In vivo fate tracking of degradable nanoparticles for lung gene transfer using PET and Cerenkov imaging.

Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(dl-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Cerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.

DAP12 expression in lung macrophages mediates ischemia/reperfusion injury by promoting neutrophil extravasation.

Neutrophils are critical mediators of innate immune responses and contribute to tissue injury. However, immune pathways that regulate neutrophil recruitment to injured tissues during noninfectious inflammation remain poorly understood. DAP12 is a cell membrane-associated protein that is expressed in myeloid cells and can either augment or dampen innate inflammatory responses during infections. To elucidate the role of DAP12 in pulmonary ischemia/reperfusion injury (IRI), we took advantage of a clinically relevant mouse model of transplant-mediated lung IRI. This technique allowed us to dissect the importance of DAP12 in tissue-resident cells and those that infiltrate injured tissue from the periphery during noninfectious inflammation. Macrophages in both mouse and human lungs that have been subjected to cold ischemic storage express DAP12. We found that donor, but not recipient, deficiency in DAP12 protected against pulmonary IRI. Analysis of the immune response showed that DAP12 promotes the survival of tissue-resident alveolar macrophages and contributes to local production of neutrophil chemoattractants. Intravital imaging demonstrated a transendothelial migration defect into DAP12-deficient lungs, which can be rescued by local administration of the neutrophil chemokine CXCL2. We have uncovered a previously unrecognized role for DAP12 expression in tissue-resident alveolar macrophages in mediating acute noninfectious tissue injury through regulation of neutrophil trafficking.

PEGylation of cationic, shell-crosslinked-knedel-like nanoparticles modulates inflammation and enhances cellular uptake in the lung.

The airway provides a direct route for administration of nanoparticles bearing therapeutic or diagnostic payloads to the lung, however optimization of nanoplatforms for intracellular delivery remains challenging. Poly(ethylene glycol) (PEG) surface modification improves systemic performance but less is known about PEGylated nanoparticles administered to the airway. To test this, we generated a library of cationic, shell crosslinked knedel-like nanoparticles (cSCKs), including PEG (1.5 kDa PEG; 2, 5, 10 molecules/polymer arm) on the outer shell. Delivery of PEGylated cSCK to the mouse airway showed significantly less inflammation in a PEG dose-dependent manner. PEGylation also enhanced the entry of cSCKs in lung alveolar epithelial cells and improved surfactant penetration. The PEGylation effect could be explained by the altered mechanism of endocytosis. While non-PEGylated cSCKs used the clathrin-dependent route for endocytosis, entry of PEGylated cSCK was clathrin-independent. Thus, nanoparticle surface modification with PEG represents an advantageous design for lung delivery. FROM THE CLINICAL EDITOR: In this study, the effects of PEGylation were studied on cross linked knedel-like nanoparticles in drug delivery through the lungs, demonstrating less airway inflammation in the studied model than with non-PEGylated nanoparticles, which suggests an overall favorable profile of PEGylated nanoparticles for alveolar delivery.

Degradable cationic shell cross-linked knedel-like nanoparticles: synthesis, degradation, nucleic acid binding, and in vitro evaluation.

In this work, degradable cationic shell cross-linked knedel-like (deg-cSCK) nanoparticles were developed as an alternative platform to replace similar nondegradable cSCK nanoparticles that have been utilized for nucleic acids delivery. An amphiphilic diblock copolymer poly(acrylamidoethylamine)(90)-block-poly(DL-lactide)(40) (PAEA(90)-b-PDLLA(40)) was synthesized, self-assembled in aqueous solution, and shell cross-linked using a hydrolyzable cross-linker to afford deg-cSCKs with an average core diameter of 45 ± 7 nm. These nanoparticles were fluorescently labeled for in vitro tracking. The enzymatic- and hydrolytic-degradability, siRNA binding affinity, cell uptake and cytotoxicity of the deg-cSCKs were evaluated. Esterase-catalyzed hydrolysis of the nanoparticles resulted in the degradation of ca. 24% of the PDLLA core into lactic acid within 5 d, as opposed to only ca. 9% degradation from aqueous solutions of the deg-cSCK nanoparticles in the absence of enzyme. Cellular uptake of deg-cSCKs was efficient, while exhibiting low cytotoxicity with LD50 values of ca. 90 and 30 µg/mL in RAW 264.7 mouse macrophages and MLE 12 cell lines, respectively, ca. 5- to 6-fold lower than the cytotoxicity observed for nondegradable cSCK analogs. Additionally, deg-cSCKs were able to complex siRNA at an N/P ratio as low as 2, and were efficiently able to facilitate cellular uptake of the complexed nucleic acids.

Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection.

Acute lung injury (ALI) is a complex syndrome with many aetiologies, resulting in the upregulation of inflammatory mediators in the host, followed by dyspnoea, hypoxemia and pulmonary oedema. A central mediator is inducible nitric oxide synthase (iNOS) that drives the production of NO and continued inflammation. Thus, it is useful to have diagnostic and therapeutic agents for targeting iNOS expression. One general approach is to target the precursor iNOS mRNA with antisense nucleic acids. Peptide nucleic acids (PNAs) have many advantages that make them an ideal platform for development of antisense theranostic agents. Their membrane impermeability, however, limits biological applications. Here, we report the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabelled antisense PNA-YR9 · oligodeoxynucleotide (ODN) hybrid and a cationic shell-cross-linked knedel-like nanoparticle (cSCK). The Y (tyrosine) residue was used for (123)I radiolabelling, whereas the R9 (arginine9) peptide was included to facilitate cell exit of untargeted PNA. Complete binding of the antisense PNA-YR9 · ODN hybrid to the cSCK was achieved at an 8 : 1 cSCK amine to ODN phosphate (N/P) ratio by a gel retardation assay. The antisense PNA-YR9 · ODN · cSCK nanocomplexes efficiently entered RAW264.7 cells, whereas the PNA-YR9 · ODN alone was not taken up. Low concentrations of (123)I-labelled antisense PNA-YR9 · ODN complexed with cSCK showed significantly higher retention of radioactivity when iNOS was induced in lipopolysaccharide+interferon-γ-activated RAW264.7 cells when compared with a mismatched PNA. Moreover, statistically, greater retention of radioactivity from the antisense complex was also observed in vivo in an iNOS-induced mouse lung after intratracheal administration of the nanocomplexes. This study demonstrates the specificity and sensitivity by which the radiolabelled nanocomplexes can detect iNOS mRNA in vitro and in vivo and their potential for early diagnosis of ALI.

Polarized alloantigen presentation by airway epithelial cells contributes to direct CD8+ T cell activation in the airway.

Activated T lymphocytes are abundant in the airway during lung allograft rejection. Based on respiratory viral studies, it is the current paradigm that T cells cannot divide in the airway, and that their accumulation in the lumen of the respiratory tract is the exclusive result of recruitment from other sites, such as mediastinal lymph nodes. Here, we show that CD8(+) T cell activation and proliferation can occur in the airway after orthotopic lung transplantation. We also demonstrate that airway epithelium expresses major histocompatibility class I predominantly on the apical surface, both in vitro and in vivo, and initiates CD8(+) T cell responses in a polarized fashion, favoring luminal activation. Our data identify a unique site for CD8(+) T cell activation after lung transplantation, and suggest that attenuating these responses may provide a clinically relevant target.

Effect of polarized release of CXC-chemokines from wild-type and cystic fibrosis murine airway epithelial cells.

The respiratory epithelium lining the airway relies on mucociliary clearance and a complex network of inflammatory mediators to protect the lung. Alterations in the composition and volume of the periciliary liquid layer, as occur in cystic fibrosis (CF), lead to impaired mucociliary clearance and persistent airway infection. Moreover, the respiratory epithelium releases chemoattractants after infection, inciting airway inflammation. However, characterizing the inflammatory response of primary human airway epithelial cells to infection can be challenging because of genetic heterogeneity. Using well-characterized, differentiated, primary murine tracheal cells grown at an air-liquid interface, which provides an in vitro polarized epithelial model, we compared inflammatory gene expression and secretion in wild-type and ΔF508 CF airway cells after infection with Pseudomonas aeruginosa. The expression of several CXC-chemokines, including macrophage inflammatory protein-2, small inducible cytokine subfamily member 2, lipopolysaccharide-induced chemokine, and interferon-inducible cytokine-10, was markedly increased after infection, and these proinflammatory mediators were asymmetrically released from the airway epithelium, predominantly from the basolateral surface. Equal amounts of CXC-chemokines were released from wild-type and CF cells. Secreted mediators were concentrated in the thin, periciliary fluid layer, and the dehydrated apical microenvironment of CF airway epithelial cells amplified the inflammatory signal, potentially resulting in high chemokine concentration gradients across the epithelium. Consistent with this observation, the enhanced chemotaxis of wild-type neutrophils was detected in CF airway epithelial cultures, compared with wild-type cells. These data suggest that P. aeruginosa infection of the airway epithelium induces the expression and polarized secretion of CXC-chemokines, and the increased concentration gradient across the CF airway leads to an exaggerated inflammatory response.

ATP7B detoxifies silver in ciliated airway epithelial cells.

Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B(-/-) mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag+/Cu+ transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

Impact of hydrogel nanoparticle size and functionalization on in vivo behavior for lung imaging and therapeutics.

Polymer chemistry offers the possibility of synthesizing multifunctional nanoparticles which incorporate moieties that enhance diagnostic and therapeutic targeting of cargo delivery to the lung. However, since rules for predicting particle behavior following modification are not well-defined, it is essential that probes for tracking fate in vivo are also included. Accordingly, we designed polyacrylamide-based hydrogel particles of differing sizes, functionalized with a nona-arginine cell-penetrating peptide (Arg(9)), and labeled with imaging components to assess lung retention and cellular uptake after intratracheal administration. Radiolabeled microparticles (1-5 microm diameter) and nanoparticles (20-40 nm diameter) without and with Arg(9) showed diffuse airspace distribution by positron emission tomography imaging. Biodistribution studies revealed that particle clearance and extrapulmonary distribution was, in part, size dependent. Microparticles were rapidly cleared by mucociliary routes but, unexpectedly, also through the circulation. In contrast, nanoparticles had prolonged lung retention enhanced by Arg(9) and were significantly restricted to the lung. For all particle types, uptake was predominant in alveolar macrophages and, to a lesser extent, lung epithelial cells. In general, particles did not induce local inflammatory responses, with the exception of microparticles bearing Arg(9). Whereas microparticles may be advantageous for short-term applications, nanosized particles constitute an efficient high-retention and non-inflammatory vehicle for the delivery of diagnostic imaging agents and therapeutics to lung airspaces and alveolar macrophages that can be enhanced by Arg(9). Importantly, our results show that minor particle modifications may significantly impact in vivo behavior within the complex environments of the lung, underscoring the need for animal modeling.

In vitro and murine efficacy and toxicity studies of nebulized SCC1, a methylated caffeine-silver(I) complex, for treatment of pulmonary infections.

The expanding clinical challenge of respiratory tract infections due to resistant bacteria necessitates the development of new forms of therapy. The development of a compound composed of silver coupled to a methylated caffeine carrier (silver carbene complex 1 [SCC1]) that demonstrated in vitro efficacy against bacteria, including drug-resistant organisms, isolated from patients with respiratory tract infections was described previously. The findings of current in vitro studies now suggest that bactericidal concentrations of SCC1 are not toxic to airway epithelial cells in primary culture. Thus, it was hypothesized that SCC1 could be administered by the aerosolized route to concentrate delivery to the lung while minimizing systemic toxicity. In vivo, aerosolized SCC1 delivered to mice resulted in mild aversion behavior, but it was otherwise well tolerated and did not cause lung inflammation following administration over a 5-day period. The therapeutic efficacy of SCC1 compared to that of water was shown in a 3-day prophylaxis protocol, in which mice infected with a clinical strain of Pseudomonas aeruginosa had increased survival, decreased amounts of bacteria in the lung, and a lower prevalence of bacteremia. Similarly, by using an airway infection model in which bacteria were impacted in the airways by agarose beads, the administration of SCC1 was significantly superior to water in decreasing the lung bacterial burden and the levels of bacteremia and markers of airway inflammation. These observations indicate that aerosolized SCC1, a novel antimicrobial agent, warrants further study as a potential therapy for bacterial respiratory tract infections.

Experimental evolution of human influenza virus H3 hemagglutinin in the mouse lung identifies adaptive regions in HA1 and HA2.

The genetic basis for virulence and host switching in influenza A viruses (FLUAV) is largely unknown. Because the hemagglutinin (HA) protein is a determinant of these properties, HA evolution was mapped in an experimental model of mouse lung adaptation. Variants of prototype A/Hong Kong/1/68 (H3N2) (wild-type [wt] HK) human virus were selected in both longitudinal and parallel studies of lung adaptation. Mapping of HA mutations found in 11 independently derived mouse-adapted populations of wt HK identified 27 mutations that clustered within two distinct regions in or near the globular frameworks of the HA1 and HA2 subunits. The adaptive mutations demonstrated multiple instances of convergent evolution involving four amino acid positions (162, 210, and 218 in HA1 and 154 in HA2). By use of reverse genetics, convergent HA mutations were shown to affect cell tropism by enhancing infection and replication in primary mouse tracheal epithelial cells in vitro and mouse lung tissue in vivo. Adaptive HA mutations were multifunctional, affecting both median pH of fusion and receptor specificity. Specific mutations within both adaptive regions were shown to increase virulence in a mouse lung model. The occurrence of mutations in the HA1 and HA2 adaptive regions of natural FLUAV host range and virulent variants of avian and mammalian viruses is discussed. This study has identified adaptive sites and regions within the HA1 and HA2 subunits that may guide future studies of viral adaptation and evolution in nature.

Autosomal dominant polycystic kidney disease is associated with an increased prevalence of radiographic bronchiectasis.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common disease with several known extrarenal manifestations, although no known pulmonary features. The formation of renal cysts in ADPKD has been attributed to dysfunction of primary cilia and the primary cilia-related proteins polycystin-1 (in 85% of cases) and polycystin-2 in renal epithelial cells. The goals of this study were to characterize the normal expression of polycystin-1 in the motile cilia of airway epithelial cells and to evaluate lung structure in ADPKD patients. METHODS: Airway epithelium from non-ADPKD patients was immunostained to localize polycystin-1 expression, and lung tissue from ADPKD patients was examined for pathologic changes. CT scans from ADPKD patients (n = 95) and a control group of non-ADPKD chronic kidney disease patients (n = 95) were retrospectively reviewed for the presence of bronchiectasis using defined criteria. RESULTS: Immunostaining revealed polycystin-1 expression in the motile cilia of non-ADPKD airway epithelial cells. Lung tissue from one of five available ADPKD patient autopsies revealed histologic changes of bronchiectasis. Review of CT scans revealed a threefold-increased prevalence of bronchiectasis in the ADPKD group compared to the control group (37% vs 13%, p = 0.002). CONCLUSIONS: ADPKD patients demonstrate an increased prevalence of radiographic bronchiectasis, a previously unrecognized manifestation of the disease. This association suggests that patients with primary cilia-associated diseases may be at risk for airway disease.

Maintenance of airway epithelium in acutely rejected orthotopic vascularized mouse lung transplants.

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the anti-apoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli may be required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide a new and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts.

Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells.

Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an alpha2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an alpha2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the alpha2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The alpha2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, alpha2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the alpha2,3-linked SA receptor.