Chong Hyun Chang

Toxicity Mechanisms in Escherichia coli Vary for Silver Nanoparticles and Differ from Ionic Silver

Silver nanoparticles (Ag NPs) are commonly added to various consumer products and materials to impair bacterial growth. Recent studies suggested that the primary mechanism of antibacterial action of silver nanoparticles is release of silver ion (Ag(+)) and that particle-specific activity of silver nanoparticles is negligible. Here, we used a genome-wide library of Escherichia coli consisting of ∼4000 single gene deletion mutants to elucidate which physiological pathways are involved in how E. coli responds to different Ag NPs. The nanoparticles studied herein varied in both size and surface charge. AgNO3 was used as a control for soluble silver ions. Within a series of differently sized citrate-coated Ag NPs, smaller size resulted in higher Ag ion dissolution and toxicity. Nanoparticles functionalized with cationic, branched polyethylene imine (BPEI) exhibited equal toxicity with AgNO3. When we used a genome-wide approach to investigate the pathways involved in the response of E. coli to different toxicants, we found that only one of the particles (Ag-cit10) exhibited a pattern of response that was statistically similar to that of silver ion. By contrast, the pathways involved in E. coli response to Ag-BPEI particles were more similar to those observed for another cationic nanoparticle that did not contain Ag. Overall, we found that the pathways involved in bacterial responses to Ag nanoparticles are highly dependent on physicochemical properties of the nanoparticles, particularly the surface characteristics. These results have important implications for the regulation and testing of silver nanoparticles.

Use of Coated Silver Nanoparticles to Understand the Relationship of Particle Dissolution and Bioavailability to Cell and Lung Toxicological Potential

Since more than 30% of consumer products that include engineered nanomaterials contain nano-Ag, the safety of this material is of considerable public concern. In this study, Ag nanoparticles (NPs) are used to demonstrate that 20 nm polyvinylpyrrolidone (PVP or P) and citrate (C)-coated Ag NPs induce more cellular toxicity and oxidative stress than larger (110 nm) particles due to a higher rate of dissolution and Ag bioavailability. Moreover, there is also a higher propensity for citrate 20 nm (C20) nanoparticles to generate acute neutrophilic inflammation in the lung and to produce chemokines compared to C110. P110 has less cytotoxic effects than C110, likely due to the ability of PVP to complex released Ag(+) . In contrast to the more intense acute pulmonary effects of C20, C110 induces mild pulmonary fibrosis at day 21, likely as a result of slow but persistent Ag(+) release leading to a sub-chronic injury response. Interestingly, the released metallic Ag is incorporated into the collagen fibers depositing around airways and the lung interstitium. Taken together, these results demonstrate that size and surface coating affect the cellular toxicity of Ag NPs as well as their acute versus sub-chronic lung injury potential.

Use of a Lipid-Coated Mesoporous Silica Nanoparticle Platform for Synergistic Gemcitabine and Paclitaxel Delivery to Human Pancreatic Cancer in Mice

Recently, a commercial albumin-bound paclitaxel (PTX) nanocarrier (Abraxane) was approved as the first new drug for pancreatic ductal adenocarcinoma in almost a decade. PTX improves the pharmaceutical efficacy of the first-line pancreatic cancer drug, gemcitabine (GEM), through suppression of the tumor stroma and inhibiting the expression of the GEM-inactivating enzyme, cytidine deaminase (CDA). We asked, therefore, whether it was possible to develop a mesoporous silica nanoparticle (MSNP) carrier for pancreatic cancer to co-deliver a synergistic GEM/PTX combination. High drug loading was achieved by a custom-designed coated lipid film technique to encapsulate a calculated dose of GEM (40 wt %) by using a supported lipid bilayer (LB). The uniform coating of the 65 nm nanoparticles by a lipid membrane allowed incorporation of a sublethal amount of hydrophobic PTX, which could be co-delivered with GEM in pancreatic cells and tumors. We demonstrate that ratiometric PTX incorporation and delivery by our LB-MSNP could suppress CDA expression, contemporaneous with induction of oxidative stress as the operating principle for PTX synergy. To demonstrate the in vivo efficacy, mice carrying subcutaneous PANC-1 xenografts received intravenous (IV) injection of PTX/GEM-loaded LB-MSNP. Drug co-delivery provided more effective tumor shrinkage than GEM-loaded LB-MSNP, free GEM, or free GEM plus Abraxane. Comparable tumor shrinkage required coadministration of 12 times the amount of free Abraxane. High-performance liquid chromatography analysis of tumor-associated GEM metabolites confirmed that, compared to free GEM, MSNP co-delivery increased the phosphorylated DNA-interactive GEM metabolite 13-fold and decreased the inactivated and deaminated metabolite 4-fold. IV injection of MSNP-delivered PTX/GEM in a PANC-1 orthotopic model effectively inhibited primary tumor growth and eliminated metastatic foci. The enhanced in vivo efficacy of the dual delivery carrier could be achieved with no evidence of local or systemic toxicity. In summary, we demonstrate the development of an effective LB-MSNP nanocarrier for synergistic PTX/GEM delivery in pancreatic cancer.

Surface Interactions with Compartmentalized Cellular Phosphates Explain Rare Earth Oxide Nanoparticle Hazard and Provide Opportunities for Safer Design

Growing international exploitation of rare earth oxides (REOs) for commercial and biological use has increased the possibility of human exposure and adverse health effects. Occupational exposure to rare earth materials in miners and polishers leads to a severe form of pneumoconiosis, while gadolinium-containing MRI contrast agents cause nephrogenic systemic fibrosis in patients with renal impairment. The mechanisms for inducing these adverse pro-fibrogenic effects are of considerable importance for the safety assessment of REO particles as well as presenting opportunities for safer design. In this study, using a well-prepared REO library, we obtained a mechanistic understanding of how REOs induce cellular and pulmonary damage by a compartmentalized intracellular biotransformation process in lysosomes that results in pro-fibrogenic growth factor production and lung fibrosis. We demonstrate that rare earth oxide ion shedding in acidifying macrophage lysosomes leads to biotic phosphate complexation that results in organelle damage due to stripping of phosphates from the surrounding lipid bilayer. This results in nanoparticle biotransformation into urchin shaped structures and setting in motion a series of events that trigger NLRP3 inflammasome activation, IL-1β release, TGF-β1 and PDGF-AA production. However, pretreatment of REO nanoparticles with phosphate in a neutral pH environment prevents biological transformation and pro-fibrogenic effects. This can be used as a safer design principle for producing rare earth nanoparticles for biological use.

Engineering an effective immune adjuvant by designed control of shape and crystallinity of aluminum oxyhydroxide nanoparticles.

Adjuvants based on aluminum salts (Alum) are commonly used in vaccines to boost the immune response against infectious agents. However, the detailed mechanism of how Alum enhances adaptive immunity and exerts its adjuvant immune effect remains unclear. Other than being comprised of micrometer-sized aggregates that include nanoscale particulates, Alum lacks specific physicochemical properties to explain activation of the innate immune system, including the mechanism by which aluminum-based adjuvants engage the NLRP3 inflammasome and IL-1β production. This is putatively one of the major mechanisms required for an adjuvant effect. Because we know that long aspect ratio nanomaterials trigger the NLRP3 inflammasome, we synthesized a library of aluminum oxyhydroxide (AlOOH) nanorods to determine whether control of the material shape and crystalline properties could be used to quantitatively assess NLRP3 inflammasome activation and linkage of the cellular response to the materials adjuvant activities in vivo. Using comparison to commercial Alum, we demonstrate that the crystallinity and surface hydroxyl group display of AlOOH nanoparticles quantitatively impact the activation of the NLRP3 inflammasome in human THP-1 myeloid cells or murine bone marrow-derived dendritic cells (BMDCs). Moreover, these in vitro effects were correlated with the immunopotentiation capabilities of the AlOOH nanorods in a murine OVA immunization model. These results demonstrate that shape, crystallinity, and hydroxyl content play an important role in NLRP3 inflammasome activation and are therefore useful for quantitative boosting of antigen-specific immune responses. These results show that the engineered design of aluminum-based adjuvants in combination with dendritic cell property-activity analysis can be used to design more potent aluminum-based adjuvants.

Two-Wave Nanotherapy to Target the Stroma and Optimize Gemcitabine Delivery to a Human Pancreatic Cancer Model in Mice

Pancreatic ductal adenocarcinoma (PDAC) elicits a dense stromal response that blocks vascular access because of pericyte coverage of vascular fenestrations. In this way, the PDAC stroma contributes to chemotherapy resistance in addition to causing other problems. In order to improve the delivery of gemcitabine, a first-line chemotherapeutic agent, a PEGylated drug-carrying liposome was developed, using a transmembrane ammonium sulfate gradient to encapsulate the protonated drug up to 20% w/w. However, because the liposome was precluded from entering the xenograft site due to the stromal interference, we developed a first-wave nanocarrier that decreases pericyte coverage of the vasculature through interference in the pericyte recruiting TGF-β signaling pathway. This was accomplished using a polyethyleneimine (PEI)/polyethylene glycol (PEG)-coated mesoporous silica nanoparticle (MSNP) for molecular complexation to a small molecule TGF-β inhibitor, LY364947. LY364947 contains a nitrogen atom that attaches, through H-bonding, to PEI amines with a high rate of efficiency. The copolymer coating also facilitates systemic biodistribution and retention at the tumor site. Because of the high loading capacity and pH-dependent LY364947 release from the MSNPs, we achieved rapid entry of IV-injected liposomes and MSNPs at the PDAC tumor site. This two-wave approach provided effective shrinkage of the tumor xenografts beyond 25 days, compared to the treatment with free drug or gemcitabine-loaded liposomes only. Not only does this approach overcome stromal resistance to drug delivery in PDAC, but it also introduces the concept of using a stepwise engineered approach to address a range of biological impediments that interfere in nanocancer therapy in a spectrum of cancers.

NADPH Oxidase-Dependent NLRP3 Inflammasome Activation and its Important Role in Lung Fibrosis by Multiwalled Carbon Nanotubes.

The purpose of this paper is to elucidate the key role of NADPH oxidase in NLRP3 inflammasome activation and generation of pulmonary fibrosis by multi-walled carbon nanotubes (MWCNTs). Although it is known that oxidative stress plays a role in pulmonary fibrosis by single-walled CNTs, the role of specific sources of reactive oxygen species, including NADPH oxidase, in inflammasome activation remains to be clarified. In this study, three long aspect ratio (LAR) materials (MWCNTs, single-walled carbon nanotubes, and silver nanowires) are used to compare with spherical carbon black and silver nanoparticles for their ability to trigger oxygen burst activity and NLRP3 assembly. All LAR materials but not spherical nanoparticles induce robust NADPH oxidase activation and respiratory burst activity in THP-1 cells, which are blunted in p22(phox) -deficient cells. The NADPH oxidase is directly involved in lysosomal damage by LAR materials, as demonstrated by decreased cathepsin B release and IL-1β production in p22(phox) -deficient cells. Reduced respiratory burst activity and inflammasome activation are also observed in bone marrow-derived macrophages from p47(phox) -deficient mice. Moreover, p47(phox) -deficient mice have reduced IL-1β production and lung collagen deposition in response to MWCNTs. Lung fibrosis is also suppressed by N-acetyl-cysteine in wild-type animals exposed to MWCNTs.

Zebrafish High‐Throughput Screening to Study the Impact of Dissolvable Metal Oxide Nanoparticles on the Hatching Enzyme, ZHE1

The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2 O3 , and NiO) could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose-dependent fashion. A peptide-based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine-based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.

Irinotecan Delivery by Lipid-Coated Mesoporous Silica Nanoparticles Shows Improved Efficacy and Safety over Liposomes for Pancreatic Cancer

Urgent intervention is required to improve the 5 year survival rate of pancreatic ductal adenocarcinoma (PDAC). While the four-drug regimen, FOLFIRINOX (comprising irinotecan, 5-fluorouracil, oxaliplatin, and leucovorin), has a better survival outcome than the more frequently used gemcitabine, the former treatment platform is highly toxic and restricted for use in patients with good performance status. Since irinotecan contributes significantly to FOLFIRINOX toxicity (bone marrow and gastrointestinal tract), our aim was to reduce the toxicity of this drug by a custom-designed mesoporous silica nanoparticle (MSNP) platform, which uses a proton gradient for high-dose irinotecan loading across a coated lipid bilayer (LB). The improved stability of the LB-coated MSNP (LB-MSNP) carrier allowed less drug leakage systemically with increased drug concentrations at the tumor sites of an orthotopic Kras-derived PDAC model compared to liposomes. The LB-MSNP nanocarrier was also more efficient for treating tumor metastases. Equally important, the reduced leakage and slower rate of drug release by the LB-MSNP carrier dramatically reduced the rate of bone marrow, gastrointestinal, and liver toxicity compared to the liposomal carrier. We propose that the combination of high efficacy and reduced toxicity by the LB-MSNP carrier could facilitate the use of irinotecan as a first-line therapeutic to improve PDAC survival.

Use of a pro-fibrogenic mechanism-based predictive toxicological approach for tiered testing and decision analysis of carbonaceous nanomaterials

Engineered carbonaceous nanomaterials (ECNs), including single-wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), graphene, and graphene oxide (GO), are potentially hazardous to the lung. With incremental experience in the use of predictive toxicological approaches, seeking to relate ECN physicochemical properties to adverse outcome pathways (AOPs), it is logical to explore the existence of a common AOP that allows comparative analysis of broad ECN categories. We established an ECN library comprising three different types of SWCNTs, graphene, and graphene oxide (two sizes) for comparative analysis according to a cell-based AOP that also plays a role in the pathogenesis of pulmonary fibrosis. SWCNTs synthesized by Hipco, arc discharge and Co-Mo catalyst (CoMoCAT) methods were obtained in their as-prepared (AP) state, following which they were further purified (PD) or coated with Pluronic F108 (PF108) or bovine serum albumin (BSA) to improve dispersal and colloidal stability. GO was prepared as two sizes, GO-small (S) and GO-large (L), while the graphene samples were coated with BSA and PF108 to enable dispersion in aqueous solution. In vitro screening showed that AP- and PD-SWCNTs, irrespective of the method of synthesis, as well as graphene (BSA) and GO (S and L) could trigger interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) production in myeloid (THP-1) and epithelial (BEAS-2B) cell lines, respectively. Oropharyngeal aspiration in mice confirmed that AP-Hipco tubes, graphene (BSA-dispersed), GO-S and GO-L could induce IL-1β and TGF-β1 production in the lung in parallel with lung fibrosis. Notably, GO-L was the most pro-fibrogenic material based on rapid kinetics of pulmonary injury. In contrast, PF108-dispersed SWCNTs and -graphene failed to exert fibrogenic effects. Collectively, these data indicate that the dispersal state and surface reactivity of ECNs play key roles in triggering a pro-fibrogenic AOP, which could prove helpful for hazard ranking and a proposed tiered testing approach for large ECN categories.