Pioneering High-Efficiency Extracellular Nanocarrier(s) Isolation in Biofluids

As per the latest World Health Organization statistics, the staggering toll of over 16 million lives lost to cancer and cardiovascular diseases worldwide underscores the urgent necessity for pioneering solutions for early diagnostics and therapeutics. One such approach is to characterize extracellular nanocarriers, including extracellular vesicles (EVs), lipoproteins (LLPs), and ribonucleoproteins (RNPs) that are present in various biofluids. Changes in the composition or quantity of extracellular nanocarriers can serve as biomarkers for diseases such as cancer, cardiovascular diseases, and neurodegenerative disorders. This makes them valuable for early disease detection and monitoring. Moreover, these nanocarriers play a vital role in intercellular communication. They carry and transfer biomolecules like proteins, lipids, and RNAs between cells and by studying them, scientists can have a better understanding of the mechanisms of various biological processes, including immune responses, cancer progression, and tissue regeneration. For example, the study of EVs in cancer can reveal how tumor cells manipulate their microenvironment to promote tumor growth and metastasis. Furthermore, because of their natural origin and ability to encapsulate and protect their cargo, EVs are currently being explored as potential drug delivery systems. They can be engineered to carry therapeutic agents, targeting specific cells or tissues, thus improving the efficiency and reducing the side effects of treatments. In fact, currently the https://www.clinicaltrials.gov site has listed as many as 224 ongoing clinical studies which contain the word “exosome” showcasing the enormous potential of extracellular nanocarriers.

To advance this field, a new study published in ACS Nano Journal by Dr. Himani Sharma, Dr. Vivek Yadav, Professor Crislyn D’Souza-Schorey, Professor David Go, Associate Professor Satyajyoti Senapati and Professor Hsueh-Chia Chang from the Department of Chemical and Biomolecular Engineering at University of Notre Dame in the United States, developed a novel technique for isolating extracellular nanocarriers, including ribonucleoproteins, from various biofluids such as plasma, urine, and saliva. The new technique is centered around a scalable high-throughput continuous isoelectric fractionation platform (CIF), which enables the bias-free and high-yield separation of nanocarriers based on their distinct isoelectric points. This platform is significantly more efficient than traditional methods, achieving high purity and yield in a fraction of the time. The team developed an innovative system that generates a linear pH gradient using a bipolar membrane. This membrane facilitates water splitting, resulting in the formation of H₃O⁺ and OH⁻ ions that create the pH gradient. The gradient is stabilized by a flow mechanism without the need for ampholytes, which are typically used to maintain pH gradients in other methods. The platform was automated with machine learning procedures to allow recalibration for different physiological fluids and nanocarriers, thus enhancing its precision and versatility.

The researchers utilized the CIF platform to separate nanocarriers in biofluids based on their distinct isoelectric points. The method was tested on ribonucleoproteins, lipoproteins, and extracellular vesicles. The authors conducted experiments using plasma, urine, and saliva samples. This involved processing these fluids through the CIF platform to isolate and collect the nanocarriers. The team measured the purity and yield of the isolated nanocarriers. The new bias-free isolation method demonstrated a throughput about 1000 times higher than previous methods, achieving separation in as little as 30 minutes, as opposed to the day-long protocols required by existing standards. They also demonstrated that their method could achieve high purity which is critical in clinical diagnostics and biomarker discovery (>93% for plasma, >95% for urine, and >97% for saliva) and high yield (>78% for plasma, >87% for urine, and >96% for saliva) which makes it highly versatile and applicable to a broad range of clinical and research settings. They compared the efficiency and effectiveness of the CIF platform with traditional methods, which often involve multistage ultracentrifugation and ultrafiltration.

In conclusion, the new study demonstrated that the CIF platform could efficiently and effectively isolate nanocarriers from biofluids, outperforming existing gold-standard methods in terms of speed, purity, and yield. Moreover, the new platform’s high efficiency and precision make it a valuable tool for personalized medicine, enabling rapid analysis of patient samples for specific biomarkers. Future developments could include further automation, integration with other analytical techniques for direct analysis of isolated nanocarriers, and adaptation for other types of nanoscale biomolecules. This platform has the potential to significantly accelerate research in extracellular communication and advance the field of liquid biopsy in cancer diagnostics.