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Technical Program TRACK 1

clinic. In summary, CBN represent a promising solution for the identification first examples of active targeting of graphene-based nanomaterials. In ad-
of inflamed vasculature through a variety of imaging modalities. dition, we reported the first chelator-free radiolabeling of RGO nanosheets
with Cu, which provided important guidance for the future research on the
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10:20am Graphene: Tumor Targeting and Chelator-Free Radio- radiolabeling and in vivo applications of graphene-based nanomaterials.
labeling
10:40am Improving the Diagnostic Sensitivity for Infectious
Technical Presentation. NEMB2016-5964 Pathogens by Combinatorial Quantum Dot Barcoding

Sixiang Shi, Cheng Xu, University of Wisconsin-Madison, Madison, Technical Presentation. NEMB2016-5912
WI, United States, Kai Yang, Soochow University, Suzhou, Jiangsu,
China, Robert J. Nickles, University of Wisconsin-Madison, Madi- Jisung Kim, University of Toronto, Toronto, ON, Canada, Mia Bion-
son, WI, United States, Zhuang Liu, Soochow University, Suzhou, di, Jordan Feld, University Health Network, Toronto, ON, Canada,
Jiangsu, China, Weibo Cai, University of Wisconsin-Madison, Madi- Warren Chan, University of Toronto, Toronto, ON, Canada
son, WI, United States
Detection of asymptomatic infections requires high diagnostic accuracy in
Objectives: Graphene, an emerging nanomaterial with single-layered car- order to promptly quarantine infected individuals, prevent further spread of
bon atoms in two dimensions has attracted tremendous interest, due to its the disease, and deliver effective treatment to those with clinical disease.
unique electronic, optical, mechanical and chemical properties, and has While sensitivity is one of the important measures of diagnostic accuracy,
been applied as a versatile platform for cancer imaging and therapy. Our many studies have focused on improving the analytical sensitivity, the min-
goal was to employ graphene oxide (GO) and reduced graphene oxide imum amount of detectable analyte, to lower the limit of detection (LOD).
(RGO) for in vivo tumor vasculature targeting via conjugating different tar- However, strategies to improve the clinical sensitivity, the probability of a test
geting ligands, and to quantitatively evaluate the tumor targeting efficacy to correctly identify diseased patients as positive, have only been explored
with positron emission tomography (PET). In addition, novel chelator-free to a limited extent. These two parameters relate to each other as the level
radiolabeling was also investigated to directly label 64Cu onto the graphene of clinical sensitivity may be reduced by poor analytical sensitivity because
surface based on transition metal-pi interactions. By eliminating the influ- some diseases, especially at the early stage of infection, present insufficient
ences of chelator, chelator-free radiolabeling can maintain the native prop- level of biomarker and can lead to false-negative test results. Apart from
erties (e.g. size, structure, drug loading capacity and pharmacokinetics) of the poor analytical sensitivity, viruses exist as quasi-species and present
nanoparticles, which enables a more precise control over their in vivo fate. sequence variations accumulated from high mutation rates, representing a
major challenge to the development of nucleic acid-based diagnostic tests.
Methods: RGO and GO nanosheets, with amino group-terminated PEG
chains on the surface, were conjugated to NOTA (1,4,7-triazacyclo- Here, we combined recent advances in Quantum Dot (QD) barcoding tech-
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nonane-1,4,7-triacetic acid) for Cu (t = 12. 7 h) labeling and TRC105 (an an- nology with Recombinase Polymerase Amplification (RPA) to demonstrate
1/2
tibody that binds to CD105, a receptor overexpressed on tumor vasculature) enhancement in diagnostic sensitivity with Hepatitis B Virus (HBV) infected
or VEGF121 (a naturally occurring protein that bind to VEGFR, another vas- patient samples. RPA was used to improve analytical sensitivity by enriching
cular marker of tumor angiogenesis) for tumor vasculature targeting. FACS the amount of target DNA, while multiple regions within the genome were
analysis, size measurements, and serum stability studies were performed detected via QD barcode-based multiplexed assay to further improve clini-
to characterize the RGO and GO conjugates. In vivo serial PET imaging and cal sensitivity. As opposed to polymerase chain reaction (PCR) that requires
ex vivo biodistribution studies were carried out to evaluate tumor targeting expensive thermocycling steps, RPA operates at a low constant tempera-
efficacy and pharmacokinetics of the nanoconjugates. Chelator-free radio- ture, which will be advantageous in resource-limited settings. Furthermore,
labeling of RGO and GO nanosheets was conducted by directly mixing the QD barcodes offer a promising diagnostic platform for simultaneous detec-
nanosheets with 64Cu at different concentrations and temperatures. The la- tion of multiple amplification regions within HBV genome due to their unique
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beling stability and imaging capacity of Cu-RGO-PEG was confirmed by se- optical signatures.
rum stability studies and in vivo PET imaging. Anti-cancer drug doxorubicin
(DOX)-loaded RGO-PEG was also investigated to understand the influence A total of 72 clinical samples with a diverse background were used to rep-
of drug loading on the labeling efficiency. resent various phases of HBV infection and disease course. The viral DNA
was first extracted from patient serum using magnetic microbeads, four
conserved regions of the extracted genome were amplified by RPA, ampli-
Results: RGO and GO nanosheets had sizes of ~20-100 nm and superb
stability after PEGylation. Serial PET imaging showed rapid and persistent fied products were detected by multiplexed QD barcode assay, and finally
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uptake of Cu-NOTA-RGO-TRC105 (5.0±0.6, 5.6±0.2, 5.7±0.2, 4.5±0.4, and fluorescence signals were measured via flow cytometry. In a blinded exper-
4.0±0.5 %ID/g at 0.5, 3, 6, 24, and 48 h post-injection; n = 4) in 4T1 tumors iment, clinical sensitivity was compared between single-plexed and multi-
and Cu-NOTA-GO-VEGF121 (6.5±1.7, 8.2±1.4, 7.7±1.4, 5.7±0.8, and 4.7±0.7 plexed detection schemes, and our combinatorial analysis demonstrates a
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%ID/g at 0.5, 3, 6, 16, and 24 post-injection; n=4) in U87MG tumors, demon- systematic increase in clinical sensitivity from 54.9-66.7% to 80.4-90.5% with
strating excellent tumor contrast and was several fold higher than the multiplexed detection for diagnostic purposes. We also demonstrate devel-
non-targeted RGO and GO conjugates. Various in vivo (e.g. blocking with opment of Receiver Operating Characteristic (ROC) curves, which identified
TRC105 or VEGF121), in vitro (e.g. flow cytometry), and ex vivo (e.g. histolo- cutoff intensity levels to achieve 100% specificity for all four regions. Thus,
gy) studies further confirmed the targeting specificity. By avoiding the use these results suggest that we can achieve near perfect clinical diagnosis of
of NOTA, 40-80% Cu was intrinsically labeled onto RGO-PEG at different patients infected with HBV by using multiple QD barcodes in the detection
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concentrations and temperatures, while the labeling yield of GO-PEG was process.
only 5-20% due to incomplete graphene structure with less pi-bonds. The
labeling stability of Cu-RGO-PEG (>> 75% after 24 h incubation) was com-
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parable to Cu-NOTA-RGO-PEG as indicated by the serums stability studies.
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Prominent tumor uptake was achieved via passive targeting alone, suggest-
ing excellent imaging capacity of 64Cu-RGO-PEG. After loading DOX, the
labeling yield of RGO(DOX)-PEG was slightly decreased (>> 30 %) due to the
competition between DOX and 64Cu for pi-bonds on the surface of RGO,
further validating the existence of Cu-pi interactions.
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Conclusions: We reported the vasculature targeting of RGO and GO conju-
gates, with enhanced tumor uptake and excellent specificity, providing the