Quantitative Detection of Biological Nanovesicles in Drops of Saliva Using Microcantilevers

Extracellular nanovesicles (EVs) are lipid-based vesicles secreted by cells and are present in all bodily fluids. They play a central role in communication between distant cells and have been proposed as potential indicators for the early detection of a wide range of diseases, including different types of cancer. However, reliable quantification of a specific subpopulation of EVs remains challenging. The process is typically lengthy and costly and requires purification of relatively large quantities of biopsy samples. Here, we show that microcantilevers operated with sufficiently small vibration amplitudes can successfully quantify a specific subpopulation of EVs directly from a drop (0.1 mL) of unprocessed saliva in less than 20 min. Being a complex fluid, saliva is highly non-Newtonian, normally precluding mechanical sensing. With a combination of standard rheology and microrheology, we demonstrate that the non-Newtonian properties are scale-dependent, enabling microcantilever measurements with a sensitivity identical to that in pure water when operating at the nanoscale. We also address the problem of unwanted sensor biofouling by using a zwitterionic coating, allowing efficient quantification of EVs at concentrations down to 0.1 μg/mL, based on immunorecognition of the EVs’ surface proteins. We benchmark the technique on model EVs and illustrate its potential by quantifying populations of natural EVs commonly present in human saliva. The method effectively bypasses the difficulty of targeted detection in non-Newtonian fluids and could be used for various applications, from the detection of EVs and viruses in bodily fluids to the detection of molecular clusters or nanoparticles in other complex fluids

Optical microscopy images showing membrane swelling, granulation and blebbing of gastric and colorectal cancer cells after the addition of melittin.

A) AGS, B) COLO205 and C) HCT-15 cells were grown on poly-D-lysine coated coverslips overnight and imaged in PBS using a light microscope before (T0) and after (T0.5, 30 seconds; T1, 1 minute; T5, 5 minutes; T10, 10 minutes; T15, 15 minutes) the addition of 20 μg/mL melittin. After melittin addition, the effects on the cells can be seen almost immediately. The yellow arrow points to membrane changes. Scale bars represent 50 microns.</p

Florescence images showing Live/Dead staining of gastric and colorectal cancer cell lines following short melittin treatments.

A) AGS, B) COLO205 and C) HCT-15 cells were grown on poly-D-lysine coated coverslips overnight and treated with melittin at 10 μg/mL or 20 μg/mL for either 1 minute or 15 minutes. All cells were stained with Calcein-AM live stain (green) and EthD-1 dead stain (red). Average viability was determined after 1 minute for AGS (53% for 10 μg/mL and 1% for 20 μg/mL), COLO205 (59% for 10 μg/mL and 12% for 20 μg/mL), and HCT-15 (62% for 10 μg/mL and 51% for 20 μg/mL), and after 15 minutes for AGS (3% for 10 μg/mL and 0% for 20 μg/mL), COLO205 (38% for 10 μg/mL and 3% for 20 μg/mL), and HCT-15 (34% for 10 μg/mL and 0% for 20 μg/mL). Viability for all negative controls was 96–99%. Scale bars represent 100 microns.</p

The membrane effects of melittin on gastric and colorectal cancer - Fig 4

Membrane changes before and after melittin treatment at different time points for A) AGS, B) COLO205 and C) HCT-15. After the addition of melittin, cells undergo membrane swelling followed by membrane blebbing. Cells were grown on poly-D-lysine coated coverslips overnight and stained with DIL membrane dye (red) and imaged by confocal microscopy before (T0) and after (T0.5, 30 seconds; T1, 1 minute; T5, 5 minutes; T10, 10 minutes; T15, 15 minutes) the addition of 20 μg/mL melittin. The yellow arrow points to membrane changes. Scale bars represent 10 microns.</p

The membrane effects of melittin on gastric and colorectal cancer

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6–24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 μg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin.</div

The membrane effects of melittin on gastric and colorectal cancer - Fig 6

SEM images of cancer cells showing differences in cellular morphology before and after melittin treatment (10 μg/mL and 20 μg/mL) for A) AGS, B) COLO205 and C) HCT-15. Cells were grown on poly-D-lysine coated silicon chips overnight and treated with melittin for 1 minute or 15 minutes. Yellow arrows point to cellular and membrane damage. Scale bars are as indicated in the respective images.</p

The membrane effects of melittin on gastric and colorectal cancer - Fig 5

AFM images showing the morphology of AGS cells A) under normal conditions (no treatment) and B) after 20 μg/mL melittin treatment. AGS cells were grown on poly-D-lysine coated coverslips overnight, treated with 20 μg/mL melittin treatment for 1 minute and then fixed in 2.5% gluteraldehyde or 8% formaldehyde. Cells were imaged in PBS on the Asylum MFP-3D. Blue arrows indicate cellular damage. Scale bars are as indicated in the respective images.</p

The membrane effects of melittin on gastric and colorectal cancer - Fig 1

(i) Propidium iodide (PI) uptake showing death of gastric and colorectal cancer cell lines following a 4-hour melittin treatment. Cells were treated with 0.5–20 μg/mL melittin and the positive control was treated with 0.1% Triton X-100. Data shown as mean ± SD where n = 4. *** represents p = (ii) Florescence images showing Live/Dead staining of gastric and colorectal cancer cell lines following a 4-hour melittin treatment. A) AGS, B) COLO205 and C) HCT-15 cells were grown on poly-D-lysine coated coverslips overnight and treated with melittin at 5 μg/mL, 10 μg/mL and 20 μg/mL for 4 hours. The positive control was treated with Triton X-100. All cells were stained with Calcein-AM live stain (green) and EthD-1 dead stain (red). Average viability was determined for AGS (78% for 5 μg/mL, 17% for 10 μg/mL and 0% for 20 μg/mL), COLO205 (86% for 5 μg/mL, 40% for 10 μg/mL and 0% for 20 μg/mL), and HCT-15 (73% for 5 μg/mL, 50% for 10 μg/mL and 4% for 20 μg/mL). Viability for all negative and positive controls was 99–100% and 0% respectively. Scale bars represent 100 microns.</p

Characterizing the Dynamic Disassembly/Reassembly Mechanisms of Encapsulin Protein Nanocages

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterize the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: T = 1 (24 nm), T = 3 (32 nm), and T = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all three encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest T = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins showed a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins’ disassembly/reassembly dynamics, thus informing their future design, modification, and application

Compositional Design of Surface Oxides in Gallium–Indium Alloys

Room-temperature liquid metal alloys encompass a highly versatile family of materials possessing a unique set of chemical, electronic, biological, and mechanical properties. The surface oxide of liquid metals has a direct influence on these properties and is often composed of one of the major alloy components (i.e., gallium or indium). However, this is not a foregone conclusion, as the identity of the surface oxide can be altered by the addition of minority elements into the liquid metal. Through judicious choice of a minority alloying metal, the composition of the oxide and therefore the resulting molten alloy’s properties are significantly modified. We demonstrate this by adding a small amount (∼5%) of several thermodynamically favorable alloying elements (X = Zn, Mg, Al) to eutectic gallium indium (EGaIn), resulting in a new class of alloys with designed surface oxide compositions that we term XGaIn. Using both STEM-EDS and XPS, XGaIn alloys are shown to form oxide layers enriched in the lowest-redox element as expected based on the thermodynamics of the alloy system. This approach is shown to be generalizable across both Ga and non-Ga-based liquid metal alloy compositions. XGaIn alloys with added Zn and Mg are shown to have strong antimicrobial activity, which has exciting implications for the development of flexible electronic medical devices and sensors