Extracellular vesicles (EVs) have entered the field of drug delivery as biological alternatives to synthetic nanocarriers, liposomes, and polymeric nanoparticles. These natural vehicles are being extensively investigated in therapeutic settings to treat various diseases, including cancer, [1][2][3][4][5][6][7][8][9][10] neurological disorders (Alzheimer's and Parkinson's diseases, stroke), [8,11] infectious diseases (meningitis, human immunodeficiency virus [HIV], and HIV-related dementia), [12][13][14][15][16] joint diseases (inflammatory arthritis), [17] as well as autoimmune [18] and cardiovascular diseases (atherosclerosis and heart attack). [19][20][21][22][23][24] EVs are known to be released by the most types of cells; their functions vary from waste disposal to transport of nucleic acids, lipids, and proteins to neighboring cells and distant organs. [25] Regarding using these vesicles for drug delivery, EVs are commonly considered as a combination of two types of vesicles, exosomes and microvesicles, that display size and compositional heterogeneity dependent on their subcellular origin. Exosomes (30-120 nm) are smaller in size and generated in multivesicular bodies. Microvesicles (50-500 nm) are larger and generated by outward budding of the plasma membrane. [26] Due to their overlapping sizes, surface markers, and lipid content, these two types of vesicles generally used together for drug delivery as a heterogeneous population.Recently, our laboratories developed novel drug delivery systems using EVs for the transport of different therapeutic molecules, including the small molecule anticancer agents paclitaxel and doxorubicin for the treatment of pulmonary metastatic cancer [27,28] and triple-negative breast cancer (TNBC). [29] We also

Tip-enhanced Raman spectroscopy (TERS) is a promising technique for structural studies of biological systems and biomolecules, owing to its ability to provide a chemical fingerprint with sub-diffraction-limit spatial resolution. This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. The high sensitivity achievable through TERS arises from the excitation of a localized surface plasmon resonance in a noble metal atomic force microscope (AFM) tip, which in combination with a metallic surface can produce huge enhancements in the local optical field. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements. Herein, a novel, biocompatible, highly enhancing surface is designed and fabricated based on few-monolayer mica flakes, mechanically exfoliated on a metal surface. These surfaces allow the formation of coupled plasmon enhancements for TERS imaging, while maintaining the biocompatibility and atomic flatness of the mica surface for high resolution AFM. The capability of these substrates for TERS is confirmed numerically and experimentally. We demonstrate up to five orders of magnitude improvement in TERS signals over conventional mica surfaces, expanding the sensitivity of TERS to a wide range of non-resonant biomolecules with weak Raman cross-sections. The increase in sensitivity obtained through this approach also enables the collection of nanoscale spectra with short integration times, improving hyperspectral mapping for these applications. These mica/metal surfaces therefore have the potential to revolutionize spectromicroscopy of complex, heterogeneous biological systems such as DNA and protein complexes.Tip-enhanced Raman scattering (TERS) combines atomic force microscopy (AFM) with Raman vibrational spectroscopy. AFM is a powerful technique for imaging on the single-molecule level, [1] and Raman spectroscopy can distinguish biological constituents and their organization and conformation through their vibrational signatures. [2][3] TERS therefore has significant potential for resolving open questions in heterogeneous, multicomponent biological systems such as DNA-protein complexes for transcription and repair. [4][5][6][7][8][9][10][11][12] However, there remain significant challenges to its widespread implementation and capacity to reliably address biological and clinical questions.The sensitivity of TERS arises from the excitation of a localized plasmon resonance in a noble metal AFM tip, which can provide an order of magnitude enhancement in the optical field close to the tip. Most TERS studies are additionally performed on noble metal surfaces, [8,[12][13][14] where plasmonic coupling between tip and surface further increases the optical field enhancement. This approach yields single-molecule and even intra-molecular sensitivity in small organic molecules. [15...

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