Nitin

Distribution of encapsulated materials in colloidal particles and its impact on oxidative stability of encapsulated materials.

The oxidative stability of encapsulated product is a critical parameter in many products from food to pharmaceutical to cosmetic industries. The overall objective of this study was to correlate differences in the distribution pattern of encapsulated material within solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) with the relative susceptibility of these materials to undergo oxidation. The distribution of an encapsulated lipid soluble dye (Nile Red) in SLNs and NLCs was quantitatively measured using fluorescence imaging. The relative susceptibility of the encapsulated material to react with free radicals generated in the aqueous phase and oxygen from the ambient environment was measured using peroxyl radical and oxygen sensitive fluorescent dyes encapsulated in the lipid phase of colloidal particles respectively. Imaging measurements demonstrate a significant exclusion of the encapsulated dye molecules from the lipid core of SLNs as compared to NLCs. Imaging results also showed significant differences in the intraparticle distribution of encapsulated dye between NLCs containing 1 and 10% liquid lipid. On the basis of these differences in distribution, we hypothesized that the relative susceptibility of encapsulated material to peroxyl radicals and oxygen would be in the order SLNs > 1% NLC > 10% NLC. Measurement of relative susceptibility of peroxyl radical sensitive dye encapsulated in SLNs and NLCs to peroxyl radicals generated in the aqueous phase validated the proposed hypotheses. However, the susceptibility of encapsulated oxygen sensitive dye to ambient oxygen was not significantly different between SLNs and NLCs. The results of this study demonstrate that difference in distribution pattern of encapsulated material within colloidal particles can significantly influence the susceptibility of encapsulated material to react with free radicals. Overall, this study demonstrates a comprehensive approach to characterize the susceptibility of encapsulated materials in colloidal particles to oxidation processes.

Optical molecular imaging detects changes in extracellular pH with the development of head and neck cancer

Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa‐647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor® 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa‐647 labeled pHLIP in intact fresh human tissue specimens using widefield and high‐resolution fluorescence imaging. Using topical delivery, Alexa‐647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa‐647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.

Cellulose nanofibrils improve dispersibility and stability of silver nanoparticles and induce production of bacterial extracellular polysaccharides

Many polymer-stabilized silver nanoparticles (AgNPs) with enhanced antibacterial properties have been synthesized, but very little is known about the fate of these materials and their interactions with microbes in physiological solutions. In this study, we evaluated the role of cellulose nanofibrils (CNFs) in stabilizing AgNPs (CNF-AgNPs) in a bacterial growth medium, determined the antibacterial properties of CNF-AgNPs and assessed the CNF and CNF-AgNP interactions with bacteria. The attachment of AgNPs to CNFs significantly improved the stability and dispersibility of AgNPs in the bacterial growth medium compared to colloidal AgNPs. The CNF-AgNPs exerted a concentration-dependent growth-inhibitory effect on E. coli. An extracellular polysaccharide (EPS)-like structure was formed around the E. coli bacterium when incubated with a sub-lethal CNF-AgNP concentration, which led to the clustering of neighboring bacteria. No EPS-like structure was observed around the bacterium incubated with high concentration of CNF-AgNPs. Overall, this study demonstrated that CNFs significantly improved the stability and dispersibility of AgNPs in physiological medium, validated the antimicrobial potential of CNF-AgNPs, and provided an insight into the physio-chemical interactions between bacteria and CNF-AgNPs in a physiological growth medium.

Widefield Optical Imaging of Changes in Uptake of Glucose and Tissue Extracellular pH in Head and Neck Cancer

The overall objective of this study was to develop an optical imaging approach to simultaneously measure altered cell metabolism and changes in tissue extracellular pH with the progression of cancer using clinically isolated biopsies. In this study, 19 pairs of clinically normal and abnormal biopsies were obtained from consenting patients with head and neck cancer at University of California, Davis Medical Center. Fluorescence intensity of tissue biopsies before and after topical delivery of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) and Alexa 647-pHLIP [pH (low) insertion peptide] was measured noninvasively by widefield imaging, and correlated with pathologic diagnosis. The results of widefield imaging of clinical biopsies demonstrated that 2-NBDG and pHLIP peptide can accurately distinguish the pathologically normal and abnormal biopsies. The results also demonstrated the potential of this approach to detect subepithelial lesions. Topical application of the contrast agents generated a significant increase in fluorescence contrast (3- to 4-fold) in the cancer biopsies as compared with the normal biopsies, irrespective of the patient and location of the biopsy within a head and neck cavity. This unpaired comparison across all the patients with cancer in this study highlights the specificity of the imaging approach. Furthermore, the results of this study indicated that changes in intracellular glucose metabolism and cancer acidosis are initiated in the early stages of cancer, and these changes are correlated with the progression of the disease. In conclusion, this novel optical molecular imaging approach to measure multiple biomarkers in cancer has a significant potential to be a useful tool for improving early detection and prognostic evaluation of oral neoplasia. Cancer Prev Res; 7(10); 1035–44. ©2014 AACR.

Image Analysis of Microstructural Changes in Almond Cotyledon as a Result of Processing

Release of oil from nuts due to damaged cellular structures can degrade the quality of products incorporating nuts. The aim of this study was to investigate the effects of different processing conditions on microstructure of almond tissue and to quantify these changes using image processing. Spinning disk confocal fluorescence microscopy was used for imaging changes in microstructure of almonds as a function of different thermal processing of almonds. Multiple staining of Nile Red and Calcofluor White was applied to differentiate cell wall structures and oil bodies within individual almond cells without chemical fixation. An algorithm for image processing, included image preprocessing, segmentation, and determination of morphological features of segmented objects, was developed. Oil-roasting processes (140 °C and 150 °C) were found to have a significant impact on microstructure of almonds when compared to the hot air-roasting and blanching processes. Oil-roasted almond at 150 °C had a greater cellular damage due to cell wall and membrane rupture. These changes in microstructure of almonds would make them slightly more susceptible to release oil during storage. The image analysis presented allows quantitative evaluation for the effect of different processing on almond microstructure.

Capture and detection of T7 bacteriophages on a nanostructured interface

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible.

Antifungal activity against Candida albicans of starch Pickering emulsion with thymol or amphotericin B in suspension and calcium alginate films

Conventional antifungal treatments against Candida albicans in the oral cavity often result in increased cytotoxicity. The goal of this study was to determine the potential of starch Pickering emulsion as a delivery vehicle for an antifungal natural phenolic compound such as thymol in simulated saliva fluid (SSF) compared to amphotericin B. An oil-in-water (o/w) emulsion was stabilized using starch particles. Physical stability of the emulsion and disruption induced by α-amylase activity in SSF was evaluated. Encapsulated thymol in o/w emulsion was compared to encapsulated amphotericin B for antifungal activity against C. albicans in suspension using emulsions or zone inhibition assay on agar plates using emulsions dispersed in alginate films. Results showed that the emulsions were stable for at least three weeks. Digestion of the emulsion by α-amylase led to coalescence of emulsion droplets. The antifungal activity of thymol and amphotericin B in emulsion formulation was enhanced upon incubation with α-amylase. Results from the zone inhibition assay demonstrated efficacy of the emulsions dispersed in alginate films. Interestingly, addition of α-amylase to the alginate films resulted in a decreased inhibitory effect. Overall, this study showed that starch Pickering emulsions have a potential to deliver hydrophobic antifungal compounds to treat oral candidiasis.

Distribution of a model bioactive within solid lipid nanoparticles and nanostructured lipid carriers influences its loading efficiency and oxidative stability

The overall goal of this study was to characterize the distribution of a model bioactive encapsulant in the lipid domain of SLNs and NLCs and its relationship with loading efficiency and reactivity of the model encapsulant with oxidative stress agents. Distribution of a model bioactive (beta-carotene) was compared to that of a fluorescent dye (Nile red) in SLNs, 10% NLC, 30% NLC, 50% NLC, 70% NLC (the number represents the percentage of liquid lipid within the total lipid amount) and emulsions. Fluorescence imaging shows that the distribution of Nile red in the lipid domain of colloidal carriers was similar to that of beta-carotene in all formulations. Based on the combination of imaging observations and loading efficiency measurements, the results demonstrate that beta-carotene was excluded from the lipid domain in both SLNs and NLCs. The extent of exclusion decreased, while uniformity in the distribution of encapsulant in the lipid domain of colloidal carrier increased with an increase in percentage of liquid lipid content of NLCs. Oxidative stability of the encapsulated beta-carotene in SLN and NLCs (at least until 30% liquid lipid composition) was significantly lower compared to that in emulsion. Only for the NLCs with 50 and 70% liquid lipid content, oxidative stability of the encapsulated compound was significantly higher than that in emulsions. Overall, the results demonstrate that differences in loading efficiency and oxidative stability of beta-carotene in SLNs and NLCs may be explained by the differences in the distribution of beta-carotene.

Enhanced antimicrobial activity based on a synergistic combination of sublethal levels of stresses induced by UV-A light and organic acids

ABSTRACT The reduction of microbial load in food and water systems is critical for their safety and shelf life. Conventionally, physical processes such as heat or light are used for the rapid inactivation of microbes, while natural compounds such as lactic acid may be used as preservatives after the initial physical process. This study demonstrates the enhanced and rapid inactivation of bacteria based on a synergistic combination of sublethal levels of stresses induced by UV-A light and two food-grade organic acids. A reduction of 4.7 ± 0.5 log CFU/ml in Escherichia coli O157:H7 was observed using a synergistic combination of UV-A light, gallic acid (GA), and lactic acid (LA), while the individual treatments and the combination of individual organic acids with UV-A light resulted in a reduction of less than 1 log CFU/ml. Enhanced inactivation of bacteria on the surfaces of lettuce and spinach leaves was also observed based on the synergistic combination. Mechanistic investigations suggested that the treatment with a synergistic combination of GA plus LA plus UV-A (GA+LA+UV-A) resulted in significant increases in membrane permeability and intracellular thiol oxidation and affected the metabolic machinery of E. coli. In addition, the antimicrobial activity of the synergistic combination of GA+LA+UV-A was effective only against metabolically active E. coli O157:H7. In summary, this study illustrates the potential of simultaneously using a combination of sublethal concentrations of natural antimicrobials and a low level of physical stress in the form of UV-A light to inactivate bacteria in water and food systems. IMPORTANCE There is a critical unmet need to improve the microbial safety of the food supply, while retaining optimal nutritional and sensory properties of food. Furthermore, there is a need to develop novel technologies that can reduce the impact of food processing operations on energy and water resources. Conventionally, physical processes such as heat and light are used for inactivating microbes in food products, but these processes often significantly reduce the sensory and nutritional properties of food and are highly energy intensive. This study demonstrates that the combination of two natural food-grade antimicrobial agents with a sublethal level of physical stress in the form of UV-A light can greatly increase microbial load inactivation. In addition, this report elucidates the potential mechanisms for this synergistic interaction among physical and chemical stresses. Overall, these results provide a novel approach to develop antimicrobial solutions for food and water systems.

Click chemistry approach for imaging intracellular and intratissue distribution of curcumin and its nanoscale carrier.

This study was aimed at developing a fluorescence imaging approach to simultaneously characterize the delivery and distribution of a bioactive molecule, curcumin, and its micelle based nanoscale carrier in cells and tissue models. To enable imaging of curcumin, a monoalkyne derivative of curcumin was synthesized and purified using LC-MS. Intracellular uptake of curcumin was characterized using a click chemistry reaction between a monoalkyne modified curcumin and Alexa-488 azide fluorescent dye in cells and tissues. Fluorescence images of cells and tissues incubated with monoalkyne curcumin showed specific detection of intracellular delivered monoalkyne curcumin using the click chemistry reaction. The fluorescence imaging results also demonstrated significant improvement in detection sensitivity of intracellular delivered curcumin as compared to measurements based on native fluorescence of unmodified curcumin. Intracellular uptake of monoalkyne curcumin was characterized as a function of incubation time and concentration. The results show a rapid uptake of monoalkyne curcumin during the first 4 h of incubation. Modification of curcumin to its monoalkyne derivative did not impact its apoptotic activity in cancer cells. DSPE-PEG micelles labeled with Alexa-647 were selected as a representative nanoscale carrier to enhance the solubility and delivery of monoalkyne curcumin. Fluorescence images of cells and tissues incubated with fluorescently labeled micelles containing monoalkyne curcumin clearly illustrate significant differences in intracellular and intratissue localization of DSPE-PEG and encapsulated monoalkyne curcumin. The imaging approach developed in this study can be used to understand delivery and distribution of diverse bioactive compounds and their nanocarrier systems as well as in situ measurement of interactions of bioactives with cellular and tissue targets.