In Vitro Analysis of Nanoparticle Effects on the Zymosan Uptake by Phagocytic Cells.

This chapter provides a protocol for analysis of nanoparticle effects on the function of phagocytic cells. The protocol relies on luminol chemiluminescence to detect zymosan uptake. Zymosan is an yeast particle which is typically eliminated by phagocytic cells via the complement receptor pathway. The luminol, co-internalized with zymosan, is processed inside the phagosome to generate a chemiluminescent signal. If a test nanoparticle affects the phagocytic function of the cell, the amount of phagocytosed zymosan and, proportionally, the level of generated chemiluminescent signal change. Comparing the zymosan uptake of untreated cells with that of cells exposed to a nanoparticle provides information about the nanoparticle's effects on the normal phagocytic function. This method has been described previously and is presented herein with several changes. The revised method includes details about nanoparticle concentration selection, updated experimental procedure, and examples of the method performance.

In Vitro Assessment of Nanoparticle Effects on Blood Coagulation.

Blood clotting is a complex process which involves both cellular and biochemical components. The key cellular players in the blood clotting process are thrombocytes or platelets. Other cells, including leukocytes and endothelial cells, contribute to clotting by expressing the so-called pro-coagulant activity (PCA) complex on their surface. The biochemical component of blood clotting is represented by the plasma coagulation cascade, which includes plasma proteins also known as coagulation factors. The coordinated interaction between platelets, leukocytes, endothelial cells, and plasma coagulation factors is necessary for maintaining hemostasis and for preventing excessive bleeding. Undesirable activation of all or some of these components may lead to pathological blood coagulation and life-threatening conditions such as consumptive coagulopathy or disseminated intravascular coagulation (DIC). In contrast, unintended inhibition of the coagulation pathways may lead to hemorrhage. Thrombogenicity is the property of a test material to induce blood coagulation by affecting one or more elements of the clotting process. Anticoagulant activity refers to the property of a test material to inhibit coagulation. The tendency to cause platelet aggregation, perturb plasma coagulation, and induce leukocyte PCA can serve as an in vitro measure of a nanomaterial's likelihood to be pro- or anticoagulant in vivo. This chapter describes three procedures for in vitro analyses of platelet aggregation, plasma coagulation time, and activation of leukocyte PCA. Platelet aggregation and plasma coagulation procedures have been described earlier. The revision here includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, and updated details about assay controls. The chapter is expanded to describe a method for the leukocyte PCA analysis and case studies demonstrating the performance of these in vitro assays.

Analysis of Pro-inflammatory Cytokine and Type II Interferon Induction by Nanoparticles.

Cytokines, chemokines, and interferons are released by the immune cells in response to cellular stress, damage and/or pathogens, and are widely used as biomarkers of inflammation. Certain levels of cytokines are needed to stimulate an immune response in applications such as vaccines or immunotherapy where immune stimulation is desired. However, undesirable elevation of cytokine levels, as may occur in response to a drug or a device, may lead to severe side effects such as systemic inflammatory response syndrome or cytokine storm. Therefore, preclinical evaluation of a test material's propensity to cause cytokine secretion by healthy immune cells is an important parameter for establishing its safety profile. Herein, we describe in vitro methods for analysis of cytokines, chemokines, and type II interferon in whole blood cultures derived from healthy donor volunteers. First, whole blood is incubated with controls and tested nanomaterials for 24 h. Then, culture supernatants are analyzed by ELISA to detect IL-1ß, TNFα, IL-8, and IFNγ. The culture supernatants can also be analyzed for the presence of other biomarkers secreted by the immune cells. Such testing would require additional assays not covered in this chapter and/or optimization of the test procedure to include relevant positive controls and/or cell types.

Autophagy Monitoring Assay II: Imaging Autophagy Induction in LLC-PK1 Cells Using GFP-LC3 Protein Fusion Construct.

Autophagy is a catabolic process involved in the degradation and recycling of long-lived proteins and damaged organelles for maintenance of cellular homeostasis, and it has also been proposed as a type II cell death pathway. The cytoplasmic components targeted for catabolism are enclosed in a double-membrane autophagosome that merges with lysosomes, to form autophagosomes, and are finally degraded by lysosomal enzymes. There is substantial evidence that several nanomaterials can cause autophagy and lysosomal dysfunction, either by prevention of autophagolysosome formation, biopersistence or inhibition of lysosomal enzymes. Such effects have emerged as a potential mechanism of cellular toxicity, which is also associated with various pathological conditions. In this chapter, we describe a method to monitor autophagy by fusion of the modifier protein MAP LC3 with green fluorescent protein (GFP; GFP-LC3). This method enables imaging of autophagosome formation in real time by fluorescence microscopy without perturbing the MAP LC3 protein function and the process of autophagy. With the GFP-LC3 protein fusion construct, a longitudinal study of autophagy can be performed in cells after treatment with nanomaterials.

Anticoagulants Influence the Performance of In Vitro Assays Intended for Characterization of Nanotechnology-Based Formulations.

The preclinical safety assessment of novel nanotechnology-based drug products frequently relies on in vitro assays, especially during the early stages of product development, due to the limited quantities of nanomaterials available for such studies. The majority of immunological tests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an anticoagulant into blood collection tubes. Heparin, ethylene diamine tetraacetic acid (EDTA), and citrate are the most commonly used anticoagulants. Novel anticoagulants such as hirudin are also available but are not broadly used. Despite the notion that certain anticoagulants may influence assay performance, a systematic comparison between traditional and novel anticoagulants in the in vitro assays intended for immunological characterization of nanotechnology-based formulations is currently not available. We compared hirudin-anticoagulated blood with its traditional counterparts in the standardized immunological assay cascade, and found that the type of anticoagulant did not influence the performance of the hemolysis assay. However, hirudin was more optimal for the complement activation and leukocyte proliferation assays, while traditional anticoagulants citrate and heparin were more appropriate for the coagulation and cytokine secretion assays. The results also suggest that traditional immunological controls such as lipopolysaccharide (LPS ) are not reliable for understanding the role of anticoagulant in the assay performance. We observed differences in the test results between hirudin and traditional anticoagulant-prepared blood for nanomaterials at the time when no such effects were seen with traditional controls. It is, therefore, important to recognize the advantages and limitations of each anticoagulant and consider individual nanoparticles on a case-by-case basis.

Nanoparticle Effects on Human Platelets in Vitro: A Comparison between PAMAM and Triazine Dendrimers.

Triazine and PAMAM dendrimers of similar size and number of cationic surface groups were compared for their ability to promote platelet aggregation. Triazine dendrimers (G3, G5 and G7) varied in molecular weight from 8 kDa-130 kDa and in surface groups 16-256. PAMAM dendrimers selected for comparison included G3 (7 kDa, 32 surface groups) and G6 (58 kDa, 256 surface groups). The treatment of human platelet-rich plasma (PRP) with low generation triazine dendrimers (0.01-1 µM) did not show any significant effect in human platelet aggregation in vitro; however, the treatment of PRP with larger generations promotes an effective aggregation. These results are in agreement with studies performed with PAMAM dendrimers, where large generations promote aggregation. Triazine dendrimers promote aggregation less aggressively than PAMAM dendrimers, a factor attributed to differences in cationic charge or the formation of supramolecular assemblies of dendrimers.

Synergistic combination therapy with nanoliposomal C6-ceramide and vinblastine is associated with autophagy dysfunction in hepatocarcinoma and colorectal cancer models.

Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6-ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with C6-ceramide. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagy mechanism.

Dendrimer-induced leukocyte procoagulant activity depends on particle size and surface charge.

AIMS: Thrombogenicity associated with the induction of leukocyte procoagulant activity (PCA) is a common complication in sepsis and cancer. Since nanoparticles are increasingly used for drug delivery, their interaction with coagulation systems is an important part of the safety assessment. The purpose of this study was to investigate the effects of nanoparticle physicochemical properties on leukocyte PCA, and to get insight into the mechanism of PCA induction. MATERIALS & METHODS: A total of 12 formulations of polyamidoamine (PAMAM) dendrimers, varying in size and surface charge, were studied in vitro using recalcification time assay. RESULTS: Irrespective of their size, anionic and neutral dendrimers did not induce leukocyte PCA in vitro. Cationic particles induced PCA in a size- and charge-dependent manner. The mechanism of PCA induction was similar to that of doxorubicin. Cationic dendrimers were also found to exacerbate endotoxin-induced PCA. CONCLUSION: PAMAM dendrimer-induced leukocyte PCA depends on particle size, charge and density of surface groups.

Autophagy monitoring assay: qualitative analysis of MAP LC3-I to II conversion by immunoblot.

Lysosomal dysfunction is a recognized toxic mechanism for xenobiotics, which can result in various pathological states. There is concern that nanoparticles, in particular, may cause lysosomal pathologies, since they are likely to accumulate within lysosomes. Dysregulation of the autophagy-lysosomal degradation pathway is an example of lysosomal dysfunction associated with exposure to some nanomaterials. Here, we present a method to monitor autophagy by measurement the autophagosome marker LC3-II, a phosphatidylethanolamine (PE)-conjugated form of microtubule-associated protein 1 light chain 3-I (MAP LC3-I). As other conditions could potentially result in LC3-II expression, treatment-related changes in expression should be further evaluated by morphological assessment, using techniques such as electron microscopy, to confirm autophagosome involvement.

Psychological morbidity in ALS: the importance of psychological assessment beyond depression alone.

The assessment of psychological morbidity in patients with ALS has centered around depression, hopelessness, and anxiety. The Brief Symptom Inventory (BSI) offers an opportunity to explore psychological morbidity more broadly. We administered this instrument to 111 patients with ALS as part of a larger study of quality of life. Scores of ALS patients on the Global Severity Index and Positive Symptom Distress Index were comparable to the majority of distressed psychiatric outpatients and significantly higher than those of non-patient adults. Among BSI subscales, scores on the Anxiety, Depression, Phobic Anxiety, and Somatization subscales also were not significantly different from distressed adult psychiatric outpatients, and were greater than normal mean scores for a non-patient population sample. Based on these data, ALS patients appear to be significantly more distressed than non-patients in the identified areas, and as distressed as approximately 68% of a distressed psychiatric outpatient sample. In conclusion, a substantial number of individuals with ALS experience psychological distress of various types. Because psychological health impacts lifespan and quality of life in these individuals, broadly-based mental health assessment and treatment should remain an important part of care for patients with ALS. The effects of physical symptoms on responses to questions used to assess psychological distress must be considered.