Second Harmonic Generation Spectroscopy and Microscopy of Liposomes, Nanoparticles, and Cells

Second harmonic generation (SHG) is used to investigate the factors that impact nanoparticle-based drug-delivery applications. In the first study, molecular adsorption and transport kinetics of a positively-charged dye, malachite green isothiocyanate (MGITC), is characterized at the surface of different colloidal liposomes in water using SHG spectroscopy. The molecular interactions of MGITC is compared to our previous investigations with malachite green (MG). In comparison to MG, MGITC demonstrates stronger adsorption and faster transport through lipid membranes. Correspondingly, the SHG experimental results are in excellent agreement with the molecular dynamics (MD) simulations results. A key finding illustrates the importance of functional groups, such as isothiocyanate, in controlling molecular translocation across the phospholipid-water interface. In a related study, temperature-dependent SHG measurements are performed to investigate the thermodynamics associated with the adsorption and transport kinetics of MG at the surface of 1,2-dioleoyl-sn-glycero-3phospho-(1’rac-glycerol) (DOPG) liposomes. The molecular transport is determined to be approximately 5 times faster at 40 ⁰C in comparison to the molecular transport at 25 ⁰C. Additionally, the changes in adsorption enthalpy and entropy are determined. The change in adsorption entropy is positive and the change in adsorption enthalpy is negative, indicating that the adsorption process is spontaneous at all aqueous temperatures. Similarly, SHG microscopy is used to probe the molecular interactions of MG and MGITC molecules at the surface of living human nonsmall adenosquamous lung cancer cells (H596 cells). The observed molecular translocation in living H596 cells is significantly faster for MGITC in comparison to MG. SHG microscopy is also used to probe fixed, dead H596 cells with MGITC dye molecules. In comparison to fixed cells, living cells have pronounced fluctuations of SHG intensity which is attributed to more complicated interactions, including active transport and cell regulation. Finally, gold, silver, and gold-silver-gold core-shell-shell (CSS) plasmonic nanoparticles having size of 10-100 nm are synthesized and functionalized with miRNA molecules using Diels-Alder chemistry. The retro Diels-Alder thermal release of miRNA from the surface of novel plasmonic nanoparticles is investigated at their corresponding plasmon resonances using surface-specific SHG spectroscopy. In summary, these time-resolved studies highlight the importance of SHG as a sensitive, powerful, and versatile tool to monitor the real-time surface chemistry of colloidal nanoparticle-based drug-delivery systems

Quantifying the potential epidemiological impact of a 2-year active case finding for tuberculosis in rural Nepal: a model-based analysis

Objectives: Active case finding (ACF) is an important tuberculosis (TB) intervention in high-burden settings. However, empirical evidence garnered from field data has been equivocal about the long-term community-level impact, and more data at a finer geographic scale and data-informed methods to quantify their impact are necessary. Methods: Using village development committee (VDC)-level data on TB notification and demography between 2016 and 2017 in four southern districts of Nepal, where ACF activities were implemented as a part of the IMPACT-TB study between 2017 and 2019, we developed VDC-level transmission models of TB and ACF. Using these models and ACF yield data collected in the study, we estimated the potential epidemiological impact of IMPACT-TB ACF and compared its efficiency across VDCs in each district. Results: Cases were found in the majority of VDCs during IMPACT-TB ACF, but the number of cases detected within VDCs correlated weakly with historic case notification rates. We projected that this ACF intervention would reduce the TB incidence rate by 14% (12–16) in Chitwan, 8.6% (7.3–9.7) in Dhanusha, 8.3% (7.3–9.2) in Mahottari and 3% (2.5–3.2) in Makwanpur. Over the next 10 years, we projected that this intervention would avert 987 (746–1282), 422 (304–571), 598 (450–782) and 197 (172–240) cases in Chitwan, Dhanusha, Mahottari and Makwanpur, respectively. There was substantial variation in the efficiency of ACF across VDCs: there was up to twofold difference in the number of cases averted in the 10 years per case detected. Conclusion: ACF data confirm that TB is widely prevalent, including in VDCs with relatively low reporting rates. Although ACF is a highly efficient component of TB control, its impact can vary substantially at local levels and must be combined with other interventions to alter TB epidemiology significantly

Business Plan for a Nepalese Restaurant in Finland

Purpose of this thesis project is to create a feasible business plan for a Nepalese restaurant in Finland. In this paper the business plan for Ravintola Z is created which will be located in the capital region of Finland. Ravintola Z will be a rising star of the ethnic restaurant scene by bringing Nepalese culture and heritage into the fore through excellent Nepalese traditional cuisine and hospitality. The idea of Ravintola Z is to create, produce and serve great, full-flavored Nepalese cuisine that has a beginning, middle and finish to every bite. The popularity of Nepalese cuisine and people’s desire to taste typical Nepalese food has inspired the authors to develop this plan and implement the outcome in future. The plan is based on both theoretical and project based methods. The theoretical approach of the report is based on a number of books, internet resources and literature review. The importance of the business plan as well as its forms are mentioned and defined. Different factors and the stages of creating a good business plan are discussed. For example, Executive summary, company’s summary, product description, SWOT analysis, strategic planning through Porters five forces, financial analysis, Market analysis and marketing plan are defined and well executed throughout the plan. Primarily, quantitative research method was conducted by designing survey questionnaires for a deeper understanding of the customer view and expectation towards Nepalese restaurant. A result of the survey was in favor of Nepalese restaurant with most of respondents showing a positive attitude towards Nepalese cuisine. Secondly, an interview with restaurant owners was also taken which helped authors to analyze success and failure factors for operating a restaurant business. Opening a new Nepalese restaurant is risky due to the competitive environment; however, there is always room for a new entrepreneur to enter with an innovative and unique product. Selection of good location and choosing suitable business attitude might result the restaurant business profitable

Growth Dynamics of Colloidal Silver-Gold Core-Shell Nanoparticles Studied by Second Harmonic Generation and Extinction Spectroscopy

The growth dynamics of colloidal silver-gold core-shell (Ag@Au CS) nanoparticles (NPs) in water are monitored in a stepwise synthesis approach using time-dependent second harmonic generation (SHG) and extinction spectroscopy. Three sequential additions of chloroauric acid, sodium citrate, and hydroquinone are added to the silver nanoparticle solution to grow a gold shell around a silver core. The first addition produces a stable urchin-like surface morphology, while the second and third additions continue to grow the gold shell thickness as the surface becomes more smooth and uniform, as determined using transmission electron microscopy. The extinction spectra after each addition are compared to finite-difference time-domain (FDTD) calculations, showing large deviations for the first and second additions due to the bumpy surface morphology and plasmonic hotspots while showing general agreement after the third addition reaches equilibrium. The SHG signal is dominated by the NP surface, providing complementary information on the growth time scales due to changes to the surface morphology. This combined approach of synthesis and characterization of Ag@Au CS nanoparticles with SHG spectroscopy, extinction spectroscopy, and FDTD calculations provides a detailed foundation for investigating complex colloidal nanoparticle growth mechanisms and dynamics in developing enhanced plasmonic nanomaterial technologies

Molecular Adsorption and Transport at Liposome Surfaces Studied by Molecular Dynamics Simulations and Second Harmonic Generation Spectroscopy

© 2019 American Chemical Society. A fundamental understanding of the factors that determine the interactions with and transport of small molecules through phospholipid membranes is crucial in developing liposome-based drug delivery systems. Here we combine time-dependent second harmonic generation (SHG) measurements with molecular dynamics simulations to elucidate the events associated with adsorption and transport of the small molecular cation, malachite green isothiocyanate (MGITC), in colloidal liposomes of different compositions. The molecular transport of MGITC through the liposome bilayer is found to be more rapid in 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPG and DOPS, respectively) liposomes, while the molecular transport is slower in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Interestingly, MGITC is observed to neither adsorb nor transport in trimethyl quinone-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (QPADOPE) liposomes due to shielding by the quinone group. The modified Langmuir adsorption isotherm model is used to determine the free energy of adsorption for MGITC, which is found to be less negative in DOPC than in DOPG and DOPS, caused by lower electrostatic interactions between the positively charged dye and the zwitterionic DOPC liposome surface. The results are compared to our previous investigations, which showed that malachite green (MG) adsorbs and transports in DOPG and DOPS liposomes but not in DOPC and QPADOPE liposomes. Molecular dynamics simulations are used to investigate the adsorption and transport properties of MG and MGITC in DOPC and DOPG liposomes using umbrella sampling to determine the free energy profiles and interfacial molecular orientations. Together, these time-resolved SHG studies and corresponding molecular dynamics simulations characterize the complicated chemical interactions at different lipid membranes to provide key molecular-level insights for potential drug delivery applications. The results also point toward understanding the role of chemical functional groups, in this case isothiocyanate, in controlling molecular adsorption at and transport through lipid bilayers

Near-Infrared Photothermal Release of siRNA from the Surface of Colloidal Gold-Silver-Gold Core-Shell-Shell Nanoparticles Studied with Second-Harmonic Generation

© 2018 American Chemical Society. Photothermal release of oligonucleotides from the surface of plasmonic nanoparticles represents a promising platform for spatiotemporal controlled drug delivery. Here we demonstrate the use of novel gold-silver-gold core-shell-shell (CSS) nanoparticles to study the photothermal cleaving and release of micro-RNA (miRNA) mimics or small interfering RNA (siRNA) under near-infrared (NIR) irradiation. The furan-maleimide-based Diels-Alder adduct cleaves thermally above 60 °C and is used to bind siRNA to the colloidal nanoparticle surface in water. We investigate the photothermal cleaving kinetics of siRNA under different NIR laser powers using surface-sensitive time-dependent second-harmonic generation (SHG) spectroscopy. The photothermal release of siRNA from the surface of CSS nanoparticles is significantly higher than that from the surface of gold nanoparticles (GNPs) under similar experimental conditions. These results demonstrate that plasmonic CSS nanoparticles with photothermal cleaving linkers have important potential applications for nanoparticle-based NIR-mediated drug-delivery systems