Role of Doxorubicin on the Loading Efficiency of ICG within Silk Fibroin Nanoparticles.

The effective loading or encapsulation of multimodal theranostic agents within a nanocarrier system plays an important role in the clinical development of cancer therapy. In recent years, the silk fibroin protein-based delivery system has been drawing significant attention to be used in nanomedicines due to its biocompatible and biodegradable nature. In this study, silk fibroin nanoparticles (SNPs) have been synthesized by a novel and cost-effective ultrasonic atomizer-based technique for the first time. The fabricated SNPs were coencapsulated by the FDA-approved indocyanine green (ICG) dye and the chemotherapeutic drug doxorubicin (DOX). The synthesized SNPs are spherical, with an average diameter of ∼37 ± 4 nm, and the ICG-DOX-coencapsulated SNPs (ID-SNPs) have a diameter size of ∼47 ± 6 nm. For the first time, here we demonstrate that DOX helps in the higher loading of ICG within the ID-SNPs, which enhances the encapsulation efficiency of ICG by ∼99%. This could be attributed to the interaction of ICG and DOX molecules with the silk fibroin protein, which helps ICG to get loaded more efficiently within these nanoparticles. The overall finding of this study suggests that the ID-SNPs could be utilized for enhanced ICG-complemented multimodal deep-tissue bioimaging and synergistic chemo-photothermal therapy.

Effect of Doxorubicin on the Near-Infrared Optical Properties of Indocyanine Green.

In recent years, chemo-photothermal therapy (chemo-PTT) has been extensively studied for the upgradation of cancer treatment. The combined therapeutic approach reduces the overall cytotoxicity and enhances the therapeutic effect against the cancerous cells. In chemo-PTT, Indocyanine green (ICG) dye, a near-infrared chromophore, is used for PTT in combination with doxorubicin (DOX), a chemotherapeutic drug. ICG and DOX work very efficiently in synergy against cancer. However, the effect of DOX on the optical properties of ICG has not been studied yet. Here, for the first time, we report the effect of DOX on the optical properties of ICG in detail. DOX interacts with ICG and induces the aggregation of ICG even at a low concentration. The coincubation of both the molecules causes H and J aggregations in ICG. However, the J aggregation becomes more prominent with an increasing DOX concentration. These findings suggest that the optical properties of ICG change upon incubation with the DOX, which might affect the efficacy of PTT.

Optical-Property-Enhancing Novel Near-Infrared Active Niosome Nanoformulation for Deep-Tissue Bioimaging.

Indocyanine green (ICG) is a clinically approved near-infrared (NIR) contrast agent used in medical diagnosis. However, ICG has not been used to its fullest for biomedical imaging applications due to its low fluorescence quantum yield, aqueous instability, concentration-dependent aggregation, and photo and thermal degradations, leading to quenching of its fluorescence emission. In the present study, a nanosized niosomal formulation, ICGNiosomes (ICGNios), is fabricated to encapsulate and protect ICG from degradation. Interestingly, compared to free ICG, the ICGNios exhibited higher fluorescence quantum yield and fluorescence emission with a bathochromic shift. Also, ICGNios nanoparticles are biocompatible, biodegradable, and readily uptaken by the cells. Furthermore, ICGNios show more enhanced fluorescence intensity through ∼1 cm thick chicken breast tissue compared to free ICG, which showed minimal emission through the same thickness of tissue. Our results suggest that ICGNios could offer a promising platform for deep-tissue NIR in vivo imaging to visualize inaccessible tissue microstructures for disease diagnosis and therapeutics.

Fluorescence photobleaching of urine for improved signal to noise ratio of the Raman signal - An exploratory study.

Urine analysis is an important clinical test routinely performed in pathology labs for disease diagnosis and prognosis. In recent years, near-infrared Raman spectroscopy has drawn considerable attention for urine analysis as it can provide rapid, reliable, and reagent-free analysis of urine samples. However, one important practical problem encountered in such Raman measurements is the orders of magnitude stronger spectral background preventing one to utilize the full dynamic range of the detector which is required for the measurement of Raman signal with good signal-to-noise ratio (SNR). We report here the results of an exploratory study carried out on human urine samples to show that the photobleaching, which is a major disadvantage during the fluorescence measurement, could be utilized for suppressing the measured background to improve the SNR of the Raman peaks. It was found that once the photobleaching reached its plateau, there were improvements by ~67% and ~47% in the SNR and the signal to background ratio (SBR), respectively, of the Raman signals as compared to the spectra measured at the start of acquisition. Further, the reduced background also allowed us to utilize the full dynamic range of the detector at increased integration time without saturating the detector indicating the possibility of obtaining an improved detection limit.

Green synthesis of near-infrared absorbing eugenate capped iron oxide nanoparticles for photothermal application.

Nanomaterials exhibit different interesting physical, chemical, electronic and magnetic properties that can be used in a variety of biomedical applications such as molecular imaging, cancer therapy, biosensing, and targeted drug delivery. Among various types of nanoparticles, super paramagnetic iron oxide nanoparticles (SPIONs) have emerged as exogenous contrast agents for in vitro and in vivo deep tissue imaging. Here, we propose a facile, rapid, non-toxic, and cost-effective single step green synthesis method to fabricate eugenate (4-allyl-2-methoxyphenolate) capped iron oxide nanoparticles (E-capped IONPs). The magnetic E-capped IONPs are first time synthesized using a medicinal aromatic plant, Pimenta dioica. The Pimenta dioica leaf extract was used as a natural reducing agent for E-capped IONPs synthesis. The crystalline structure and size of the synthesized spherical nanoparticles were confirmed using the x-ray diffraction and electron microscopic images respectively. In addition, the presence of the functional groups, responsible for capping and stabilizing the synthesized nanoparticles, were identified by the Fourier transform infra-red spectrum. These nanoparticles were found to be safe for human cervical cancer (HeLa) and human embryonic kidney 293 (HEK 293) cell lines and their safety was established using MTT[3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide] assay. These green synthesized E-capped IONPs display a distinct absorbance in the tissue transparent near-infrared (NIR) wavelength region. This property was used for the NIR photothermal application of E-capped IONPs. The results suggest that these E-capped IONPs could be used for deep tissue photothermal therapy along with its application as an exogenous contrast agent in biomedical imaging.

Fluorescence photo-bleaching of urine and its applicability in oral cancer diagnosis.

Photo-stability of urine is of crucial importance for the applicability of fluorescence spectroscopy of urine samples for diagnosis of cancer. We report the results of a detailed study on fluorescence photo-bleaching of human urine samples. We also present the results of a preliminary investigation on evaluation of the applicability of photo-bleaching characteristics of urine for discriminating patients with oral cancer from healthy volunteers. The time-lapse fluorescence induced by continuous shining of 405 nm radiation from a diode laser was recorded from the urine samples obtained from 18 patients with oral cancer as well as from 22 healthy volunteers with history of no known major illness in the past two months. The integrated fluorescence intensity (ΣI), calculated for each spectrum, was found to decrease with time till a point after which no further decrease was observed. Further, while significant differences were observed in the spectra of cancerous patients and healthy volunteers, these differences were found to be varying with time till the intensities of the observed fluorescence spectra corresponding to the two categories of urine samples became stable. The curve, generated by plotting ΣI vs. time, was found to be best fitted (R2 > 0.95) with a double-exponential decay function. The photo-bleaching constants, obtained from curve-fitting, were found to have statistically significant differences corresponding to the urine samples of cancerous patients and healthy volunteers. A classification algorithm developed based on nearest-mean classifier (NMC) and applied on the photo-bleaching constants in leave-one-subject-out cross-validation mode was found to provide a sensitivity and specificity of up to ∼ 86% in discriminating the two categories of urine samples.

Nanotrap-Enhanced Raman Spectroscopy: An Efficient Technique for Trace Detection of Bioanalytes.

Reliable diagnosis of disease using body fluids requires sensitive and accurate detection of disease-specific analytes present in the fluid. In recent years, there has been increasing interest in using surface-enhanced Raman spectroscopy (SERS) for this purpose. The demonstrable signal enhancement and sensitivity of SERS makes it ideally suited for detection of a trace quantity of any analyte. However, lack of reproducibility along with large spatial variability in the measured Raman intensities due to differential (and often random) distribution of surface "hot spots" limits its routine clinical use. We propose here a technique, nanotrap-enhanced Raman spectroscopy (NTERS), for overcoming these long-standing limitations and challenges of SERS. In this technique, hot spots are formed by drying up a microvolume drop of the liquid, containing the mixture of nanoparticles and analytes in the focal volume of the Raman excitation laser, and the Raman signal is detected from these spots containing the analytes localized within the nanoparticle aggregates. The performance of the technique was evaluated in detecting trace quantities of two Raman-active analytes, Rhodamine 6G (R6G) and urea. It was found that R6G and urea could be detected down to a concentration of 50 nM with signal-to-noise ratio (SNR) value of ∼75 and 4 mM with SNR value of ∼500, respectively. A comparison with SERS revealed that NTERS not only had significantly superior (around 2 orders of magnitude) signal enhancement but also had high reproducibility because of its intrinsic ability to form nanoparticle aggregates with high repetitiveness. Another advantage of NTERS is its simplicity and cost effectiveness as it does not require any specialized substrate.