in particle internalization by infected cells. Microparticles of different sizes (0.5 - 2 μm) were internalized in large numbers by Mtb-infected THP-1 macrophages and murine primary Bone Marrow Derived Macrophages in vitro. Drastic improvement in particle uptake was observed with cationic particles in vitro and in mice lungs. Rapid uptake of rifampicin-loaded cationic microparticles allowed high intracellular accumulation of the drug and lead to enhanced anti-bacterial function when compared to non-modified rifampicin-loaded microparticles. Cytocompatibility assay and histological analysis in vivo confirmed that the formulations were safe and did not elicit any adverse reaction. Additionally, pulmonary delivery of cationic particles in mice resulted in two-fold higher uptake in resident alveolar macrophages compared to non-modified particles. This study provides a framework for future design of drug carriers to improve delivery of anti-TB drugs inside Mtb-infected cells.

771 Novel lipid nanoparticle vaccine platform for efficient delivery of high- and low-affinity epitopes

BackgroundTherapeutic cancer vaccines represent an intriguing approach to cancer immunotherapy and they have been widely explored for the last decade. As opposed to standard modalities, such as surgery and chemotherapy, an effective vaccine-based immune response may provide protection against metastatic disease. Peptide based vaccines can elicit a highly targeted immune response and include a simple, fast and cost-effective production due to recent developments in solid phase peptide synthesis. Recent development within the field of COVID-19 vaccines has highlighted the use of lipid nanoparticles as an effective drug delivery system for vaccination. Incorporation of peptide antigens into engineered micro- and nanoparticles enables induction of a potent T cell response, partly attributed to prolonged and improved antigen presentation by dendritic cells after particle internalization. Peptide-based vaccines are often based on delivery of high-affinity T cell model epitopes. However, the therapeutic relevance of vaccination with low-affinity epitopes is gaining increasing support following the observation that high-affinity epitopes can promote T cell exhaustion resulting from excessive T cell receptor stimulation. Here, we characterize and evaluate a novel lipid nanoparticle (LNP) vaccine platform that is suited for delivery of both high- and low-affinity epitopes in the setting of therapeutic cancer vaccination.MethodsLNPs were formulated to carry high- or low-affinity peptide epitopes from Ovalbumin (OVA) in conjunction with the TLR7 agonist 1V270. The peptides were anchored to the surface of the LNPs via a reducible DSPE-PEG2000 linker system. The therapeutic vaccine platform was evaluated in vivo both as a monotherapy and in combination with adoptive transfer of OT-I T cells in the syngeneic B16-OVA murine melanoma model.ResultsThe LNP vaccine promotes efficient antigen-release and ensures high, continuous antigen-presentation by antigen-presenting cells. While the LNPs can be administered via multiple routes, intratumoral vaccination favors enhanced particle uptake in dendritic cells in the tumor. Formulated with either high- or low-affinity epitopes, intratumorally delivered vaccine particles promote superior tumor-infiltration of adoptively transferred T cells, which translates into potent anti-tumor efficacy in vivo. Finally, we show that vaccination with both CD8+ and CD4+ epitopes can delay tumor growth and prolong survival in an antigen-dependent manner.ConclusionsThis study presents a versatile and multi-purpose LNP vaccine platform that ensures effective delivery of high- and low-affinity epitopes. Intratumoral administration promotes vaccine particle uptake by intratumoral dendritic cells, which is followed by T cell infiltration and anti-tumor efficacy in vivo.Ethics ApprovalAll animal procedures were approved by the Danish National Animal Experiments Inspectorate.

Initial Biological Assessment of Upconversion Nanohybrids

Nanoparticles for medical use should be non-cytotoxic and free of bacterial contamination. Upconversion nanoparticles (UCNPs) coated with cucurbit[7]uril (CB[7]) made by combining UCNPs free of oleic acid, here termed bare UCNPs (UCn), and CB[7], i.e., UC@CB[7] nanohybrids, could be used as photoactive inorganic-organic hybrid scaffolds for biological applications. UCNPs, in general, are not considered to be highly toxic materials, but the release of fluorides and lanthanides upon their dissolution may cause cytotoxicity. To identify potential adverse effects of the nanoparticles, dehydrogenase activity of endothelial cells, exposed to various concentrations of the UCNPs, was determined. Data were verified by measuring lactate dehydrogenase release as the indicator of loss of plasma membrane integrity, which indicates necrotic cell death. This assay, in combination with calcein AM/Ethidium homodimer-1 staining, identified induction of apoptosis as main mode of cell death for both particles. The data showed that the UCNPs are not cytotoxic to endothelial cells, and the samples did not contain endotoxin contamination. Higher cytotoxicity, however, was seen in HeLa and RAW 264.7 cells. This may be explained by differences in lysosome content and particle uptake rate. Internalization of UCn and UC@CB[7] nanohybrids by cells was demonstrated by NIR laser scanning microscopy.

Particle Uptake Driven Phagocytosis in Macrophages and Neutrophils Enhances Bacterial Clearance

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

The Roles of Phospholipase A2 in Phagocytes

Phagocytic cells, such as macrophages, neutrophils, and dendritic cells, ingest particles larger than about 0.5 μM and thereby clear microbial pathogens and malignant cells from the body. These phagocytic cargoes are proteolytically degraded within the lumen of phagosomes, and peptides derived from them are presented on Major Histocompatibility Complexes (MHC) for the activation of T cells. Mammalian PLA2 isozymes belong to a large family of enzymes that cleave phospholipids at the second position of the glycerol backbone, releasing a free fatty acid and a lysolipid moiety. In human macrophages, at least 15 different PLA2 forms are expressed, and expression of many of these is dependent on pathogenic stimulation. Intriguing questions are why so many PLA2 forms are expressed in macrophages, and what are the functional consequences of their altered gene expression after encountering pathogenic stimuli. In this review, we discuss the evidence of the differential roles of different forms of PLA2 in phagocytic immune cells. These roles include: lipid signaling for immune cell activation, initial phagocytic particle uptake, microbial action for the killing and degradation of ingested microbes, and the repair of membranes induced by oxygen radicals. We also discuss the roles of PLA2 in the subsequent digestion of ingested phagocytic cargoes for antigen presentation to T cells.

Adsorption of bio-organic eco-corona molecules reduces the toxic response to metallic nanoparticles in Daphnia magna

As the use of engineered nanomaterials increases, so does the risk of them spreading to natural ecosystems. Hitherto, knowledge regarding the toxic properties of nanoparticles (NP’s) and their potential interactions with natural bio-organic molecules adsorbed to them, and thereby forming surface coronas, is limited. However, we show here that the toxic effect of NPs of tungsten carbide cobalt (WC–Co) and cobalt (Co) on the crustacean Daphnia magna is postponed in the presence of natural biological degradation products (eco-corona biomolecules). For Daphnia exposed to WC–Co NPs the survival time increased with 20–25% and for Co NPs with 30–47% after mixing the particles with a solution of eco-corona biomolecules before exposure. This suggests that an eco-corona, composed of biomolecules always present in natural ecosystems, reduces the toxic potency of both studied NPs. Further, the eco-coronas did not affect the particle uptake, suggesting that the reduction in toxicity was related to the particle-organism interaction after eco-corona formation. In a broader context, this implies that although the increasing use and production of NPs may constitute a novel, global environmental threat, the acute toxicity and long-term effects of some NPs will, at least under certain conditions, be reduced as they enter natural ecosystems.

Activated Fc receptor supramolecular complex

Phagocytosis is a part of immune response. IgG opsonized particles of greater than 1 um are recognized by Fcy receptors on the surface of professional phagocytes such as macrophages and neutrphils. IgGs are part of the immune system and is a cognate ligand of the Fc receptor. Live Cell Affinity Receptor chromatography (LARC) was used to capture an activated Fcy receptor supramolecular complex from the surface of live human neutrophils, by allowing IgG opsonized microbeads to bind to the cell surface. The cells were burst in PBS, collected and digested along side with controls. Isolated FcyR complex was analysed by LC-MS/MS. Fc and control experiment lists of SEQUEST correlated proteins were screened for a total cumulative score of at least 2400 and a minimum of three different peptides. This served as the basis of protein involvement in the FcyR mediated phagocytosis, which were then searched with iHOP for their interaction partners. Gathered interactions were then exported and Cytoscape, Osprey and String algorithms were used to generate network of interacting proteins. PAKs2-4 and PAK6 were detected with LARC. PAK2 and PAK4 were predicted by algorithms to have a central role in particle uptake. From Western Blotting, endogenous PAKs2-4 and PAK6 were detected in murine macrophages. Immunofluorescent staining was then used to verify the presence of these proteins in the forming phagosome and showed localization of PAKs to the phagosome. The same effect was observed with transfection of GFP constructs of PAKs. Upon transfection with dominant negative PAKs reduction in phagocytosis was observed.

Activated Fc receptor supramolecular complex

Phagocytosis is a part of immune response. IgG opsonized particles of greater than 1 um are recognized by Fcy receptors on the surface of professional phagocytes such as macrophages and neutrphils. IgGs are part of the immune system and is a cognate ligand of the Fc receptor. Live Cell Affinity Receptor chromatography (LARC) was used to capture an activated Fcy receptor supramolecular complex from the surface of live human neutrophils, by allowing IgG opsonized microbeads to bind to the cell surface. The cells were burst in PBS, collected and digested along side with controls. Isolated FcyR complex was analysed by LC-MS/MS. Fc and control experiment lists of SEQUEST correlated proteins were screened for a total cumulative score of at least 2400 and a minimum of three different peptides. This served as the basis of protein involvement in the FcyR mediated phagocytosis, which were then searched with iHOP for their interaction partners. Gathered interactions were then exported and Cytoscape, Osprey and String algorithms were used to generate network of interacting proteins. PAKs2-4 and PAK6 were detected with LARC. PAK2 and PAK4 were predicted by algorithms to have a central role in particle uptake. From Western Blotting, endogenous PAKs2-4 and PAK6 were detected in murine macrophages. Immunofluorescent staining was then used to verify the presence of these proteins in the forming phagosome and showed localization of PAKs to the phagosome. The same effect was observed with transfection of GFP constructs of PAKs. Upon transfection with dominant negative PAKs reduction in phagocytosis was observed.

Screening for Effects of Inhaled Nanoparticles in Cell Culture Models for Prolonged Exposure

Respiratory exposure of humans to environmental and therapeutic nanoparticles repeatedly occurs at relatively low concentrations. To identify adverse effects of particle accumulation under realistic conditions, monocultures of Calu-3 and A549 cells and co-cultures of A549 and THP-1 macrophages in the air–liquid interphase culture were exposed repeatedly to 2 µg/cm2 20 nm and 200 nm polystyrene particles with different functionalization. Particle accumulation, transepithelial electrical resistance, dextran (3–70 kDa) uptake and proinflammatory cytokine secretion were determined over 28 days. Calu-3 cells showed constant particle uptake without any change in barrier function and cytokine release. A549 cells preferentially ingested amino- and not-functionalized particles combined with decreased endocytosis. Cytokine release was transiently increased upon exposure to all particles. Carboxyl-functionalized demonstrated higher uptake and higher cytokine release than the other particles in the A549/THP-1 co-cultures. The evaluated respiratory cells and co-cultures ingested different amounts and types of particles and caused small (partly transient) effects. The data suggest that the healthy cells can adapt to low doses of non-cytotoxic particles.