KEYWORDS: Laser irradiation, Fluorescence, Monte Carlo methods, Fluorescence intensity, Diffusion, Molecules, Data modeling, Gold nanoparticles, Time metrology, Pulsed laser operation
SignificanceResealing time based loading efficiency of optoporation is the key parameter for drug or gene delivery. This work describes a comparatively simple optical approach to directly measure the cell membrane resealing time of the gold nanoparticle mediated photoporation.AimTo establish a membrane potential detection optical system, which can provide a direct measurement of resealing time of the optoporated cells.ApproachVoltage sensitive dye has been used to label the gold nanoparticle covered cell before laser activation and the resealing time was estimated from the voltage change due to the fluorescence light intensity change before and after laser activation. The approach has been validated by the simulated data based on diffusion model and Monte Carlo simulation and the experimental data obtained from a flow cytometry analysis.ResultsThe measured resealing time after perforation varied from 28.6 to 163.8 s on Hela cells when the irradiation fluence was increased, with a correlation coefficient (R2) of 0.9938. This result is in agreement with the resealing time (1-2 min) of photothermal porated Hela cells measured by electrical impedance method. The intracellular delivery efficiency of extracellular macromolecular under the same irradiation fluence depends mainly on diffusion velocity rather than pore size.ConclusionThe method described here can be used to directly measure resealing time of optoporated cells for accurately estimating the loading efficiency on discovering the mechanism of optoporation.
There are many situations in medicine and biology when it is desirable to introduce a macromolecule into mammalian cells. Gold nanoparticles mediated photoporation is a promising technique that is an efficient, relatively high-throughput and virus-free method. Up to now different pulse laser and kinds of gold nanoparticles with different size, different shape were employed to achieve photoporation for macromolecule delivery such as dextran, protein and plasmid. Here, gold nanosphere and nanorod activated by nanosecond laser were used to improve permeability of membrane, fluorescence labeled dextran and antibody were delivered into cells. The influence of laser parameters and the micro-environment around cells were analyzed, the results showed that concentration of gold nanoparticles is important for photoporation, the cells is easy to die with higher concentration above 104 gold nanoparticles per cell that corresponding to a narrow irradiation fluence. Results also demonstrated that for cells incubated in PBS, successful permeabilization was observed at lower irradiation fluence than cells incubated in RPMI medium. During irradiation the micro-phenomena was observed by homemade plat, we found that the vapor bubble generation around gold nanoparticles is the main mechanism of the photoporation, and the properties of these nanobubbles are highly dependent on the size of the nanoparticles and the properties of the laser pulse according to extended two-temperature model. In addition, a diffusion model that based on measuring resealing time invasively, was established to assess the loading efficiency, results showed that this approach is an ideal and accurate estimation of the loading efficiency of cells by photoporation for accounting hole-resealing dynamics.
Combining photodynamic therapy (PDT) and immunotherapy modalities has shown encouraging therapeutic efficacy against various metastasis cancers. Developing a functional nanoparticle for tumor-targeting and on-demand release of drug is still a major focus for advancing therapeutic approach. Herein, we assembled a β-cyclodextrin (β-CD) modified MMP-2 responsive peptide with a photosensitizer-loaded liposome to construct a tumor immune microenvironment and laser dual-triggered nano system (matrix metalloproteinase 2 (MMP-2) responsive peptide liposome, MR@Lip) for melanoma therapy. The β-CDs encapsulating SB-3CT were released due to the cleavage of the peptide substrate by MMP-2 which is highly expressed in tumor stroma. The localized released SB-3CT was kept in the stroma and inhibited the expression of MMP, down-regulating the soluble NKG2D ligands. The liposome loading photosensitizer Ce6 targeted and killed melanoma cells under laser irradiation, while induced the expression of NKG2D ligands and finally leading to an increase in sensitivity of A375 cells to NK cells. This study might provide novel insight into the development of a new nanomedicine to achieve programmed release of antitumor drugs and better integration of PDT and immune therapy for melanoma.
Firstly, gold nanosphere and 5-aminolevulinic acid (ALA) conjugant was used to study the photodynamic therapy efficiency and its mechanism. We found that the conjugant can improve the cell killing efficiency that mainly attribute to the carriers function of the gold nanoparticles. In order to shift the absorption peak to red light that offer deeper penetration of tissues, gold nanorod and Hematoporphyrin monomethyl Ether (HMME) was conjugated to employ photodynamic therapy of KB cells. During the research, the 808 nm laser light and xenon lamp were used to irradiate the sample, which offered better therapy efficiency than gold nanosphere. Since TiO2 can be used to effectively generate reactive oxygen species (ROS) for photodynamic application with the absorption in the ultraviolet range without oxygen, TiO2 nanoparticles (NPs) are sensitized by linking with the photosensitizer, HMME, to form HMME-TiO2 nanocomposites (NCs) for demonstrating the photodynamic effects under the illumination of white light. The HMME-TiO2 NCs of different composition ratios are prepared for maximizing the generation of ROS and optimizing the inactivation effect of KB cells. The material characteristics and the ROS generation capability of the HMME-TiO2 NCs with the optimized combination ratio show their merits in a photodynamic process under white light irradiation. The application of such NCs to KB cell experiments results in a higher inactivation efficiency when compared to pure HMME of the same concentration. In general, different nanoparticles can improve PDT efficiency with different increasement, mechanism, advantage and disadvantage, we should choose different nonaparticle according to different applications.
Photoacoustic responses induced by laser-excited photothermal bubbles (PTBs) in colloidal gold solutions are relevant to the theranostics quality in biomedical applications. Confined to the complexity of nonstationary, multiscale events, and multiphysical parameters of PTBs, systematic studies of the photoacoustic effects remain obscure. Photoacoustic effects mediated by PTB dynamics and a physical mechanism are studied based on a proof-of-principle multimodal platform integrating side-scattering imaging, time-resolved optical response, and acoustic detection. Results show excitation energy, nanoparticle (NP) size, and NP concentration have strong influence on photoacoustic responses. Under the characteristic enhancement regime, the photoacoustic signal amplitude increases linearly with excitation energy and increases quadratically with the NP diameter. As for the effects of the NP concentration (characterized by absorption coefficient), a higher photoacoustic signal amplitude is generally induced by a dense NP distribution. However, with an increase in the NP size, the shielding effect of NP swarm prevents the increase of photoacoustic responses. This study presents experimental evidence of some key physical phenomena governing the PTB-induced photoacoustic signal generation in gold NP suspensions, which may help enrich theranostic approaches in clinical applications by rationalizing operation parameters.
Although TiO2 can be used to effectively generate reactive oxygen species (ROS) for photodynamic application, its absorption in the ultraviolet range makes the excitation harmful to tissue. Based on the concept of a sensitized solar cell, TiO2 nanoparticles (NPs) are sensitized by linking with the photosensitizer, HMME, to form HMME-TiO2 nanocomposites (NCs) for demonstrating the photodynamic effects under the illumination of white light. The HMME-TiO2 NCs of different composition ratios are prepared for maximizing the generation of ROS and optimizing the inactivation effect of KB cells. The material characteristics and the ROS generation capability of the HMME-TiO2 NCs with the optimized combination ratio show their merits in a photodynamic process under white light irradiation. The application of such NCs to KB cell experiments results in a higher inactivation efficiency when compared to pure HMME of the same concentration.
There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function, singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding. Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX–GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP’s size. The LFE increased with an increase of the GNP size (2R ≤50 nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.
In this paper, HMME-TiO2 nanocomposites was synthesized and characterized through TEM, Uv-vis spectra, Zeta potential, FTIR spectra. The characterization results show that HMME was successfully conjugated onto the surface of TiO2. It can be seen from the TEM images the average size of HMME-TiO2 conjugation is nearly spherical and the particle size range from 20 to 28 nm. Compared with HMME, the B bands of HMME-TiO2 were much broader and lower while in the region of Q bands the absorption peaks of HMME-TiO2 are higher than that of HMME. Encapsulation efficiency of HMME-loaded TiO2 was assessed and calculated as 45.46%. FTIR spectra show the bonding between TiO2 and HMME was through the hydrogen-bonding between COOH and OH bonds. Fluorescence microscope results demonstrated HMME-TiO2 mainly distributed in the membrane and cytoplasm of SCC cells and its best incubation time is six hours. After treated with HMME-TiO2 plus light irradiation (1.8J/cm2 , 632nm), the viability of SCC cells turned to 32.96% is much lower than that treated with HMME plus light irradiation. It can be concluded that the combination of HMME and TiO2 will enhance the PDT efficiency of HMME. In the process of HMME-TiO2 mediated PDT, as a kind of photosensitizer HMME can induce the death of SCC cells, meanwhile it can transform electron to the conductive band of TiO2 stimulating the photocatalytic activity of TiO2 under visible light. The photocatalytic of TiO2 can also induce the death of SCC cells. The combination of these two effects lead to more SCC cells died.
Light-absorbing nanoparticles that are heated by short laser pulses can transiently increase membrane permeability. We evaluate the membrane permeability by flow cytometry assaying of propidium iodide and fluorescein isothiocyanate dextran (FITC-D) using different laser sources. The dependence of the transfection efficiency on laser parameters such as pulse duration, irradiant exposure, and irradiation mode is investigated. For nano- and also picosecond irradiation, we show a parameter range where a reliable membrane permeabilization is achieved for 10-kDa FITC-D. Fluorescent labeled antibodies are able to penetrate living cells that are permeabilized using these parameters. More than 50% of the cells are stained positive for a 150-kDa IgG antibody. These results suggest that the laser-induced permeabilization approach constitutes a promising tool for targeted delivery of larger exogenous molecules into living cells.
Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkin's disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.
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