Development of PEGylated PLGA nanoparticle for controlled and sustained drug delivery in cystic fibrosis

<p>Abstract</p> <p>Background</p> <p>The mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in CF. The most common mutation, ΔF508-CFTR, is a temperature-sensitive, trafficking mutant with reduced chloride transport and exaggerated immune response. The ΔF508-CFTR is misfolded, ubiquitinated, and prematurely degraded by proteasome mediated- degradation. We recently demonstrated that selective inhibition of proteasomal pathway by the FDA approved drug PS-341 (pyrazylcarbonyl-Phe-Leuboronate, a.k.a. Velcade or bortezomib) ameliorates the inflammatory pathophysiology of CF cells. This proteasomal drug is an extremely potent, stable, reversible and selective inhibitor of chymotryptic threonine protease-activity. The apprehension in considering the proteasome as a therapeutic target is that proteasome inhibitors may affect proteostasis and consecutive processes. The affect on multiple processes can be mitigated by nanoparticle mediated PS-341 lung-delivery resulting in favorable outcome observed in this study.</p> <p>Results</p> <p>To overcome this challenge, we developed a nano-based approach that uses drug loaded biodegradable nanoparticle (PLGA-PEG^PS-341) to provide controlled and sustained drug delivery. The <it>in vitro </it>release kinetics of drug from nanoparticle was quantified by proteasomal activity assay from days 1-7 that showed slow drug release from day 2-7 with maximum inhibition at day 7. For <it>in vivo </it>release kinetics and biodistribution, these drug-loaded nanoparticles were fluorescently labeled, and administered to C57BL6 mice by intranasal route. Whole-body optical imaging of the treated live animals demonstrates efficient delivery of particles to murine lungs, 24 hrs post treatment, followed by biodegradation and release over time, day 1-11. The efficacy of drug release in CF mice (<it>Cftr^-/-</it>) lungs was determined by quantifying the changes in proteasomal activity (~2 fold decrease) and ability to rescue the <it>Pseudomonas aeruginosa </it>LPS (<it>Pa</it>-LPS) induced inflammation, which demonstrates the rescue of CF lung disease in murine model.</p> <p>Conclusion</p> <p>We have developed a novel drug delivery system to provide sustained delivery of CF "correctors" and "anti-inflammatories" to the lungs. Moreover, we demonstrate here the therapeutic efficacy of nano-based proteostasis-modulator to rescue <it>Pa-LPS </it>induced CF lung disease.</p

Critical Role of VCP/p97 in the Pathogenesis and Progression of Non-Small Cell Lung Carcinoma

Valosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression.Our results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05).Thus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes

Early-Age-Related Changes in Proteostasis Augment Immunopathogenesis of Sepsis and Acute Lung Injury

adult) mechanisms that augment immunopathogenesis of sepsis and acute lung injury. model to standardize the efficacy of salubrinal (inhibitor of eIF2α de-phosphorylation) in controlling the accumulation of ubiquitinated proteins and the NFκB levels. Finally, we evaluated the therapeutic efficacy of salubrinal to correct proteostasis-imbalance in the adult mice based on its ability to control CLP induced IL-6 secretion or recruitment of pro-inflammatory cells.Our data demonstrate the critical role of early-age-related proteostasis-imbalance as a novel mechanism that augments the NFκB mediated inflammation in sepsis and ALI. Moreover, our data suggest the therapeutic efficacy of salubrinal in restraining NFκB mediated inflammation in the adult or older subjects

Inhibition of histone-deacetylase activity rescues inflammatory cystic fibrosis lung disease by modulating innate and adaptive immune responses

Abstract Background Chronic lung disease resulting from dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and NFκB-mediated neutrophilic-inflammation forms the basis of CF-related mortality. Here we aimed to evaluate if HDAC inhibition controls Pseudomonas-aeruginosa-lipopolysaccharide (Pa-LPS) induced airway inflammation and CF-lung disease. Methods For in vitro experiments, HEK293-cells were transfected with IL-8 or NFκB-firefly luciferase, and SV40-renilla- luciferase reporter constructs or ΔF508-CFTR-pCEP, followed by treatment with suberoylanilide hydroxamic acid (SAHA), Trichostatin-A (TSA) and/or TNFα. For murine studies, Cftr +/+ or Cftr −/− mice (n = 3) were injected/instilled with Pa-LPS and/or treated with SAHA or vehicle control. The progression of lung disease was monitored by quantifying changes in inflammatory markers (NFκB), cytokines (IL-6/IL-10), neutrophil activity (MPO, myeloperoxidase and/or NIMP-R14) and T-reg numbers. Results SAHA treatment significantly (p < 0.05) suppresses TNFα-induced NFκB and IL-8 reporter activities in HEK293-cells. Moreover, SAHA, Tubacin (selective HDAC6-inhibitor) or HDAC6-shRNAs controls CSE-induced ER-stress activities (p < 0.05). In addition, SAHA restores trafficking of misfolded-ΔF508-CFTR, by inducing protein levels of both B and C forms of CFTR. Murine studies using Cftr +/+ or Cftr −/− mice verified that SAHA controls Pa-LPS induced IL-6 levels, and neutrophil (MPO levels and/or NIMP-R14), NFκB-(inflammation) and Nrf2 (oxidative-stress marker) activities, while promoting FoxP3+ T-reg activity. Conclusion In summary, SAHA-mediated HDAC inhibition modulates innate and adaptive immune responses involved in pathogenesis and progression of inflammatory CF-lung disease

Early-age-related changes in VCP expression correlates with ubiquitin accumulation and hyper-inflammatory response.

<p>The longitudinal lungs sections from control, CLP or i.t. <i>Pa</i>-LPS (1 µg/gm bw) induced murine model (n = 3–4) were processed for histological evaluation. (<b>A</b>) We verified by TUNEL assay that accumulation of damaged- or misfolded- proteins leads to an increase in lung cell apoptosis in the adult mice by quantifying the changes in number of TUNEL positive cells. This is further exacerbated by <i>Pa</i>-LPS- or CLP- induced injury, verifying the correlation of ubiquitinated protein accumulation to increased apoptosis. The bottom panel of each staining shows the spearman's correlation coefficient analysis of the number of apoptotic cells in pediatric- and adult- mice (10 uniform representative areas from each mouse). (<b>B</b>) The adult mice show a constitutive increase in inflammation as compared to the pediatric group (H&E) that correlates with an increase in NFκB (C) and VCP (D) protein levels. The right panel of each staining shows the spearman's correlation coefficient analysis of fluorescence intensity in pediatric- and adult- mice (10 uniform representative areas from each mouse). (<b>E</b>) The <i>Pa</i>-LPS and CLP treatments induce the accumulation of ubiquitinated proteins that correlates with further increase in protein levels of NFκB (C) and VCP (D). <i>Data confirms that early-age-related proteostasis-imbalance augments Pa-LPS or sepsis induced injury</i>.</p

Proteasome inhibition regulates protein turnover rates.

<p>The human bronchial epithelial cells (HBE) were treated for 2 hours (2 h, partial) or overnight (O/N, broad) with low dose proteasome inhibitor (MG-132, 1 µM) and accumulation of newly synthesized ubiquitinated protein was monitored over time by metabolic labeling and immunoprecipitation. Lower levels of ubiquitinated proteins in overnight MG132 treated cells during protein synthesis indicate that decreased proteasomal activity inhibits protein synthesis as a feedback inhibition loop. <i>Lower proteasomal activity inhibits protein turnover rates (proteostasis-imbalance) by inhibiting both protein- synthesis and degradation</i>.</p

Critical role of CFTR-dependent lipid rafts in cigarette smoke-induced lung epithelial injury

Apoptosis of lung epithelial and endothelial cells by exposure to cigarette smoke (CS) severely damages the lung tissue, leading to the pathogenesis of emphysema, but the underlying mechanisms are poorly understood. We have recently established a direct correlation between decreased lipid raft CFTR expression and emphysema progression through increased ceramide accumulation. In the present work, we investigated the role of membrane CFTR in regulating apoptosis and autophagy responses to CS exposure. We report a constitutive and CS-induced increase in the number of TUNEL-positive apoptotic cells in Cftr−/− murine lungs compared with Cftr+/+ murine lungs that also correlated with a concurrent increase in the expression of ceramide, NF-κB, CD95/Fas, lipid raft proteins, and zonula occludens (ZO)-1/2 (P < 0.001). We also verified that stable wild-type CFTR expression in CFBE41o− cells controls constitutively elevated caspase-3/7 activity (−1.6-fold, P < 0.001). Our data suggest that membrane CFTR regulates ceramide-enriched lipid raft signaling platforms required for the induction of Fas-mediated apoptotic signaling. In addition, lack of membrane CFTR also modulates autophagy, as demonstrated by the significant increase in constitutive (P < 0.001) and CSE-induced (P < 0.005) perinuclear accumulation of green fluorescent protein-microtubule-associated protein 1 light chain-3 (LC3) in the absence of membrane CFTR (CFBE41o− cells). The significant constitutive and CS-induced increase (P < 0.05) in p62 and LC3β expression in CFTR-deficient cells and mice corroborates these findings and suggest a defective autophagy response in the absence of membrane CFTR. Our data demonstrate the critical role of membrane-localized CFTR in regulating apoptotic and autophagic responses in CS-induced lung injury that may be involved in the pathogenesis of severe emphysema

Early-age-related proteostasis-imbalance is critical for immunopathogenesis of chronic or fatal disease.

<p>Our data suggest that early-age-dependent decrease in proteasomal activity (PSMB6) results in accumulation of damaged- or misfolded- ubiquitinated proteins leading to an increase in VCP activity as a cytoprotective ER stress response. The sustained increase in VCP expression augments NFκB activation that mediates chronic inflammatory- and apoptotic- signaling. Moreover, accumulation of ubiquitinated proteins has a synergistic effect on these detrimental processes, leading to chronic or potentially fatal injury. Our data suggest the therapeutic potential of salubrinal and selective VCP inhibition in controlling the accumulation of ubiquitinated proteins (proteostasis-imbalance) and NFκB mediated chronic or fatal disease. <i>We identify here a promising therapeutic strategy to restrain the immunopathogenesis of chronic or fatal injury in older subjects</i>.</p

Salubrinal controls CLP induced inflammatory response.

<p>The peritoneal lavage from age- and sex- matched control, CLP and/or intraperitoneal (i.p.) salubrinal (1 mg/kg/bw) groups of C57BL6 mice (n = 3–4) were used to quantify changes in IL-6 (A) levels, and number and pro-inflammatory state of inflammatory cells (B). (<b>A</b>) Salubrinal is effective in significantly (p = 0.05) controlling the CLP induced pro-inflammatory cytokine, IL-6 levels. (<b>B</b>) The flow cytometry analysis shows the number (x-axis) of peritoneal neutrophils (NIMP-R14), macrophages (Mac3) and CD4+ T cells (CD4) isolated from control- and CLP- groups after 24 hrs, with- and without- salubrinal treatment. The pro-inflammatory state of individual cell population was verified by co-staining for intracellular IFNγ (y-axis). Data shows increase in numbers of neutrophils (upper panel) and macrophages (middle panel) with decreasing IFNγ levels after CLP while number of CD4⁺ T cells (lower panel) decrease and have increased IFNγ levels. <i>Salubrinal is effective in controlling CLP induced inflammatory response</i>.</p