This study reports a deep-reactive-ion-etched microneedle device (ExoNeedle chip) coated with a hybrid hydrogel that can directly capture cancer-associated extracellular vesicles (EVs) in interstitial fluid (ISF) in a minimally invasive manner, as opposed to conventional liquid biopsy methods involving sample extraction and EV isolation in-vitro or tissue biopsies for melanoma. Tumor derived EVs give insight into a tumor’s mutational burden and provide both prognostic and diagnostic value as a liquid biopsy tool. Melanoma is an aggressive cancer with a lack of promising markers for early detection, onset of metastasis, and monitoring of recurrence. The ExoNeedle chip is intended to pierce through the epidermis and parts of the dermis to isolate melanoma secreted EVs from the ISF to alleviate these unmet needs. The microneedle arrays were created through the Deep Reactive Ion Etching (DRIE) process and a subsequent wet etching step to sharpen the needles. A 4 inch silicon wafer spin coated with 3 microns of positive photoresist was lithographically patterned and etched by DRIE using the standard Bosch processes to expose arrays of cylindrical pillars. Next, the cylindrical features were sharpened using an isotropic wet etch consisting of a mixture of hydrofluoric and nitric acid. The microneedle arrays were made and cleaved into individual patches. Meanwhile, hybrid hydrogel solution was made from Polyvinyl alcohol (PVA) and alginate by dissolving each of them separately in water under heat, followed by mixing the solutions. After centrifuging and filtering the hydrogel, it was conjugated with Annexin V (Av) protein to give it affinity for cancerous exosomes. Lastly, this hydrogel complex was added onto the microneedle patches and gelated with the help of a humidifier that deposited Ca2+ ion vapor over the hydrogel. The microneedles were first optimized and validated using melanoma cell line secreted EVs. Melanoma EVs were successfully isolated and quantified to evaluate the efficacy of the microneedle patches yielding above an 80% capture efficiency from purified serum samples. The microneedle patches were DiO stained, fluorescently imaged, visualized in a scanning electron microscope, and protein concentration was determined respectfully. Additionally, the functional hydrogel coatings on the microneedles are dissolved in ethylenediaminetetraacetic acid (EDTA), subsequently unbinding EVs from Av due to EDTA-based Ca2+ chelation. The dissolved solution is then used for enumeration and quantification of EVs by nanoparticle tracking analysis. We are further validating these patches in skin mimicking models and patient derived xenograft models of melanoma. The technology developed in this study for the isolation of melanoma EVs can broadly pave the way for minimally invasive point of care cancer diagnostics and tracking for melanoma patients. Citation Format: Scott M. Smith, Abha Kumari, Thiago Reis, Yoon-Tae Kang, Sunitha Nagrath. Deep reactive ion etched microneedle array for in-vivo melanoma cancer monitoring via cancer exosome isolation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3297.

communication. Their membrane contains and protects proteins, nucleic acids, and metabolites. Hence, they can serve as active cargo delivery vehicles and messengers of genetic information. [1] There has been an increased interest in the clinical use of EVs as biomarkers due to their significance in cellular signaling, disease progression, and therapeutics. The isolation and characterization of exosomes allow their use as reliable biomarkers for minimally invasive disease diagnosis, called liquid biopsy. One of the promising opportunities for disease diagnosis is the diagnosis of cancer since EV secretion is increased by many cancer types. Their presence in a variety of biosamples -that is, blood, urine, saliva -makes them an attractive avenue for exploration for liquid biopsy to provide a simple, in vitro analysis of a patient's tumor status. [2,3] Deregulated cellular metabolism has been established as a hallmark of cancer which supports the use of metabolite characterization as a sensitive, inexpensive, and origin-agnostic tool for minimally-invasive diagnosis of cancer. [4] However, the large amounts of biosample contaminants and the size of the desired endosome-derived small EVs (sEV), also called exosomes, at just 30-150 nm diameter makes isolation difficult and challenging [5] using conventional EV isolation methods.Polydimethylsiloxane (PDMS) is an inexpensive robust polymer that is commonly used as the fundamental fabrication material for soft-lithography-based microfluidic devices. Owing to its versatile material properties, there are some attempts to use PDMS as a porous 3D structure for sensing. However, reliable and easy fabrication has been challenging along with the inherent hydrophobic nature of PDMS hindering its use in biomedical sensing applications. Herein, a cleanroom-free inexpensive method to create 3D porous PDMS structures, "ExoSponge" and the effective surface modification to functionalize its 3D porous structure is reported. The ability of ExoSponge to recover cancer-associated extracellular vesicles (EVs) from complex biological samples of up to 10 mL in volume is demonstrated. When compared to ultracentrifugation, the ExoSponge shows a significant increase in cancer EV isolation of more than 210%. Targeted ultra-high pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further employed to profile 70 metabolites in the EVs recovered from the lung cancer patient's plasma. The resulting profiles reveal the potential intraexosomal metabolite biomarker, phenylacetylglutamine (PAG), in non-small cell lung cancer. The high sensitivity, simple usage, and cost-effectiveness of the ExoSponge platform creates huge potential for rapid, economical and yet specific isolation of exosomes enabling future diagnostic applications of EVs in cancers.

Small extracellular vesicles, often termed as “exosomes” carry informative cargo containing proteins and lipids, reflective of their cellular origin. Thus, they are promising biomarkers for early diagnosis of cancer. However, conventional profiling methods like quantitative polymerase chain reaction (qPCR) require complex procedures, thereby limiting the analytical sensitivity of exosomes for liquid biopsies. Here, we demonstrate a sensitive microfluidic device (CDEXO) that isolates and profiles cancer-associated exosomes directly from blood plasma using assembled chiral gold nanoparticles (AuNPs). The unique changes in chiral signals are associated with specific biomolecules on the exosomes’ membranes. Thus, we can distinguish exosomes of lung cancer patients from those of healthy individuals and detect mutated EGFR proteins on the membrane. Hence, this low-cost microfluidic device is an attractive technique for rapid, sensitive, and versatile profiling of various extracellular vesicles. Methods: The top layer of CDEXO devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The bottom glass slide was functionalized with a layer-by-layer assembly of cationic poly(dimethyl diallyl ammonium chloride) and anionic polystyrene sulfonate. AuNPs were prepared by adding gold nanoplates to a growth solution. Next, AuNPs were conjugated with biotinylated Annexin-V, using Neutravidin-biotin chemistry. Exosomes were harvested from lung cancer cell lines (A549, H1650, H3255) and lung fibroblasts (MRC5) and spiked into the CDEXO chip. EDTA was used to release the captured exosomes and quantified using nanoparticle tracking analysis (NTA). CD spectra were measured by spectrometry. Imaging of AuNPs and exosomes were done using scanning electron microscope (SEM). Results: CDEXO captures cancer-associated exosomes with an efficiency of 81.1±1.5%. H3255 derived exosomes that exhibited EGFR point mutation showed the greatest change in spectral signals from the baseline, followed by A549 (wild type), H1650, (EGFR exon19 deletion) and MRC5 (healthy). The CD responses were measurable at exosome numbers as low as 100. Further validation with 19 lung cancer patients showed an average 40% change in chiral signals from isolated exosomes that were 5.6 times higher in patients than healthy donors. Conclusions: A microfluidic device with chiral AuNPs allows sensitive and accurate detection of lung cancer-associated exosomes by conjugation with Annexin V. The resulting strong CD peaks that arise from specific interactions between exosomal surface proteins and chiral AuNPs facilitate in-depth profiling of target exosomes, including EGFR mutation expression. Citation Format: Yoon-Tae Kang, Ji-Young Kim, Emine Sumeyra Turali-Emre, Hee-Jeong Jang, Minjeong Cha, Abha Kumari, Colin Palacios-Rolston, Chitra Subramanian, Emma Purcell, Sarah Owen, Chung-Man Lim, Rishindra Reddy, Shruthi Jolly, Nithya Ramnath, Nicholas A. Kotov, Sunitha Nagrath. Chiroptical detection and mutation analysis of cancer-associated extracellular vesicles in microfluidic devices with oriented chiral nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 994.