Sarabjeet Singh Suri

Nanotechnology-based drug delivery systems

Nanoparticles hold tremendous potential as an effective drug delivery system. In this review we discussed recent developments in nanotechnology for drug delivery. To overcome the problems of gene and drug delivery, nanotechnology has gained interest in recent years. Nanosystems with different compositions and biological properties have been extensively investigated for drug and gene delivery applications. To achieve efficient drug delivery it is important to understand the interactions of nanomaterials with the biological environment, targeting cell-surface receptors, drug release, multiple drug administration, stability of therapeutic agents and molecular mechanisms of cell signalling involved in pathobiology of the disease under consideration. Several anti-cancer drugs including paclitaxel, doxorubicin, 5-fluorouracil and dexamethasone have been successfully formulated using nanomaterials. Quantom dots, chitosan, Polylactic/glycolic acid (PLGA) and PLGA-based nanoparticles have also been used for in vitro RNAi delivery. Brain cancer is one of the most difficult malignancies to detect and treat mainly because of the difficulty in getting imaging and therapeutic agents past the blood-brain barrier and into the brain. Anti-cancer drugs such as loperamide and doxorubicin bound to nanomaterials have been shown to cross the intact blood-brain barrier and released at therapeutic concentrations in the brain. The use of nanomaterials including peptide-based nanotubes to target the vascular endothelial growth factor (VEGF) receptor and cell adhesion molecules like integrins, cadherins and selectins, is a new approach to control disease progression.

Expression of Toll‐Like Receptor 9 in Horse Lungs

Toll‐like receptor 9 (TLR9) has been found to be the main receptor to respond to bacterial DNA in a wide variety of species. Recent work has shown that TLR9 is expressed in a diverse set of cells within the lung. However, much of this data has been centered on human and mouse cell culture lines or primary cultures and very little is known of TLR9 expression in intact lung, especially that of the horse. Here we show that TLR9 is expressed in the lungs of horses in a wide variety of cells. In particular, we note expression in pulmonary intravascular macrophages (PIMs), alveolar macrophages, bronchial epithelial cells, and type‐II cells amongst others. Immunogold electron microscopy localized TLR9 in nuclei, cytoplasm, and plasma membrane of various lung cells. The data also show that E. coli lipopolysaccharide significantly increased expression of TLR9 mRNA in lungs and the number of cells in the lung septa that were positive for TLR9 protein. Protein expression was seen in airway epithelium, vascular endothelium, and inflammatory cells in blood vessels. Intravenous administration of gadolinium chloride, which depletes macrophages, before the lipopolysaccharide treatment significantly inhibited the LPS‐induced increase in TLR9 mRNA in the lungs of the horses. We conclude that TLR9 is expressed in lung cells including PIMs and that the lipopolysaccharide treatment increases TLR9 mRNA expression. The increase in TLR9 mRNA is eliminated by depletion of PIMs, implicating these cells as a major source of TLR9 in the equine lung. Anat Rec, 292:1068–1077, 2009.

Correct Targeting of Plant ARF GTPases Relies on Distinct Protein Domains

Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N‐terminal myristoyl group but to different target membranes. Specifically, we investigated how ARF1 is targeted to the Golgi and post‐Golgi structures, whereas ARFB accumulates at the plasma membrane. While the subcellular localization of ARFB appears to depend on multiple domains including the C‐terminal half of the GTPase, the correct targeting of ARF1 is dependent on two domains: an N‐terminal ARF1 domain that is necessary for the targeting of the GTPase to membranes and a core domain carrying a conserved MxxE motif that influences the relative distribution of ARF1 between the Golgi and post‐Golgi compartments. We also established that the N‐terminal ARF1 domain alone was insufficient to maintain an interaction with membranes and that correct targeting is a protein‐specific property that depends on the status of the GTP switch. Finally, an ARF1–ARFB chimera containing only the first 18 amino acids from ARF1 was shown to compete with ARF1 membrane binding loci. Although this chimera exhibited GTPase activity in vitro, it was unable to recruit coatomer, a known ARF1 effector, onto Golgi membranes. Our results suggest that the targeting of ARF GTPases to the correct membranes may not only depend on interactions with effectors but also relies on distinct protein domains and further binding partners on the Golgi surface.

Role of pulmonary intravascular macrophages in endotoxin-induced lung inflammation and mortality in a rat model

BackgroundBile-duct ligated (BDL) rats recruit pulmonary intravascular macrophages (PIMs) and are highly susceptible to endotoxin-induced mortality. The mechanisms of this enhanced susceptibility and mortality in BDL rats, which are used as a model of hepato-pulmonary syndrome, remain unknown. We tested a hypothesis that recruited PIMs promote endotoxin-induced mortality in a rat model.MethodsRats were subjected to BDL to induce PIM recruitment followed by treatment with gadolinium chloride (GC) to deplete PIMs. Normal and BDL rats were treated intravenously with E. coli lipopolysaccharide (LPS) with or without GC pre-treatment followed by collection and analyses of lungs for histopathology, electron microscopy and cytokine quantification.ResultsBDL rats recruited PIMs without any change in the expression of IL-1β, TNF-α and IL-10. GC caused reduction in PIMs at 48 hours post-treatment (P < 0.05). BDL rats treated intravenously with E. coli LPS died within 3 hours of the challenge while the normal LPS-treated rats were euthanized at 6 hours after the LPS treatment. GC treatment of rats 6 hours or 48 hours before LPS challenge resulted in 80% (1/5) and 100% (0/5) survival, respectively, at 6 hours post-LPS treatment. Lungs from BDL+LPS rats showed large areas of perivascular hemorrhages compared to those pre-treated with GC. Concentrations of IL-1β, TNF-α and IL-10 were increased in lungs of BDL+LPS rats compared to BDL rats treated with GC 48 hours but not 6 hours before LPS (P < 0.05).ConclusionWe conclude that PIMs increase susceptibility for LPS-induced lung injury and mortality in this model, which is blocked by a reduction in their numbers or their inactivation.

The role of RGD-tagged helical rosette nanotubes in the induction of inflammation and apoptosis in human lung adenocarcinoma cells through the P38 MAPK pathway.

The rosette nanotubes (RNTs) are a class of biologically inspired, self-assembling, metal-free, hydrophilic nanotubes, which hold tremendous potential as targeted drug delivery vehicles. We investigated the cell signaling events caused by lysine-functionalized RNTs (K-RNT) co-assembled with Arg-Gly-Asp-Ser-Lys-functionalized RNTs (RGDSK-RNT) for induction of inflammation and apoptosis in human adenocarcinoma (Calu-3) cells. When co-assembled in a ratio of 1:10 microM these composite RNTs (referred to as RGDSK/K-RNTs) rapidly induced phosphorylation of P38 mitogen-activated protein kinase (MAPK) within 2 min. Higher concentrations of RGDSK/K-RNTs (>10:100 microM) resulted in a P38 MAPK-dependent increase in secretion of TNF-alpha. RGDSK/K-RNTs (1:10-40:400 microM) also caused a concentration- and P38 MAPK-dependent increase in caspase-3 activity and DNA fragmentation in Calu-3 cells at 18 h of exposure. Over-expression of pro-apoptotic genes including caspase-3, BAK1, CIDEB, TP53BP2, FAS, TNF and FASLG supported pro-apoptotic behaviors of these RNTs. We conclude that RGDSK/K-RNTs induce phosphorylation of P38 MAPK, which regulate secretion of TNF-alpha, activation of caspase-3 and apoptosis in Calu-3 cells. These results suggest that the RNTs could be used as a drug to induce apoptosis in cancer cells or as a versatile platform to deliver a variety of biologically active molecules for cancer therapy.

Low Inflammatory Activation by Self‐Assembling Rosette Nanotubes in Human Calu‐3 Pulmonary Epithelial Cells

Rosette nanotubes (RNT) are a new class of metal-free organic nanotubes synthesized through self-assembly. Because of the wide range of potential biomedical applications associated with these materials, it is necessary to evaluate their potential in vitro toxicity. Here the cytotoxicity of a lysine-functionalized nanotube (RNT-K) in a human Calu-3 pulmonary epithelial cell line is investigated. The cells were treated with media only (control), lysine (50 mg mL(-1)), RNT-K (1, 5, and 50 microg mL(-1)), Min-U-Sil quartz microparticles (QM; 80 microg mL(-1)), and lipopolysaccharide (LPS; 1 microg mL(-1)). The supernatants were analyzed at 1, 6, and 24 h after treatment for the expression of three proinflammatory mediators: IL-8, TNF-alpha and EMAP-II. Cellular viability determined with the Trypan blue assay is significantly reduced in the QM and high-dose RNT-treated groups. TNF-alpha and EMAP-II are undetectable by enzyme-linked-immunosorbent assay (ELISA) in the supernatant of all groups. Although IL-8 concentrations do not differ between treatments, its concentrations increase with time within each of the groups. Quantitative reverse-transcriptase polymerase chain reaction (qRTPCR) of IL-8 mRNA shows increased expression in the high-dose RNT-treated groups at both 1 and 6 h, while an adhesion molecule, ICAM-1 mRNA, shows the greatest increase at 6 h in the QM-treated group. In summary, RNT-K neither reduces cell viability at moderate doses nor does it induce a time-dependent inflammatory response in pulmonary epithelial cells in vitro.

Angiostatin inhibits acute lung injury in a mouse model

Acute lung injury is marked by profound influx of activated neutrophils, which have delayed apoptosis, along with fluid accumulation that impairs lung function and causes high mortality. Inflammatory and antimicrobial molecules, such as reactive oxygen species from activated neutrophils with prolonged lifespan, cause tissue damage and contribute to lung dysfunction. Angiostatin, an endogenous antiangiogenic molecule, is expressed in the lavage fluid of patients with acute respiratory distress syndrome and modifies neutrophil infiltration in a mouse model of peritonitis. Our aim was to investigate the therapeutic role of angiostatin in acute lung injury. We analyzed bronchoalveolar lavage and lung tissues from C57BL/6 mouse model of Escherichia coli LPS-induced acute lung injury to assess the effects of angiostatin treatment. Subcutaneous angiostatin administered at 5 h after LPS treatment reduces histological signs of inflammation, protein accumulation, lung Gr1+ neutrophils, myeloperoxidase activity, and expression of phosphorylated p38 MAPK in lung tissues and peripheral blood neutrophils, while increasing the number of apoptotic cells in the lungs without affecting the levels of macrophage inflammatory protein-1 α, IL-1β, keratinocyte chemoattractant, and monocyte chemoattractant protein-1 in lavage and lung homogenates at 9 and 24 h after LPS treatment. In contrast, angiostatin administered intravenously 5 h after LPS treatment did not reduce histological sign of inflammation, BAL cell recruitment, and protein concentration at 9 h of LPS treatment. We conclude that angiostatin administered subcutaneously after LPS challenge inhibits acute lung inflammation up to 24 h after LPS treatment.

Macrophage inflammatory response to self-assembling rosette nanotubes.

Rosette nanotubes (RNTs) are a new class of nanomaterials with significant therapeutic potential. However, societal concerns related to the potential adverse health effects of engineered nanomaterials drew attention towards the investigation of their interaction with the human U937 macrophage cell line. The cells are treated with medium only (control), lysine (50 microg mL(-1)), lysine-functionalized RNTs (RNT-K; 1, 5, and 50 microg mL(-1)), Min-U-Sil quartz microparticles (80 microg mL(-1)), or lipopolysaccharide (1 microg mL(-1)). The supernatant and cells are assayed for cell viability, cytokine protein, and mRNA expression at 1, 6, and 24 h post-treatment. The results indicate that RNT-K activate transcription of proinflammatory genes (interleukin-8 and tumor necrosis factor-alpha (TNF-alpha)) within 1 h, but this effect is not accompanied by protein secretion into the supernatant. The effect of the length of RNTs on human U937 macrophage viability is also investigated. Although both short and long RNT-K exhibit time-dependent effects on TNF-alpha transcription, only the short RNT-K (5 microg mL(-1)) increase TNF-alpha concentration at 6 h relative to the long RNT-K. Moreover, RNT-K (1 and 5 microg mL(-1)) have no effect on cell viability by 24 h. These data indicate that RNT-K do not induce a robust inflammatory response or cytotoxicity in the U937 human macrophage cell line, and therefore could be used for biomedical applications.

RGD-tagged helical rosette nanotubes aggravate acute lipopolysaccharide-induced lung inflammation.

Rosette nanotubes (RNT) are a novel class of self-assembled biocompatible nanotubes that offer a built-in strategy for engineering structure and function through covalent tagging of synthetic self-assembling modules (G∧C motif). In this report, the G∧C motif was tagged with peptide Arg-Gly-Asp-Ser-Lys (RGDSK-G∧C) and amino acid Lys (K-G∧C) which, upon co-assembly, generate RNTs featuring RGDSK and K on their surface in predefined molar ratios. These hybrid RNTs, referred to as Kx/RGDSKy-RNT, where x and y refer to the molar ratios of K-G∧C and RGDSK–G∧C, were designed to target neutrophil integrins. A mouse model was used to investigate the effects of intravenous Kx/RGDSKy-RNT on acute lipopolysaccharide (LPS)-induced lung inflammation. Healthy male C57BL/6 mice were treated intranasally with Escherichia coli LPS 80 μg and/or intravenously with K90/RGDSK10-RNT. Here we provide the first evidence that intravenous administration of K90/RGDSK10-RNT aggravates the proinflammatory effect of LPS in the mouse. LPS and K90/RGDSK10-RNT treatment groups showed significantly increased infiltration of polymorphonuclear cells in bronchoalveolar lavage fluid at all time points compared with the saline control. The combined effect of LPS and K90/RGDSK10-RNT was more pronounced than LPS alone, as shown by a significant increase in the expression of interleukin-1β, MCP-1, MIP-1, and KC-1 in the bronchoalveolar lavage fluid and myeloperoxidase activity in the lung tissues. We conclude that K90/RGDSK10-RNT promotes acute lung inflammation, and when used along with LPS, leads to exaggerated immune response in the lung.

Comparison of the response to experimentally induced short-term inflammation in the temporomandibular and metacarpophalangeal joints of horses

OBJECTIVE To investigate the relationship between inflammatory responses of the temporomandibular joint (TMJ) and the metacarpophalangeal (MCP) joint in clinically normal horses. ANIMALS 7 mature horses. PROCEDURES In each horse, 1 TMJ and 1 MCP joint were injected with lipopolysaccharide (LPS; 0.0025 μg). The contralateral TMJ and MCP joint were injected with saline (0.9% NaCl) solution. Synovial fluid samples were collected from all 4 joints over 24 hours after injection. Concentrations of interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and total protein were measured via immunoassay. Horses were assessed for clinical signs of joint inflammation at each time point. RESULTS Concentrations of interleukin-6 were not significantly different between LPS-injected MCP joints and TMJs at any time point. Transforming growth factor-β concentrations were significantly increased in MCP joints, compared with concentrations in TMJs, at 12 and 24 hours after injection. Tumor necrosis factor-α concentrations were significantly higher in LPS-injected TMJs than in LPS-injected MCP joints at 1 and 6 hours after injection. Total protein concentration did not differ significantly between LPS-injected MCP joints and TMJs. Injection of LPS induced clinical inflammation at all time points; additionally, 2 MCP joints (but no TMJs) had an inflammatory response to injection of saline solution. CONCLUSIONS AND CLINICAL RELEVANCE The inflammatory response to LPS appeared to be attenuated more quickly in TMJs than in MCP joints of horses. The difference in response suggested that a lack of clinical osteoarthritis in the TMJ of horses could be attributable to a difference in cytokine response.