Kaustabh Kumar Maiti

Surface-enhanced Raman scattering in cancer detection and imaging.

Technologies that use surface-enhanced Raman scattering (SERS) have experienced significant growth in biomedical research during the past 4 years. In this review we summarize the progress in SERS for cancer diagnostics, including multiplexed detection and identification of new biomarkers, single-nucleotide polymorphisms, and circulating tumor cells. SERS is also used as a non-invasive tool for cancer imaging with immunoSERS microscopy, histological analysis of biopsies, and in vivo detection of tumors. We discuss the future of SERS probes compatible with multiple imaging modalities and their potential for clinical translation (e.g., endoscope-based and intraoperative imaging as tools for surgical guidance). Moreover, we highlight the potential of SERS agents for targeted drug delivery and photothermal therapy.

Ultrasensitive Near‐Infrared Raman Reporters for SERS‐Based In Vivo Cancer Detection

Surface-enhanced Raman spectroscopy (SERS) has recently emerged as an alternative to fluorescence-based spectroscopy in bioimaging, as it can minimize photobleaching, peak overlapping, and low signal-to-noise ratio in complex biological systems. SERS probes are based on the 10–10-fold scattering enhancement caused by the proximity of Ramanactive signature molecules to the surface of metal nanoparticles (NPs), which can be modulated with molecular recognition motifs to render diagnostic tools for optical imaging and therapeutic studies. However, the preparation of ultrasensitive SERS probes is hampered by the limited availability, sensitivity, and reproducibility of Raman-active compounds. This drawback is particularly important at the near-infrared (NIR) region, where the availability of reporters is restricted to a few Raman-active molecules. Herein, we report the first combinatorial approach to discover novel and highly sensitive NIR SERS reporters. The synthesis and screening of an 80-member tricarbocyanine library led to the identification of CyNAMLA-381 as a NIR SERS reporter with 12-fold higher sensitivity than the standard 3,3’-diethylthiatricarbocyanine (DTTC), and we validated its advantages for the construction of ultrasensitive in vivo SERS probes. A major bottleneck in SERS probe discovery is the development of highly sensitive Raman reporters. Most of the commonly used Raman signature molecules are active in the UV/Vis range (e.g., crystal violet, malachite green isothiocyanate, rhodamine-6G, Nile blue, 2-napthalenethiol, TRITC (tetramethylrhodamine-5-isothiocyanate), and XRITC (Xrhodamine-5-(and-6)-isothiocyanate), and thus have a restricted potential for in vivo imaging. The adequacy of the NIR region for in vivo studies has raised the interest in NIR surface-enhanced resonance Raman spectroscopy (SERRS)-active molecules. Although the cyanine derivative DTTC has been regarded as a standard in NIR SERRS studies, it shows only a moderate Raman intensity, which limits the preparation of highly sensitive probes for in vivo applications. Since little is known about the correlation between the cyanine scaffold and its Raman intensity, we designed a library of structurally diverse tricarbocyanines with the aim of discovering novel NIR SERRS-active compounds that surpass the sensitivity of DTTC. The tricarbocyanine core is an accessible NIR structure, the central chlorine atom of which can be replaced with different nucleophiles. We designed the synthesis of tricarbocyanine derivatives by substitution with different amines, and acetylated the resulting alkylor benzylamino groups to obtain compounds with NIR absorption properties and good chemical stability in aqueous media (CyNA). To prepare compounds that could be chemisorbed on gold nanoparticles (AuNPs), we prepared the scaffold 1 with an aminopropyl linker that could be later coupled to a disulfide-containing lipoic acid spacer (Scheme 1). The amine group of 1 was Boc-protected prior to the derivatization of the central chlorine atomwith 80 structurally different primary amines including heterocyclic, alkyl, and aromatic groups (for structures, see Chart S1 in the Supporting Information). After acetylation, the compounds were treated with an optimized TFA/dichloromethane (1:9) solution that overcame the lability of the tricarbocyanine core in acidic conditions. The final coupling to a lipoic acidactivated ester resin yielded 80 derivatives (CyNAMLA) with an average purity of 90% (for data of HPLC-determined purities, see Table S1 in the Supporting Information). CyNAMLA compounds proved to be remarkably NIRactive with absorbance maximumwavelengths around 800 nm (Table S1 in the Supporting Information). Their SERS [*] A. Samanta, X. Liao, Prof. Y. T. Chang Department of Chemistry & MedChem Program of Life Sciences Institute, National University of Singapore 117543 Singapore (Singapore) Fax: (+65)6779-1691 E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

Development of biocompatible SERS nanotag with increased stability by chemisorption of reporter molecule for in vivo cancer detection.

Biocompatible surface-enhanced Raman scattering (SERS) nanotag has been developed by chemisorption of novel Raman reporters on gold colloid. We modified our previously published best five reporter molecules (B2, B7, C3, C7 and C9) from triphenylmethine (TM) library using lipoic acid (LA) as a linker to covalently attach the reporters on gold colloid. Among these TM-LA molecules, B2LA showed the highest SERS signal intensity and stability over time. Further, time course SERS intensity of B2LA was compared with currently popular Raman reporter malachite green isothiocyanate (MGITC). The results demonstrated that signal intensity from B2LA was even stable over a period of one month. In vitro SERS screening was performed in cancer cell lines using B2LA containing nanotag conjugated with selective antibodies recognizing HER2 and EGFR cancer proteins. We found reasonably strong SERS signals from both HER2 and EGFR positive cells whereas no signal was measured from respective negative cells. Moreover, we successfully proved this recognition by cell imaging using fluorescein isothiocyanate (FITC) labeled antibody conjugated nanotag. Both SERS and cell-imaging study further confirmed the selective binding of antibody conjugated nanotag to cancer cells over-expressing HER2 and EGFR. In addition, as a proof of concept, in vivo SERS measurement in a mouse model was carried out to detect the nanotag-anchored cancer cells that are subcutaneously injected to the animal.

Multiplex cancer cell detection by SERS nanotags with cyanine and triphenylmethine Raman reporters.

SERS nanotags have been prepared to accomplish the multiplex detection of cancer cells. Herein we evaluated the adequacy of lipoic acid-containing cyanine derivatives (Cy3LA and Cy5LA) to function as multiplex partners with a triphenylmethine Raman reporter (B2LA) under a single excitation wavelength. SERS experiments enabled the multiplex recognition of two different cancer cells with antibody-conjugated nanotags that were derivatized with optimized cyanine and triphenylmethine reporters.

Aggregation induced Raman scattering of squaraine dye: Implementation in diagnosis of cervical cancer dysplasia by SERS imaging.

The extent of squaraine dye aggregation that reflects on surface enhanced Raman signal scattering (SERS) intensity upon adsorption on nano-roughened gold surface has been investigated. Here we have synthesized a serious of six squaraine dyes consisting of two different electron donor moiety i.e. 1,1,2-trimethyl-1H-benzo[e]indole and 2-methylbenzo[d]thiazole which modulates the chemisorptions and hydrophobicity being designated as SQ1, SQ2, SQ3, SQ4, SQ5 and SQ6. Interestingly, SQ2 (mono lipoic acid appended), SQ5 and SQ6 (conjugated with hexyl and dodecyl side chain) squaraine derivatives having more tendency of aggregation in DMSO-water mixed solvent showed significant increase of Raman scattering in the fingerprint region when chemisorbed on spherical gold nanoparticles. Two sets of SERS nanotags were prepared with colloidal gold nanoparticle (Au-NPs size: 40 nm) by incorporating Raman reporters SQ2 and SQ5 followed by thiolated PEG encapsulation (SH-PEG, SH-PEG-COOH) denoted as AuNPs-SQ2-PEG and AuNPs-SQ5-PEG. Further conjugation of these nanotag with monoclonal antibodies specific to over expressed receptors, EGFR and p16/Ki-67 in cervical cancer cell, HeLa showed prominent SERS mapping intensity and selectivity towards cell surface and nucleus. The fast and accurate recognition obtained by antibody triggered SERS-nanotag has been compared with conventional time consuming immunocytochemistry technique which prompted us to extend further investigation using real patient cervical smear sample for a non-invasive, ultrafast and accurate diagnosis.

Pluronic Triblock Copolymer Encapsulated Gold Nanorods as Biocompatible Localized Plasmon Resonance-Enhanced Scattering Probes for Dark-Field Imaging of Cancer Cells

Gold nanorods (GNR) are synthesized using cetylmethylammonium bromide (CTAB) surfactants which function as structure-directing agents. However, CTAB forms a tightly bound cationic bilayer on GNR surface with the cationic trimethylammonium head group exposed to the aqueous media, which is known to be highly toxic in vitro and in vivo. Pluronic is a non-ionic triblock polymer, which can associate with CTAB and form stable CTAB–polymer complexes due to hydrophobic interactions. In this work, two types of Pluronic triblock copolymers were used to encapsulate GNR to reduce their cytotoxicity and improve colloidal and optical stability for biological applications. These formulations were characterized by UV–vis absorption spectra analysis, transmission electron microscopy, cell viability studies, differential interference contrast microscopy and dark-field imaging.

New insight of squaraine-based biocompatible surface-enhanced Raman scattering nanotag for cancer-cell imaging

AIM Development of highly sensitive diagnostic nanoprobe for cancer imaging based on surface-enhanced Raman scattering (SERS) platform. MATERIALS & METHODS Synthesis of novel squaraine dyes as a Raman signature molecule denoted as lipoic-squaraine-lipoic (LSL), propyl-squaraine-lipoic (PSL) and propyl-squaraine-propyl (PSP). The SERS-nanotag constructed with a Raman signature molecule which is attached on gold nanoparticle and further encapsulated with heterofunctionalized PEG. Antibody conjugation with best SERS-nanotag for target specific recognition. RESULTS SERS nanotag Au-LSL-PEG showed significant signal intensity and remarkable stability. Anti-EGF receptor and Her2-conjugated Au-LSL-PEG-nanotag were successfully applied for selective recognition of cancer cells like A549, OSCC and MCF7. CONCLUSION The newly developed SERS-nanotag Au-LSL-PEG serves as a valuable tool for diagnostic detection of cancer cells, and may find potential applications for cancer screening in real patient samples.

Emergence of Gold-Mesoporous Silica Hybrid Nanotheranostics: Dox-Encoded, Folate Targeted Chemotherapy with Modulation of SERS Fingerprinting for Apoptosis Toward Tumor Eradication

Strategically fabricated theranostic nanocarrier delivery system is an unmet need in personalized medicine. Herein, this study reports a versatile folate receptor (FR) targeted nanoenvelope delivery system (TNEDS) fabricated with gold core silica shell followed by chitosan-folic acid conjugate surface functionalization by for precise loading of doxorubicin (Dox), resembled as Au@SiO2 -Dox-CS-FA. TNEDS possesses up to 90% Dox loading efficiency and internalized through endocytosis pathway leading to pH and redox-sensitive release kinetics. The superior FR-targeted cytotoxicity is evaluated by the nanocarrier in comparison with US Food and Drug Administration (FDA)-approved liposomal Dox conjugate, Lipodox. Moreover, TNEDS exhibits theranostic features through caspase-mediated apoptosis and envisages high surface plasmon resonance enabling the nanoconstruct as a promising surface enhanced Raman scattering (SERS) nanotag. Minuscule changes in the biochemical components inside cells exerted by the TNEDS along with the Dox release are evaluated explicitly in a time-dependent fashion using bimodal SERS/fluorescence nanoprobe. Finally, TNEDS displays superior antitumor response in FR-positive ascites as well as solid tumor syngraft mouse models. Therefore, this futuristic TNEDS is expected to be a potential alternative as a clinically relevant theranostic nanomedicine to effectively combat neoplasia.

Unveiling NIR Aza-BODIPY Dyes as Raman Probes: SERS-Guided Selective Detection and Imaging of Human Cancer Cells

The development of new Raman reporters has attracted immense attention in diagnostic research based on surface enhanced Raman scattering (SERS) techniques, which is a well established method for ultrasensitive detection through molecular fingerprinting and imaging. Herein, for the first time, we report the unique and efficient Raman active features of the selected aza-BODIPY dyes 1-6. These distinctive attributes could be extended at the molecular level to allow detection through SERS upon adsorption onto nano-roughened gold surface. Among the newly revealed Raman reporters, the amino substituted derivative 4 showed high signal intensity at very low concentrations (ca. 0.4 μm for 4-Au). Interestingly, an efficient nanoprobe has been constructed by using gold nanoparticles as SERS substrate, and 4 as the Raman reporter (4-Au@PEG), which unexpectedly showed efficient recognition of three human cancer cells (lung: A549, cervical: HeLa, Fibrosarcoma: HT-1080) without any specific surface marker. We observed well reflected and resolved Raman mapping and characteristic signature peaks whereas, such recognition was not observed in normal fibroblast (3T3L1) cells. To confirm these findings, a SERS nanoprobe was conjugated with a specific tumour targeting marker, EGFR (Epidermal Growth Factor Receptor), a well known targeted agent for Human Fibrosarcoma (HT1080). This nanoprobe efficiently targeted the surface marker of HT1080 cells, threreby demonstrating its use as an ultrasensitive Raman probe for detection and targeted imaging, leaving normal cells unaffected.

A Ratiometric Near-Infrared Fluorogen for the Real Time Visualization of Intracellular Redox Status during Apoptosis

Direct monitoring of apoptotic progression is a major step forward for the early assessment of therapeutic efficacy of certain treatments and the accurate evaluation of the spread of a disease. Here, the regulatory role of glutathione (GSH) is explored as a potential biomarker for tracking apoptosis. For this purpose, a near- infrared (NIR) squaraine dye is introduced that is capable of sensing GSH in a ratiometric manner by switching its emission from NIR (690 nm) to visible region (560 nm). The favorable biocompatible attributes of the probe facilitated the real-time monitoring of apoptotic process in line with the conventional apoptotic assay. Furthermore, the robust nature of the probe was utilized for the quantitative estimation of GSH during different stages of apoptosis. Through this study, an easy and reliable method of assaying apoptosis is demonstrated, which can provide valuable insights in translational clinical research.