Novel opto-fluidic drug delivery system for efficient cellular transfection.

Intracellular drug delivery is at the heart of many diagnosis procedures and a key step in gene therapy. Research has been conducted to bypass cell barriers for controlled intracellular drug release and made consistent progress. However, state-of-the-art techniques based on non-viral carriers or physical methods suffer several drawbacks, including limited delivery yield, low throughput or low viability, which are key parameters in therapeutics, diagnostics and drug delivery. Nevertheless, gold nanoparticle (AuNP) mediated photoporation has stood out as a promising approach to permeabilize cell membranes through laser induced Vapour NanoBubble (VNB) generation, allowing the influx of external cargo molecules into cells. However, its use as a transfection technology for the genetic manipulation of therapeutic cells is hindered by the presence of non-degradable gold nanoparticles. Here, we report a new optofluidic method bringing gold nanoparticles in close proximity to cells for photoporation, while avoiding direct contact with cells by taking advantage of hydrodynamic focusing in a multi-flow device. Cells were successfully photoporated with [Formula: see text] efficiency with no significant reduction in cell viability at a throughput ranging from [Formula: see text] to [Formula: see text]. This optofluidic approach provides prospects of translating photoporation from an R &D setting to clinical use for producing genetically engineered therapeutic cells.

Cellular Environment and Phenotypic Heterogeneity: How Data-Driven Modeling Finds the Smoking Gun.

The cellular environment modifies cellular phenotypes, in particular, the stress response phenotype, which easily exhibits high phenotypic heterogeneity due to the common characteristics of its regulatory networks. The aim of this work is to quantify and interpret the impact of collagen type I, a major component of the cellular environment, on the phenotypic heterogeneity of the cellular response. Our approach combines in an original way the monitoring of the response of a single cell and the mathematical modeling of the network. After a detailed statistical description of the phenotypic heterogeneity of the cellular response, the mathematical modeling explains how the observed changes can be explained by an induced increase in the average expression of a central protein of the regulatory network. The predictions of the data-driven model are fully consistent with the biochemical measurements performed. The framework presented here is also a new general methodology to study phenotypic heterogeneity, although we focus here on the response to proteotoxic stress in HeLa cells.

Fine-tuned control of stress priming and thermotolerance.

A common signature of cell adaptation to stress is the improved resistance upon priming by prior stress exposure. In the context of hyperthermia, priming or preconditioning with sublethal heat shock can be a useful tool to confer thermotolerance and competitive advantage to cells. In the present study, we develop a data-driven modeling framework that is simple and generic enough to capture a broad set of adaptation behaviors to heat stress at both molecular and cellular levels. The model recovers the main features of thermotolerance and clarifies the tradeoff principles which maximize the thermotolerance effect. It therefore provides an effective predictive tool to design preconditioning and fractionation hyperthermia protocols for therapeutic purpose.

A minimal titration model of the mammalian dynamical heat shock response.

Environmental stress, such as oxidative or heat stress, induces the activation of the heat shock response (HSR) and leads to an increase in the heat shock proteins (HSPs) level. These HSPs act as molecular chaperones to maintain cellular proteostasis. Controlled by highly intricate regulatory mechanisms, having stress-induced activation and feedback regulations with multiple partners, the HSR is still incompletely understood. In this context, we propose a minimal molecular model for the gene regulatory network of the HSR that reproduces quantitatively different heat shock experiments both on heat shock factor 1 (HSF1) and HSPs activities. This model, which is based on chemical kinetics laws, is kept with a low dimensionality without altering the biological interpretation of the model dynamics. This simplistic model highlights the titration of HSF1 by chaperones as the guiding line of the network. Moreover, by a steady states analysis of the network, three different temperature stress regimes appear: normal, acute, and chronic, where normal stress corresponds to pseudo thermal adaption. The protein triage that governs the fate of damaged proteins or the different stress regimes are consequences of the titration mechanism. The simplicity of the present model is of interest in order to study detailed modelling of cross regulation between the HSR and other major genetic networks like the cell cycle or the circadian clock.

A high-power tunable Raman fiber ring laser for the investigation of singlet oxygen production from direct laser excitation around 1270 nm.

We report on the development of a tunable Raman fiber ring laser especially designed for the investigation of the 3Σ(-)(g) →1 Δg transition of molecular oxygen. Singlet oxygen (1Δg) is a reactive species of importance in the fields of biology, photochemistry, and phototherapy. Tunability of the Raman fiber ring laser is achieved without the use of an intracavity tunable bandpass filter and the laser thus achieves a slope efficiency only obtained up to now in Perot-Fabry cavities. A measurement of the action spectrum of a singlet oxygen trap is made in air-saturated ethanol and acetone to demonstrate the practical application of the tunable Raman fiber ring laser for the investigation of the 3Σ(-)(g) →1 Δg transition of molecular oxygen.

Protein level variability determines phenotypic heterogeneity in proteotoxic stress response.

Cell-to-cell variability in stress response is a bottleneck for the construction of accurate and predictive models which could guide clinical diagnosis and treatment of certain diseases, for example, cancer. Indeed, such phenotypic heterogeneity can lead to fractional killing and persistence of a subpopulation of cells which are resistant to a given treatment. The heat shock response network plays a major role in protecting the proteome against several types of injuries. Here, we combine high-throughput measurements and mathematical modeling to unveil the molecular origin of the phenotypic variability in the heat shock response network. Although the mean response coincides with known biochemical measurements, we found a surprisingly broad diversity in single-cell dynamics with a continuum of response amplitudes and temporal shapes for several stimulus strengths. We theoretically predict that the broad phenotypic heterogeneity is due to network ultrasensitivity together with variations in the expression level of chaperones controlled by the transcription factor heat shock factor 1. Furthermore, we experimentally confirm this prediction by mapping the response amplitude to chaperone and heat shock factor 1 expression levels.

Hypericin-loaded lipid nanocapsules for photodynamic cancer therapy in vitro.

Hypericin (Hy), a naturally occurring photosensitizer (PS), is extracted from Hypericum perforatum plants, commonly known as St. John's wort. The discovery of the in vitro and in vivo photodynamic activities of hypericin as a photosensitizer generated great interest, mainly to induce a very potent antitumoral effect. However, this compound belongs to the family of naphthodianthrones which are known to be poorly soluble in physiological solutions and produce non-fluorescent aggregates (A. Wirz et al., Pharmazie, 2002, 57, 543; A. Kubin et al., Pharmazie, 2008, 63, 263). These phenomena can reduce its efficiency as a photosensitizer for the clinical application. In the present contribution, we have prepared, characterized, and studied the photochemical properties of Hy-loaded lipid nanocapsule (LNC) formulations. The amount of singlet oxygen ((1)O2) generated was measured by the use of p-nitroso-dimethylaniline (RNO) as a selective scavenger under visible light irradiation. Our results showed that Hy-loaded LNCs suppressed aggregation of Hy in aqueous media, increased its apparent solubility, and enhanced the production of singlet oxygen in comparison with free drug. Indeed, encapsulation of Hy in LNCs led to an increase of (1)O2 quantum yield to 0.29-0.44, as compared to 0.02 reported for free Hy in water. Additionally, we studied the photodynamic activity of Hy-loaded LNCs on human cervical carcinoma (HeLa) and Human Embryonic Kidney (HEK) cells. The cell viability decreased radically to 10-20% at 1 µM, reflecting Hy-loaded LNC25 phototoxicity.

Cancerous cell death from sensitizer free photoactivation of singlet oxygen.

Singlet oxygen ((1)O(2)) is an electronic state of molecular oxygen which plays a major role in many chemical and biological photo-oxidation processes. It has a high chemical reactivity which is commonly harnessed for therapeutic issues. Indeed, (1)O(2) is believed to be the major cytotoxic agent in photodynamic therapy. In this treatment of cancer, (1)O(2) is created, among other reactive species, by an indirect transfer of energy from light to molecular oxygen via excitation of a photosensitizer (PS). This PS is believed to be necessary to obtain an efficient (1)O(2) production. In this paper, we demonstrate that production of (1)O(2) is achieved in living cells from PS-free 1270 nm laser excitation of molecular oxygen. The quantity of (1)O(2) produced in this way is sufficient to induce an oxidative stress leading to cell death. Other effects such as thermal stress are discriminated and we conclude that cell death is only due to (1)O(2) creation. This new simplified scheme of (1)O(2) activation can be seen as a breakthrough for phototherapies of malignant diseases and/or as a noninvasive possibility to generate reactive oxygen species in a tightly controlled manner.