Research

I. Nanoparticle/Bioaerosol Toxicology

• Molecular- and Material-based Mechanisms of Nanotoxicity

Nanomaterials have become increasingly popular in the production of a wide range of products and processes including, but not limited to, cosmetics, pharmaceuticals, medical research, semiconductor fabrication, food, and electronic manufacturing. The global market for nanomaterial-based products is estimated to reach $ 35 billion by 2030. Increased use of nanoparticles heightens the potential for human exposure, especially from airborne particles or the consumption of products containing them. However, the potential toxicity imposed by nanomaterials remains understudied. In the past 20 years, my research has deciphered the toxicity of many nanomaterials. Understanding these mechanisms of cytotoxicity informs the design of safer nanomaterials for various industry applications.

I have delineated the relationship between nanoparticles of differing compositions, the production of reactive oxygen species (ROS), and alterations of intracellular Ca²⁺ concentrations. Nanoparticles increase [Ca²⁺]_in. The moderation of this increase by agents such as nifedipine suggests that a portion of this increase reflects an influx of extracellular calcium. Membrane disruption (e.g., by the demonstrated lipid peroxidation) may play a role in this influx. Nanoparticles disrupt store-operated calcium entry. The increases in intracellular content OS may also have multiple sources. There exist synergistic relationships between intracellular [Ca²⁺] and OS, and the increases in both are reduced by antioxidants. While [Ca²⁺]_in and OS affect the activity of each other, they induce cell death by distinct pathways. These findings suggest that nanoparticles trigger cell death by multiple pathways. Recently, I discovered that nanoparticles not only kill cells, but they also suppress cell proliferation and alter cell cycle in a particle-specific manner. The molecular mechanisms contributing to these phenomena are the subject of continuing research.

To identify the physicochemical properties of nanoparticles that dictate cytotoxicity, I conducted a systematic study using oxides of transition metals (Ti, Cr, Mn, Fe, Ni, Cu, Zn) in the fourth period of the Periodic Table. Our data suggest that cytotoxicity is a function of particle surface charge, particle metal dissolution, and the number of available binding sites on the particle surface. Band-gap energy does not affect toxicity.

Representative Publications:
2020. Melissa H. Cambre, Natalie J. Holl, Bolin Wang, Lucas Harper, Han-Jung Lee, Charles Chusuei, Ethan T. Williams, Jerry D. Argo, Raja Ram Pandey, Fang Yao Stephen Hou, and Yue-Wern Huang. Cytotoxicity of NiO and Ni(OH)₂ nanoparticles is mediated by oxidative stress-induced cell death and suppression of cell proliferation. MDPI International Journal of Molecular Sciences, 21, 2355; doi:10.3390/ijms21072355.

2013. Charles C. Chusuei, Chi-Heng Wu, Shravan Mallavarapu, Jeffrey G. Winiarz, Jong-Sik Moon, Robert S. Aronstam, and Yue-Wern Huang. Cytotoxicity in the age of nano: cell toxicity reflects certain physiochemical properties of the fourth period of metal oxide nanoparticles. Chemico-Biological Interactions, 206:319-326.

2010. Chuan-Chin Huang, Robert S. Aronstam, Da-Ren Chen, and Yue-Wern Huang. Oxidative stress, calcium homeostasis, and altered gene expression in human lung epithelial cells exposed to ZnO nanoparticles. Toxicology in Vitro 24:45-55. doi: 10.1016/j.tiv.2009.09.007. (13 September 2009) (Ranked 10^th of the ScienceDirect Top 25 Hottest Articles during October 2009 – June 2011)

2006. Weisheng Lin, Yue-Wern Huang, Xiao-Dong Zhou, and Yinfa Ma. In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicology and Applied Pharmacology 217: 252-259.  doi:10.1016/j.taap.2006.10.004. (Among the top 10 of the ScienceDirect Top 25 hottest articles during April 2007 – March 2012)

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• Evolution and Behavior of Bioaerosols and Detection of Pathogens in Indoor Environments

Respiratory pathogens have tremendous public health impact and cause large economic losses. In the US, the annual economic burden of influenza alone is more than $11.2 billion. Among transmission modes of viral and bacterial respiratory diseases, airborne transmission is not well understood. In this mode, microorganisms are contained in droplet nuclei, forming bioaerosols, and thus can suspend in the air for a longer time, causing higher exposure and infection risks. In the medical field, aerosol transmission is still thought to “reflect a modern understanding of aerosol science,” which requires more fundamental studies. The ongoing pandemic of COVID-19 further highlights the urgent need to understand the transport and evolution of pathogen-containing aerosols, as there are contradictory evidence and conclusions on the potential transmission of SARS-CoV-2 via the airborne mode. In this NSF-funded project, our objectives are to 1) examine the decay of microorganisms in bioaerosols as a function of time, bioaerosol size, and environmental conditions; 2) characterize the evolution and transport of indoor bioaerosols under different ventilation configurations; and 3) establish a computational fluid dynamics (CFD) model coupled with bioaerosol evolution to optimize intervention strategies to reduce the infection risk of indoor bioaerosols.

Representative Publications:
2021. Yanxiao Li, Zhekun Peng, Natalie J. Holl, Md. Rifat Hassan, John M. Pappas, Congjie Wei, Omid Hoseini Izadi, Yang Wang, Xiangyang Dong, Cheng Wang, Yue-Wern Huang, DongHyun Kim, Chenglin Wu. MXene-graphene field effect transistor sensing influenza virus and SARS-CoV-2. ACS Omega, 6(10):6643-6653.

2020. Yang Wang, Guang Xu, and Yue-Wern Huang. Modeling the load of SARS-CoV-2 virus in human expelled particles during coughing and speaking. PLoS ONE, 15(10): e0241539. https://doi.org/10.1371/journal.pone.0241539.

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• Health Effects of Electronic Cigarette Aerosols

In 2019, 10.9 million American adults used Electronic Nicotine Delivery Systems (ENDS), such as the e-cigarette, e-hookah, and vape pen. Based upon the National Youth Tobacco Survey administered in May 2021, 2.8% (0.32 million) of U.S. middle school students and 11.3% (1.72 million) high school students reported e-cigarette use. Despite their touted benefit of reducing secondhand smoke, ENDS usage can still produce it, causing environmental health concerns. The rise in lung diseases linked to ENDS usage raises the question of the risks associated with secondhand ENDS smoke. These aerosols can also create thirdhand smoke on exposed surfaces. Both can induce oxidative stress on the airway epithelium, leading to diseases like asthma, bronchitis, and bronchiolitis obliterans. Aerosol exposure can result in posttranslational modifications in the nucleosome, potentially impacting biological processes like DNA repair and cell cycle which pertain to disease progression. While research has largely focused on direct ENDS aerosol exposure, there’s a research gap concerning secondhand and thirdhand ENDS aerosols. We aim to bridge this gap by using a simulated respiratory system for consistent aerosol generation and analysis; importantly, the system allows us to study the physical, chemical, and toxicological properties of secondhand and thirdhand ENDS aerosols under various environmental conditions. Our innovation lies in the use of a simulated respiratory system that we created, eliminating the need for human subjects. For toxicological endpoints, we focus on studying the oxidative stress and epigenetic effects of these aerosols and use a high-resolution aerosol instrument for rapid measurement to characterize their physiochemical properties.

Representative Publications:
2022. Weixing Hao, Kashala Fabrice Kapiamba, Varuni Abhayaratne, Shoaib Usman, Yue-Wern Huang, and Yang Wang. A filter-based system mimicking the particle deposition and penetration in human respiratory system for secondhand smoke generation and characterization. Inhalation Toxicology, 34(7-8): 189-199.

2022. Kashala Fabrice Kapiamba, Weixing Hao, Stephen Adom, Wenyan Liu, Yue-Wern Huang, and Yang Wang. Examining metals in primary and secondhand aerosols released by electronic cigarettes. ACS Chemical Research in Toxicology, 35(6): 954-962.

II. Delivery Platforms and Uptake Mechanisms of Biologically Active Molecules

• Cell-Penetrating Peptide (CPP)-Mediated Uptake of Biologically Active Molecules

Multiple approaches have been developed to introduce bioactive macromolecules, including DNA, RNA and protein, into living cells. The challenge is to design highly efficient and noncytotoxic carriers that can overcome the low permeability of cell membranes while promoting appropriate intracellular trafficking, and even nuclear delivery, in target cells. In the field of gene transfer, for instance, both viral and nonviral vectors have been used to deliver sense and antisense nucleic acids into mammalian cells. Nonviralsystems present several advantages over viral systems insofar as they are simple to use, easy to produce, less toxic, and do not induce specific immune responses. Among the non-viral systems, cell-penetrating peptides (CPPs, a.k.a. protein transduction domains)have been proposed to be an effective venue. However, the internalization mechanisms of CPPs remain understudied. In the past fifteen years, I have been investigating CPP-mediated cellular uptake mechanisms of cargoes such as nanomaterials, DNA plasmids, and small proteins. Depending on the sequence of the CPP and the physical and chemical properties of the carrier/cargo systems, the uptake involves direct membrane penetration, energy-dependent classical endocytosis and lipid raft- and actin-dependent macropinocytosis. It is uncertain whether clathrin- or caveolin-dependent endocytosis is involved, as results from pharmacological inhibitors and siRNA that interfere with cellular uptake processes are not entirely consistent. Because certain CPP/cargo complexes are entrapped in vesicles (i.e., endosomes and lysosomes), several sequence variations of CPPs have been designed to aid the escape of cargoes from vesicles.

Representative Publications:
2021. Natalie Holl, Han-Jung Lee, and Yue-Wern Huang. Evolutionary timeline of genetic delivery and gene therapy. Current Gene Therapy, 21(2):89-111.

2015. Yue-Wern Huang, Han-Jung Lee, Larry M. Tolliver, and Robert Aronstam. Delivery of nucleic acids and nanomaterials by cell-penetrating peptides: opportunities and challenges. BioMed Research International. Special Issue “Advances in Gene Delivery Systems”, volume 2015, article ID 834079, 16 pages. doi:10.1155/2015/834079.

2011. Betty Revon Liu, Yue-Wern Huang, Jeffrey G. Winiarz, Huey-Jenn Chiang, and Han-Jung Lee. Intracellular delivery of quantum dots mediated by histidine- and arginine-rich HR9 cell-penetrating peptides through the direct membrane translocation mechanism. PMID: 21329975. Biomaterials 32:3520-3537. (Y. H. and H. L. are corresponding authors.)

2010. Yi Xu, Betty Revon Liu, Han-Jung Lee, Katie B. Shannon, Jeffrey G. Winiarz, Tien-Chun Wang, Huey-Jenn Chiang, and Yue-Wern Huang. Nona-arginine facilitates delivery of quantum dots into cells via multiple pathways. Journal of Biomedicine and Biotechnology 2010:11. doi:10.1155/2010/948543. PMCID: PMC2965432.

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• uPA-mediated Targeted Gene Delivery System on Cancer

Breast cancer became the most common cancer in women as of 2022, accounting for 31% of all new annual cancer cases in the United States. Around 15% of breast cancers are triple-negative breast cancer (TNBC), which is the most aggressive subtype and characterized by the absence of three common receptors—estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), and therefore do not respond to hormonal or anti-HER2 therapies. It is urgent to explore targeted therapeutic strategies for TNBC due to its poor prognosis and rare effective targeting therapy. W have developed a polyamidoamine (PAMAM) dendrimer-based targeted gene delivery system to utilize urokinase-type plasminogen activator (uPA) to target uPA receptor (uPAR), which is highly expressed in both TNBC cells and cancer-associated stromal cells. The constructed system was evaluated for its ability to target cancer and its microenvironment and improve the transfection efficiency of anticancer nucleic acid. Results of flow cytometer and confocal microscopy showed that uPA-dendrimer improved the transfection efficiency of GTI-2040, an anticancer oligonucleotide, in MDA-MB-231 cell line and HCC2218 fibroblast cell line up to 6-fold compared to the GTI-2040 only group. GTI-2040 delivered by uPA-dendrimers killed cells by ~30% through knock-downing human ribonucleotide reductase component (R2) by 35%. In addition, bio-distribution and therapeutic studies showed significant inhibition of tumor growth in the TNBC orthotopic xenograft mice model with uPA-dendrimer/GTI-2040 administration for 14 days. Collectively, uPA-dendrimers improved the transfection efficiency of anticancer nucleic acids in both breast cancer cells and cancer-associated stromal cells, showing the targeted capacity and attenuating tumor growth in mice model. uPA-dendrimers have great potential in developing efficient targeted delivery systems to treat TNBC and other uPAR high-expressing cancers.

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• Tissue Engineering with Biomaterials

Bone repair using autografts or allografts is subject to several limitations. We have focused on facilitating bone repair and regeneration using a hydroxyapatite (HA) microsphere carrier in combination with bone morphogenetic protein 2 (BMP-2) and relaxin. BMP-2 is one of two proteins currently used to induce osteogenesis in clinical settings in orthopedic and dental applications. However, BMP-2 degrades rapidly when injected directly into a defect site, necessitating a high dose of the protein, which can have undesired effects, including swelling and an increased risk for cancer. Additionally, relaxin was thought to augment the effect of BMP2-induced osteogenesis as an enhancer. Accordingly, we have been investigating the use if an osteoconductive HA microsphere to control the delivery of BMP-2 and relaxin. Results from in vitro and in vivo studies reveal time-dependent release of BMP-2 and relaxin, and augmented effects on osteogenesis.

Representative Publications:
2022. Youqu Shen, Mohamed N. Rahaman, and Yue-Wern Huang. Geometry effect of open hollow hydroxyapatite microsphere on bone regeneration in rat calvarial defects. (Submitted to Bone Reports)

2020. Sahitya Injamuri, Mohamed Rahaman, Youqu Shen, and Yue-Wern Huang. Relaxin enhances bone regeneration with BMP-2 loaded hydroxyapatite microspheres. Journal of Biomedical Materials Research Part A, 108(5): 1231-1242; doi:10.1002/jbm.a.36897.

2020. Krishna C. R. Kolan, Yue-Wern Huang, Julie Semon, Ming C. Leu. 3D-printed biomimetic bioactive glass scaffolds for bone regeneration in rat calvarial defects. International Journal of Bioprinting, 6(2):82-98. doi:10.18063/ijb.v6i2.274.

III. Environmentally-Related Research Topics

• Ecotoxicological Research

My interests in ecotoxicology are broad. Recently I have partnered with an environmental engineering group at S&T to delineate the effect of algae on the combined toxicity of nanoparticles and heavy metals in C. Dubia. The focus is on modeling bioavailability and depuration rate of heavy metals influenced by the presence or absence of food and nanoparticles.

In the past, I investigated the population decline of the endangered species Ozark hellbenders. Topics of investigation include relationships among general animal health, reproductive hormones, and contaminants (pharmaceuticals, plasticizers, phytoestrogens) in animals and in their habitats. I also studied the levels of heavy metals (Cu, Zn, Cd, and Pb) in fish and sediments at a Superfund site in the Old Lead Belt in Missouri. My studies on body burden, toxicokinetics, and metabolism of PCBs using frogs in the Green Bay area revealed intricate relationships associated with unique reproductive features, as well as environmental and physiological factors.

Representative Publications:

2021. Xuesong Liu, Jianmin Wang, and Yue-Wern Huang. Quantifying the effect of nano-TiO₂ on the toxicity of lead on C. dubia using a two-compartment modeling approach. Chemosphere, 263:127958; doi:10.1016/j.chemosphere.2020.127958.

2019. Xuesong Liu, Jiamin Wang, Yue-Wern Huang, and Tao Kong. Algae (Raphidocelis) reduce combined toxicity of nano-TiO₂ and lead on C. Dubia. Science of the Total Environment, 686: 246-253.

2010. Chuan-Chin Huang, Yi Xu, Paul Nam, Jeff Briggler, Mike McKee, and Yue-Wern Huang.  Heavy metals, hematology, plasma chemistry, and parasites in adult hellbenders (Cryptobranchus alleganiensis). doi:10.1002/etc.148. Environmental Toxicology and Chemistry 29:1132-1137.

2007. Mauricio Solis, Chichin Liu, Janet Bandeff, Paul Nam, Dev Niyogi, and Yue-Wern Huang. Occurrence of organic chemicals in two rivers inhabited by Ozark hellbenders (Cryptobranchus alleganiensis bishopi). Archives of Environmental Contamination and Toxicology 53(3):426-434.

2004. Nord L. Gale,Craig D. Adams,Bobby G. Wixson,Keith A. Loftin,and Yue-Wern Huang. Lead, zinc, copper, and cadmium in fish and river sediments from the Big River and Flat River Creek of Missouri Old Lead Belt. Environmental Geochemistry and Health, 26:37-49.