Page 78 - ASME_NEMB_2016_Program

Technical Program TRACK 6

dence of endometrial carcinoma. Although considered rare, this side effect, this material for a variety of biomedical and clinical applications by using
along with other serious adverse effects (such as blood clots, strokes, and chemical functionalization of nanotextured surfaces. The silicone masks
cataracts), has resulted in a debate concerning the benefits versus risks of were placed on top of the NPS films to normalize the area of sample ex-
TMX use in cancer prevention. posure. Serum and urine samples were first spotted into each well and
then incubated at room temperature. Only small proteins and peptides can
As the key to success for breast cancer chemoprevention relies upon long- diffuse into the nanopores, while large proteins are excluded and subse-
term delivery of drugs while circumventing side effects, we have developed quently removed by washing. The small peptide fractions were extracted
a novel local delivery strategy for the constant and sustained administration by elution buffer. Using this procedure, Hep-25 can be enriched in the
of TMX. We maintain a long-term, local release of TMX in mammary tissues optimized nanopores, and then analyzed by MALDI-TOF MS. To achieve
by utilizing a novel implantable nanochannel Delivery System (nDS). The absolute quantification of our target peptide biomarker, we introduced
nDS consists of a bioinert, implantable, and mechanically robust silicon heavy Hep-25 (Glu1-Thr17-Hepcidin) into human serum samples as internal
membrane which houses an exact number of densely packed slit-nanochan- standards. Our method requires only microliter sample volume and elimi-
nels as small as 2.5 nm with tight tolerances on size, geometry, and surface nates time-consuming sample pretreatment, while still maintaining a high
properties. Providing steady levels of TMX at the mammary gland target degree of precision, accuracy, and sensitivity. In a clinical validation of our
tissue through nDS delivery maximizes the therapeutic index while limiting technique, Hep-25 levels were quantified in both serum and urine from 119
the unwanted secondary effects, which will ultimately improve patient com- healthy volunteers and 19 patients suffering from inflammation. The levels
pliance. of hepcidin were found to be gender, menopausal, and inflammation status
dependent. The application of and proof-of-concept for this new approach
In this work we chemically induced tumorigenesis in Sprague-Dawley rats could help not only to improve our understanding of iron disorder diseas-
by N-methyl-N-nitrosourea (NMU) injection to promote the development of es, but also consequently enable hepcidin to become a viable diagnostic
estrogen-dependent tumors. We then performed ovariectomies seven days biomarker for clinical uses.
after NMU injection to mimic post-menopausal biology. nDS implants loaded
with either TMX or PEG400 (negative control) were inserted under the left
abdominal mammary gland to determine the effects of nDS-TMX on tumor ILISA for the Diagnosis of Infectious Diarrhea
growth and tumor biomarkers. Utilizing LC/MS we were able to quantitatively
determine the amount of TMX released from the nDS into the plasma. Rats Poster Presentation. NEMB2016-6016
were also examined for palpable tumors to assess breast tumor incidence,
latency to onset, and multiplicity. Our results show that the nDS implant en-
ables the effective delivery of TMX in this breast tumor model. Further, this Linlin Zhang, Sheng Tong, Gang Bao, Rice University, Houston,
technology has the potential to rapidly provide long-term breast cancer pro- TX, United States
tection with significant improvement in the quality of life of patients at high
risk, thereby saving thousands of lives every year. Infectious diarrhea or gastroenteritis is one of the leading causes of death
worldwide, particularly in children. Viral, bacterial, or parasitic infection can
all lead to serious and long lasting syndrome. Early and accurate diagnosis
Nanotrap-based assay for absolute quantification of iron regula- and targeted therapy will undoubtedly improve the survival rate. A growing
tor-hepcidin from human bodily fluids list of biomarkers, including proteins, genes, and carbohydrates from both
the patients and the pathogens, has been indicated to have great potential
in early detection of infectious diarrhea. However, a more sensitive, specific,
Poster Presentation. NEMB2016-6082 and reliable method for clinical diagnosis is still lacking. In this study, we
developed an antibody-based iron oxide-linked immunosorbent assay (ILI-
Jia Fan, Houston Methodist Research Institute, Houston, TX, United SA) for the detection of virus- and bacteria-induced diarrhea. By combining
States, Hung-jen Wu, Texas A&M University, College Station, TX, nanotechnological tools with serum biomarkers for diagnosis and prognosis,
United States, Guangjun Nie, Yuliang Zhao, National Center for our method not only has the aforementioned sensitivity, specificity, and
Nanoscience and Technology, Beijing, China, Tony Y. Hu, Houston reliability, but also shows significant advantages over diagnostic methods
Methodist Research Insititute, Houston, TX, United States clinically used.
Rotavirus has been the most common cause of acute gastroenteritis requir-
Endogenous serum peptides that carry important information of disease
are considered to be great potential biomarkers for clinical diagnosis. ing hospitalization among children. However, immunity to rotavirus is still
very poorly understood. Tests including rotavirus-specific IgG, stool IgA, and
However, due to the extremely high dynamic range of protein concentra- neutralizing antibodies have been used in the past, but data in humans are
tion in serum and the interference of highly abundant and large proteins, conflicting. Serum IgA is the most widely used correlate. Here we devel-
the detection of the serum peptide biomarkers remains a challenge. Here- oped a novel assay for IgA detection, which takes advantage of the dense
in, we developed a silica nanopore-based (Nanotrap) assay to selectively
enrich and quantify a low-abundance peptide, hepcidin, from human body atom packing in iron oxide nanocrystals. By combining acid-dissolution of
nanocrystals and a ferrous ion-induced stoichiometric chromogenic reaction,
fluids (HBF). our method can easily achieve high signal amplification. To be brief, we
Hepcidin consists of 25 amino acids (Hep-25) that could down-regulating synthesized iron oxide nanoparticles of different sizes and conjugated to an-
cellular iron. Pathological excess or deficiency of hepcidin could lead to tibodies against human IgA. By optimizing various experimental conditions,
a variety of iron disorders and be used as a diagnostic tool in clinic. Thus,
we believe the measurement of hepcidin levels in HBF is urgently needed including the species of detection and capture antibodies (mouse, rabbit, or
goat), the concentration of the reducing agent (2-Mercaptoethylamine HCl)
to facilitate the personalized medicine. Several methods have been devel- for antibody reduction, the selection of antibody fragments (half IgG or Fab’)
oped for quantifying hepcidin. However, antibody-based method are lack for conjugation, the concentration of nanoparticles used for detection, and
of the selectivity to differentiate Hep-25 from the other two N-terminal trun- the incubation time, we can accurately reach the concentration of IgA as low
cated hepcidin isoforms, Hep-20 and Hep-22, which are not expected to
play significant roles in iron metabolism. In regards to the MS-based meth- as 1.5 pM with high reliability and repeatability. With nanoparticles of larger
diameter, we further increased our detection sensitivity to 0.2 pM.
ods still require large volume sample and time-consuming pre-treatment. Several bacterial pathogens can also cause serious diarrhea and lead to
In this study, we developed a high-throughput approach for peptides ex- death due to severe dehydration. Clostridium difficile (C. difficile) infection is
traction using Nanotraps with different nanotextures (pore size, surface, the most commonly reported case in hospitalized patients. Traditional stool
and structure) specifically and precisely tailored for hepcidin enrichment.
We further investigated the mechanisms of hepcidin enrichment in test measures stool leukocytes and lactoferrin levels, which provides low
78 nanopores, including size-exclusion, surface charge, and pore morphol- accuracy. In our study, we provide a novel and reliable diagnostic method
ogy effects, and provide a basis for understanding the interaction of the by assessing the level of C. difficile toxins in serum. Different from rotavirus
infection, we used biomarkers derived from the pathogens as the analyte,
target peptide with NPS thin films, which is highly useful for adapting