Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

A Smart Intracellular Self-Assembling Bioorthogonal Raman Active Nanoprobe for Targeted Tumor Imaging.

Inspired by the principle of in situ self-assembly, the development of enzyme-activated molecular nanoprobes can have a profound impact on targeted tumor detection. However, despite their intrinsic promise, obtaining an optical readout of enzyme activity with high specificity in native milieu has proven to be challenging. Here, a fundamentally new class of Raman-active self-assembling bioorthogonal enzyme recognition (nanoSABER) probes for targeted tumor imaging is reported. This class of Raman probes presents narrow spectral bands reflecting their vibrational fingerprints and offers an attractive solution for optical imaging at different bio-organization levels. The optical beacon harnesses an enzyme-responsive peptide sequence, unique tumor-penetrating properties, and vibrational tags with stretching frequencies in the cell-silent Raman window. The design of nanoSABER is tailored and engineered to transform into a supramolecular structure exhibiting distinct vibrational signatures in presence of target enzyme, creating a direct causality between enzyme activity and Raman signal. Through the integration of substrate-specific for tumor-associated enzyme legumain, unique capabilities of nanoSABER for imaging enzyme activity at molecular, cellular, and tissue levels in combination with machine learning models are shown. These results demonstrate that the nanoSABER probe may serve as a versatile platform for Raman-based recognition of tumor aggressiveness, drug accumulation, and therapeutic response.

Shining Light on Osteoarthritis: Spatially Offset Raman Spectroscopy as a Window into Cartilage Health.

Articular cartilage is a complex tissue, and early detection of osteoarthritis (OA) is crucial for effective treatment. However, current imaging modalities lack molecular specificity and primarily detect late-stage changes. In this study, we propose the use of Spatially Offset Raman Spectroscopy (SORS) for non-invasive, depth-dependent, and molecular-specific diagnostics of articular cartilage. We demonstrate the potential of SORS to penetrate deep layers of cartilage, providing a comprehensive understanding of disease progression. Our SORS measurements were characterized and validated through mechanical and histological techniques, revealing strong correlations between spectroscopic measurements and both Young's modulus and depth of cartilage damage. By longitudinally monitoring enzymatically degraded condyles, we further developed a depth-dependent damage-tracking method. Our analysis revealed distinct components related to sample depth and glycosaminoglycan (GAG) changes, offering a comprehensive picture of cartilage health. Collectively, these findings highlight the potential of SORS as a valuable tool for enhancing OA management and improving patient outcomes.

Targeted Enzyme Activity Imaging with Quantitative Phase Microscopy.

Quantitative phase imaging (QPI) is a powerful optical imaging modality for label-free, rapid, and three-dimensional (3D) monitoring of cells and tissues. However, molecular imaging of important intracellular biomolecules such as enzymes remains a largely unexplored area for QPI. Herein, we introduce a fundamentally new approach by designing QPI contrast agents that allow sensitive detection of intracellular biomolecules. We report a new class of bio-orthogonal QPI-nanoprobes for in situ high-contrast refractive index (RI) imaging of enzyme activity. The nanoprobes feature silica nanoparticles (SiO2 NPs) having higher RI than endogenous cellular components and surface-anchored cyanobenzothiazole-cysteine (CBT-Cys) conjugated enzyme-responsive peptide sequences. The nanoprobes specifically aggregated in cells with target enzyme activity, increasing intracellular RI and enabling precise visualization of intracellular enzyme activity. We envision that this general design of QPI-nanoprobes could open doors for spatial-temporal mapping of enzyme activity with direct implications for disease diagnosis and evaluating the therapeutic efficacy.

Surface-enhanced Raman scattering: An emerging tool for sensing cellular function.

Continuous long-term intracellular imaging and multiplexed monitoring of biomolecular changes associated with key cellular processes remains a challenge for the scientific community. Recently, surface-enhanced Raman scattering (SERS) has been demonstrated as a powerful spectroscopic tool in the field of biology owing to its significant advantages. Some of these include the ability to provide molecule-specific information with exquisite sensitivity, working with small volumes of precious samples, real-time monitoring, and optimal optical contrast. More importantly, the availability of a large number of novel Raman reporters with narrower full width at half maximum (FWHM) of spectral peaks/vibrational modes than conventional fluorophores has created a versatile palette of SERS-based probes that allow targeted multiplex sensing surpassing the detection sensitivity of even fluorescent probes. Due to its nondestructive nature, its applicability has been recognized for biological sensing, molecular imaging, and dynamic monitoring of complex intracellular processes. We critically discuss recent developments in this area with a focus on different applications where SERS has been used for obtaining information that remains elusive for conventional imaging methods. Current reports indicate that SERS has made significant inroads in the field of biology and has the potential to be used for in vivo human applications. This article is categorized under: Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

Broadband SERS Enhancement by DNA Origami Assembled Bimetallic Nanoantennas with Label-Free Single Protein Sensing.

Engineering hotspots in surface-enhanced Raman spectroscopy (SERS) through precisely controlled assembly of plasmonic nanostructures capable of expanding intense field enhancement are highly desirable to enhance the potentiality of SERS as a label-free optical tool for single molecule detection. Inspired by DNA origami technique, we constructed plasmonic dimer nanoantennas with a tunable gap decorated with Ag-coated Au nanostars on origami. Herein, we demonstrate the single-molecule SERS enhancements of three dyes with emission in different spectral regions after incorporation of single dye molecules in between two nanostars. The enhancement factors (EFs) achieved in the range of 109-1010 for all the single dye molecules, under both resonant and nonresonant excitation conditions, would enable enhanced photostability during time-series measurement. We further successfully explored the potential of our designed nanoantennas to accommodate and detect a single thrombin protein molecule after selective placement in the wide nanogap of 10 nm. Our results suggest that such nanoantennas can serve as a broadband SERS enhancer and enable specific detection of target biological molecules with single-molecule sensitivity.

Advancing Raman spectroscopy from research to clinic: Translational potential and challenges.

Raman spectroscopy has emerged as a non-invasive and versatile diagnostic technique due to its ability to provide molecule-specific information with ultrahigh sensitivity at near-physiological conditions. Despite exhibiting substantial potential, its translation from optical bench to clinical settings has been impacted by associated limitations. This perspective discusses recent clinical and biomedical applications of Raman spectroscopy and technological advancements that provide valuable insights and encouragement for resolving some of the most challenging hurdles.

DNA-Origami-Based Assembly of Au@Ag Nanostar Dimer Nanoantennas for Label-Free Sensing of Pyocyanin.

Early-stage detection of diseases caused by pathogens is a prerequisite for expedient patient care. Due to the limited signal-to-noise ratio, molecular diagnostics needs molecular signal amplification after recognition of the target molecule. In this present study, we demonstrate the design of plasmonically coupled bimetallic Ag coated Au nanostar dimers with controlled nanogap using rectangular DNA origami. We further report the utility of the designed nanostar dimer structures as efficient SERS substrate for the ultrasensitive and label-free detection of the pyocyanin molecule, which is a biomarker of the opportunistic pathogenic bacteria, Pseudomonas aeruginosa. The experimental results showed that the detection limit of pyocyanin with such nanoantenna based biosensor was 335 pM, which is much lower than the clinical range of detection. Thus, fast, sensitive and label-free detection of pyocyanin at ultralow concentration in an infected human body can pave a facile route for early stage warning for severe bacterial infections.

Noble copper-silver-gold trimetallic nanobowls: An efficient catalyst.

We demonstrated the design of tiny bowls of copper-silver-gold (Cu-Ag-Au) alloy type noble trimetallic nanocrystals with a unique shape. All the structural characterizations confirm the presence of copper (Cu), silver (Ag), and gold (Au) in the trimetallic nanobowls. Finally, we examined the catalytic efficiency of trimetallic Cu-Ag-Au nanobowls for reduction of 4-nitrophenol to 4-aminophenol and found that these nanobowls were 14, 23 and 43-fold more active than each of the constituent metals, Au, Cu and Ag, respectively.

White light emission from a mixture of silicon quantum dots and gold nanoclusters and its utilities in sensing of mercury(ii) ions and thiol containing amino acid.

White light emitting mixture (WLEM) was produced by controlled mixing of blue emitting silicon quantum dots (Si QDs) and orange red emitting gold nanoclusters (Au NCs). The chromaticity color co-ordinate of the WLEM studied using CIE (Commission Internationale del'Eclairage) diagram was found to be (0.33, 0.32), which was very close to that of perfect white light emitting source. The WLEM can also be achieved in the form of gel, solid and film with nearly the same CIE co-ordinates which enhances its utility as white light-emitting source in solid state devices. The reversible and thermo-responsive behaviour of the WLEM broadens its application in thermal sensing. Furthermore, the system was found to be showing fast, sensitive and selective detection of Hg2+ ions and thiol containing amino acid cysteine.

DNA Origami Directed Au Nanostar Dimers for Single-Molecule Surface-Enhanced Raman Scattering.

We demonstrate the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami. Au nanostars with uniform and sharp tips were immobilized on rectangular DNA origami dimerized structures to create nanoantennas containing monomeric and dimeric Au nanostars. Single Texas red (TR) dye was specifically attached in the junction of the dimerized origami to act as a Raman reporter molecule. The SERS enhancement factors of single TR dye molecules located in the conjunction region in dimer structures having interparticle gaps of 7 and 13 nm are 2 × 1010 and 8 × 109, respectively, which are strong enough for single analyte detection. The highly enhanced electromagnetic field generated by the plasmon coupling between sharp tips and cores of two Au nanostars in the wide conjunction region allows the accommodation and specific detection of large biomolecules. Such DNA-directed assembled nanoantennas with controlled interparticle separation distance and stoichiometry, and well-defined geometry, can be used as excellent substrates in single-molecule SERS spectroscopy and will have potential applications as a reproducible platform in single-molecule sensing.

Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.

Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPµTP). The assay was developed on the APPµTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin-HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7-170 ng mL(-1) while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL(-1) respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R(2) = 0.8081, p-value < 0.0001) and absorbance (R(2) = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5'-NH2 terminus was immobilised covalently to the APPµTP and hybridised with different samples of biotinylated single-stranded por A DNA.