Lipid-based particle engineering via spray-drying for targeted delivery of antibiotics to the lung.

Pulmonary delivery of antibiotics for the treatment of tuberculosis provides several benefits compared to conventional oral and parenteral administration. API-loaded particles delivered directly to alveolar macrophages, where Mycobacterium tuberculosis resides, can reduce the required dose and decrease the severe side effects of conventional treatment. In this work, lipid-microparticles loaded with rifampicin were engineered via spray-drying to be administered as a carrier-free dry powder for inhalation. Although, it is well-known that spray-drying of lipid-based excipients is strongly limited, a completely lipid-based formulation using diglycerol full ester of behenic acid was produced. The solid state of the lipid, providing high melting temperature, absence of polymorphism and monophasic crystallization, led to high yield of spray-dried particles (83%). Inhalable particles of mass median aerodynamic diameter of 2.36 µm, median geometric size of 2.05 µm, and negative surface (-50.03 mV) were engineered. Such attributes were defined for deep lung deposition and targeted delivery of antibiotics to alveolar macrophages. Superior aerodynamic performance as carrier-free DPI was associated to a high fine particle fraction of 79.5 %. No in vitro cytotoxic effects were found after exposing epithelial cell lines and alveolar macrophages. In vitro uptake of particles into alveolar macrophages indicated the efficiency of their targeted delivery. The use of highly processable and safe lipid-based excipients for particle engineering via spray-drying can extend the availability of materials for functionalized applications for pulmonary delivery.

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.

Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs.

Phospholipidosis (PLD), the intracellular accumulation of phospholipids, is an adaptive response to toxic stimuli and serves as an important parameter in the biological assessment of compounds. Cationic amphiphilic drugs are the main inducers of PLD and may impair the function of alveolar macrophages. In vivo and in vitro models are used for PLD screening but the choice of the cellular model may be important because PLD develops in a cell- and species-specific manner. In this study, a panel of different staining (LysoSensor, Acridine Orange, Nile Red, HCS LipidTOX, LysoID) was evaluated in murine (DMBM-2, J774, RAW264.7) and human (THP-1, monocyte-derived macrophages from peripheral blood) cells to identify the most sensitive and easy to analyze staining method and to detect species-specific differences in the reaction pattern. Amiodarone and chloroquine served as inducers of PLD. High content screening was used to compare number, area, and intensity of the staining. Due to the fast staining protocol and the sensitivity of the detection, LysoID proved to be the most suitable dye of the testing. The lower induction of PLD by chloroquine reported in vivo was also seen in this study. THP-1 macrophages, followed by DMBM-2 cells, produced the most similar reaction pattern to human monocyte-derived macrophages.

In vitro toxicity screening of polyglycerol esters of fatty acids as excipients for pulmonary formulations.

One of the main problems for the development of pulmonary formulations is the low availability of approved excipients. Polyglycerol esters of fatty acids (PGFA) are promising molecules for acting as excipient for formulation development and drug delivery to the lung. However, their biocompatibility in the deep lung has not been studied so far. Main exposed cells include alveolar epithelial cells and alveolar macrophages. Due to the poor water-solubility of PGFAs, the exposure of alveolar macrophages is expected to be much higher than that of epithelial cells. In this study, two PGFAs and their mixture were tested regarding cytotoxicity to epithelial cells and cytotoxicity and functional impairment of macrophages. Cytotoxicity was assessed by dehydrogenase activity and lactate dehydrogenase release. Lysosome function, phospholipid accumulation, phagocytosis, nitric oxide production, and cytokine release were used to evaluate macrophage function. Cytotoxicity was increased with the increased polarity of PGFA molecules. At concentrations above 1 mg/ml accumulation in lysosomes, impairment of phagocytosis, secretion of nitric oxide, and increased release of cytokines were noted. The investigated PGFAs in concentrations up to 1 mg/ml can be considered as uncritical and are promising for advanced pulmonary delivery of high powder doses and drug targeting to alveolar macrophages.

Air-liquid interface culture changes surface properties of A549 cells.

A549 cells are common models in the assessment of respiratory cytotoxicity. To provide physiologically more representative exposure conditions and increase the differentiation state, respiratory cells, for instance Calu-3 bronchial epithelial cells, are cultured at an air-liquid interface (ALI). There are indications that A549 cells also change their phenotype upon culture in ALI. The influence of culture in two variations of transwell cultures compared to conventional culture in plastic wells on the phenotype of A549 cells was studied. Cells were characterized by morphology, proliferation and transepithelial electrical resistance, whole genome transcription analysis, Western blot and immunocytochemical detection of pro-surfactant proteins. Furthermore, lipid staining, surface morphology, cell elasticity, surface tension and reaction to quartz particles were performed. Relatively small changes were noted in the expression of differentiation markers for alveolar cells but A549 cells cultured in ALI showed marked differences in lipid staining and surface morphology, surface tension and cytotoxicity of quartz particles. Data show that changes in physiological reactions of A549 cells in ALI culture were rather caused by change of surface properties than by increased expression of surfactant proteins.

Cytotoxicity screening of emulsifiers for pulmonary application of lipid nanoparticles.

INTRODUCTION: The pulmonary route is a non-invasive administration route that receives growing attention. The challenge for formulation development of orally inhaled formulations is, however, the limited number of approved excipients. Lipid nanoparticles are desired drug delivery systems for inhalation because lipids are biocompatible. However, addition of emulsifiers to stabilize the formulation may cause toxic effects. Alveolar epithelial cells and alveolar macrophages are the main cell types that get in contact with inhaled formulations in the deep lung. The different cell types are supposed to differ in the extent of particle uptake. Kolliphor RH40, Poloxamer 188, and Tween 80 are approved for use in oral formulations and widely used in the academic field for manufacturing of lipid nanoparticles. However, little is known about their pulmonary toxicity. METHODS: Cytotoxicity of Kolliphor RH40, Poloxamer 188, and Tween 80 was studied by integration into solid lipid nanoparticles loaded with itraconazole as model drug. Cytotoxicity of the formulations was assessed in human alveolar epithelial cells and human and murine macrophages and correlated to cell uptake. RESULTS: The tested emulsifiers showed overall low cytotoxicity with less pronounced adverse effects in human cells than in murine macrophages. Cellular uptake of Poloxamer 188 containing lipid nanoparticles was decreased in macrophages, while uptake of lipid nanoparticles with the other emulsifiers was similar in epithelial cells and phagocytes. CONCLUSION: The tested emulsifiers appear suitable for use in pulmonary applications. Due to larger cell size and lower proliferation rate human cells showed lower cytotoxicity than the murine cells. Being human cells, they appear more suitable for the screening of adverse effects in human lungs.

MicroRNA-451a overexpression induces accelerated neuronal differentiation of Ntera2/D1 cells and ablation affects neurogenesis in microRNA-451a-/- mice.

MiR-451a is best known for its role in erythropoiesis and for its tumour suppressor features. Here we show a role for miR-451a in neuronal differentiation through analysis of endogenous and ectopically expressed or silenced miR-451a in Ntera2/D1 cells during neuronal differentiation. Furthermore, we compared neuronal differentiation in the dentate gyrus of hippocampus of miR-451a-/- and wild type mice. MiR-451a overexpression in lentiviral transduced Ntera2/D1 cells was associated with a significant shifting of mRNA expression of the developmental markers Nestin, ßIII Tubulin, NF200, DCX and MAP2 to earlier developmental time points, compared to control vector transduced cells. In line with this, accelerated neuronal network formation in AB.G.miR-451a transduced cells, as well as an increase in neurite outgrowth both in number and length was observed. MiR-451a targets genes MIF, AKT1, CAB39, YWHAZ, RAB14, TSC1, OSR1, POU3F2, TNS4, PSMB8, CXCL16, CDKN2D and IL6R were, moreover, either constantly downregulated or exhibited shifted expression profiles in AB.G.miR-451a transduced cells. Lentiviral knockdown of endogenous miR-451a expression in Ntera2/D1 cells resulted in decelerated differentiation. Endogenous miR-451a expression was upregulated during development in the hippocampus of wildtype mice. In situ hybridization revealed intensively stained single cells in the subgranular zone and the hilus of the dentate gyrus of wild type mice, while genetic ablation of miR-451a was observed to promote an imbalance between proliferation and neuronal differentiation in neurogenic brain regions, suggested by Ki67 and DCX staining. Taken together, these results provide strong support for a role of miR-451a in neuronal maturation processes in vitro and in vivo.

Are in vivo and in vitro assessments of comparative and combined toxicity of the same metallic nanoparticles compatible, or contradictory, or both? A juxtaposition of data obtained in respective experiments with NiO and Mn3O4 nanoparticles.

Comparative and combined damaging effects of NiO and Mn3O4 nanoparticles were estimated on cultures of several established human cell lines. The cytotoxicity indices used were: (a) reduction in cellular dehydrogenase activity, (b) decrease in the ATP-content, (c) for SH-SY5Y cells also decrease in the tyrosine hydroxylase content. The combined cytotoxicity was modeled using the Response Surface Methodology. When assessing the stability of metal oxide nanoparticles (MeO-NPs) in cultural media used by us, we found that the addition of the fetal bovine serum (FBS) to them renders NiO-NPs and, to even greater extent, Mn3O4-NPs exponentially slow soluble while without FBS their dissolution was virtually undetectable. At the same time, sedimentation of these MeO-NPs noticeably slowed down in the presence of the same FBS. We have found dependence of cell damage on concentrations of MeO-NPs and higher cytotoxicity of Mn3O4-NP compared with NiO-NP. Thus, comparative assessment of the NPs unspecific toxicity obtained in our animal experiments was reproduced by the "in vitro" tests. However, with respect to manganese-specific brain damage "in vivo" discovered previously, present experiments on neurons "in vitro" showed only a certain enhancing effect of Mn3O4-NP on the action of NiO-NP, but the role of NiO-NP in the combination prevailed.