Laser-based transfection techniques have proven high applicability in several cell biologic applications. The delivery of different molecules using these techniques has been extensively investigated. In particular, new high-throughput approaches such as gold nanoparticle–mediated laser transfection allow efficient delivery of antisense molecules or proteins into cells preserving high cell viabilities. However, the cellular response to the perforation procedure is not well understood. We herein analyzed the perforation kinetics of single cells during resonant gold nanoparticle–mediated laser manipulation with an 850-ps laser system at a wavelength of 532 nm. Inflow velocity of propidium iodide into manipulated cells reached a maximum within a few seconds. Experiments based on the inflow of FM4-64 indicated that the membrane remains permeable for a few minutes for small molecules. To further characterize the cellular response postmanipulation, we analyzed levels of oxidative heat or general stress. Although we observed an increased formation of reactive oxygen species by an increase of dichlorofluorescein fluorescence, heat shock protein 70 was not upregulated in laser-treated cells. Additionally, no evidence of stress granule formation was visible by immunofluorescence staining. The data provided in this study help to identify the cellular reactions to gold nanoparticle–mediated laser manipulation.
Modulation of the cell membrane permeability by the plasmonic interaction of gold nanoparticles and short laser pulses for cell manipulation or destruction has been the objective of several recent studies. Gold nanoparticles in close vicinity to the cellular membrane are irradiated to evoke a nanoscale membrane perforation, enabling extracellular molecules to enter the cell. However, besides several basic studies no real translation from proof of concept experiments to routine usage of this approach was achieved so far.
In order to provide a reproducible and easy-to-use platform for gold nanoparticle mediated (GNOME) laser manipulation, we established an automated and encased laser setup. We demonstrate its feasibility for high-throughput cell manipulation. In particular, protein delivery into canine cancer cells is shown. The biofunctional modification of cells was investigated using the caspase 3 protein, which represents a central effector molecule in the apoptotic signaling cascade. An efficient and temporally well-defined induction of apoptosis was observed with an early onset 2 h after protein delivery by GNOME laser manipulation. Besides protein delivery, modulation of gene function using GNOME laser transfection of antisense molecules was demonstrated, showing the potential of this technique for basic science and screening purposes.
Concluding, we established GNOME laser manipulation of cells as a routine method, which can be utilized reliably for the efficient delivery of biomolecules. Its intrinsic features, being low impairment of the cell viability, high delivery efficiency and universal applicability, render this method well suited for a large variety of biomedical application.
The interaction of cell-adhered nanostructures with laser light has attracted much interest within the biomedical field. Molecular delivery using a variety of plasmonic nanostructures, such as structured surfaces, nanoparticles and particle clusters, is currently evolving from its proof-of-concept into a routine method. Here, gold represents the material of choice, as it provides unique optical properties, different surface modifications as well as biocompatibility. In addition, new materials (e.g. polypyrrole) provide interesting alternatives.
Applying this approach, a variety of molecules, such as fluorescent dyes, proteins, antisense structures, and DNA, has been transfected in order to manipulate the cellular functions in different experimental settings. Antisense structures, for example, allow the efficient down regulation of the gene activity of a target, providing insights into the gene’s function. The delivery of proteins, as executing molecules in the cell, can exhibit an immediate effect on the cell behavior, allowing a minute observation of the intracellular kinetics.
Direct cell manipulation can be achieved with this approach as well. Increasing the nanoparticle concentration and/or the radiant exposure, effective cell destruction is induced. Using targeted nanoparticles (e.g. by antibody conjugation) in combination with spatially selective laser irradiation permits well-directed cell manipulation even in mixed cultures and potentially in tissues. Furthermore, excited gold nanoparticles can directly trigger cellular reactions, which can possibly be utilized for cell stimulation. The manifold possibilities of nanostructure assisted laser manipulation are still in development.
Laser-based transfection techniques have gained significant interest during the last decade. Either single cell manipulation by focusing on the cell membrane or high-throughput can be realized with laser transfection. The latter is for example provided by gold nanoparticle mediated laser transfection. It is based on the heating of gold nanoparticles through laser irradiation, which permeabilizes the membrane. This technique satisfies most prerequisites of a reliable transfection technique, like efficiency and minimal cell impact. In order to bring it closer to routine usage, we investigated new particle configurations for gold nanoparticle mediated laser transfection. Our setup employs a 532 nm and 850 ps laser system. We immobilized gold particles on cell culture surfaces or modified silica particles with a gold particle surface coverage. Furthermore, first experiments achieving cell perforation with an organic nanoparticle based on polypyrrole were conducted. These three options achieved comparable efficiencies to the incubation of cells with free gold nanoparticles. With regard to the underlying mechanisms of perforation, we performed fluorescence microscopy based imaging of the cell state combined with holographic imaging directly after perforation. First results indicated a power dependent ion (calcium) and volume exchange with the extracellular medium in the first two minutes after perforation. In conclusion, our results can pave the way to a safer and more efficient way of high-throughput laser transfection with gold nanoparticles.
In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.
Alternative high throughput transfection methods are required to understand the molecular network of the cell, which is linked to the evaluation of target genes as therapeutic agents. Besides diagnostic purposes, the transfection of primary- and stem cells is of high interest for therapeutic use. Here, the cell release of trans- or exogene proteins is used to develop immune cancer therapies. The basic requirement to accomplish manipulation of cells is an efficient and gentle transfection method. Therefore, we developed an automatized cell manipulation platform providing high throughput by using GNOME laser transfection. Herein, the interaction of moderately focused laser pulses with gold nanoparticles in close vicinity to the cell membrane mediate transient membrane permeabilization. The exact nature of the involved permeabilization effects depends on the applied particles and laser parameters. Hereinafter, we describe investigations considering the parameter regime, the permeabilization mechanism and the safety profile of GNOME laser transfection. The experimental and calculated results imply a combined permeabilization mechanism consisting of both photochemical and photothermal effects. Furthermore, paramount spatial control achieved either by laser illumination with micrometer precision or targeted gold nanoparticle binding to the cells was demonstrated, allowing selective cell manipulation and destruction. Additionally, the possibility to manipulate difficult to transfect primary cells (neurons) is shown. These results give insights in the basic mechanisms involved in GNOME laser transfection and serve as a strong basis to deliver different molecules for therapeutic (e.g. proteins) and diagnostic (siRNA) use.
The delivery of antisense structures, like siRNA, is beneficial for new therapeutic approaches in regenerative sciences. Optical transfection techniques enable high spatial control combined with minimal invasive treatment of cells due to the use of short laser pulses. However, single cell laser transfection by a tightly focused laser beam, for example femtosecond laser transfection, has the major drawback of low throughput. Compared to this, high-throughput in laser transfection is possible by applying gold nanoparticles irradiated by a weakly focused laser beam scanning over the cell sample. Herein, we show the delivery of antisense molecules and demonstrate the minimal cytotoxicity of a method called gold nanoparticle mediated (GNOME) laser transfection. A 532 nm microchip laser in conjugation with 200 nm gold nanoparticles at a concentration of 0.5 μg/cm2 is used. In addition to antisense molecules, the uptake of dextrans of several sizes is analyzed.
The gold nanoparticle (AuNP) mediated ultrashort laser cell membrane perforation has been proven as an efficient
delivery method to bring membrane impermeable molecules into the cytoplasm. Nevertheless, the underlying
mechanisms have not been fully determined yet. Different effects may occur when irradiating a AuNP with ultrashort
laser pulses and finally enable the molecule to transfer. Depending on the parameters (pulse length, laser fluence and
wavelength, particle size and shape, etc.) light absorption or an enhanced near field scattering can lead to perforation of
the cell membrane when the particle is in close vicinity. Here we present our experimental results to clarify the
perforation initiating mechanisms. The generation of cavitation and gas bubbles due to the laser induced effects were
observed via time resolved imaging. Additionally, pump-probe experiments for bubble detection was performed.
Furthermore, in our patch clamp studies a depolarization of the membrane potential and the current through the
membrane of AuNP loaded cell during laser treatment was detected. This indicates an exchange of extra- and intra
cellular ions trough the perforated cell membrane for some milliseconds. Additionally investigations by ESEM imaging
were applied to study the interaction of cells and AuNP after co incubation. The images show an attachment of AuNP at
the cell membrane after several hours of incubation. Moreover, images of irradiated and AuNP loaded cells were taken to
visualize the laser induced effects.
Manipulation of cells requires the delivery of membrane-impermeable substances like genetic materials or
proteins into the cytoplasm. Thus delivery of molecules over the cell membrane barrier is one of the key
technologies in molecular biology. Many techniques concerning especially the delivery foreign DNA have been
developed. Notwithstanding there still is a range of applications where these standard techniques fail to raise the
desired results due to low efficiencies, high toxicity or other safety issues. Especially the transfection of sensitive
cell types like primary and stem cells can be problematic. Here we present an alternative, laser based technique to
perforate the cell membrane and thus allowing efficient delivery of extra cellular molecules: Gold nanoparticles
(GNP) are brought into close contact with the cell, were the laser-GNP interaction leads to membrane
perforation. This allows the utilisation of a weakly focused laser beam leading to fast scanning of the sample and
thus to a high throughput. To investigate the GNP-laser interaction in more detail we have compared membrane
perforation obtained by different laser pulse lengths. From our results we assume strong light absorption for ps
laser pulses and relatively small particles as the initiating perforation mechanism, whereas an enhanced near field
scattering occurs at 200 nm GNP when using fs laser pulses. SEM and ESEM imaging were applied to give a
deeper insight in the GNP-cell interaction and the effects of laser radiation on the GNP. Additionally dextran-
FITC derivatives of varying sizes were used to investigate the impact of molecule size on delivery efficiency.
The delivery of extra cellular molecules into cells is essential for cell manipulation. For this purpose genetic materials
(DNA/RNA) or proteins have to overcome the impermeable cell membrane. To increase the delivery efficiency and cell
viability of common methods different nano- and micro material based approaches were applied. To manipulate the cells,
the membrane is in contact with the biocompatible material. Due to a field enhancement of the laser light at the material
and the resulting effect the cell membrane gets perforated and extracellular molecules can diffuse into the cytoplasm.
Membrane impermeable dyes, fluorescent labelled siRNA, as well as plasmid vectors encoded for GFP expression were
used as an indicator for successful perforation or transfection, respectively. Dependent on the used material, perforation
efficiencies over 90 % with a cell viability of about 80 % can be achieved. Additionally, we observed similar efficiencies
for siRNA transfection. Due to the larger molecule size and the essential transport of the DNA into the nucleus cells are
more difficult to transfect with GFP plasmid vectors. Proof of principle experiments show promising and adequate
efficiencies by applying micro materials for plasmid vector transfection. For all methods a weakly focused fs laser beam
is used to enable a high manipulation throughput for adherent and suspension cells. Furthermore, with these alternative
optical manipulation methods it is possible to perforate the membrane of sensitive cell types such as primary and stem
cells with a high viability.
The resulting effects of the interaction between nanoparticles and laser irradiation are a current matter in research.
Depending on the laser parameters as well as the particles properties several effects may occur e.g. bubble formation,
melting, fragmentation or an optical breakdown at the surface of the nanoparticle. Besides the investigations of these
effects, we employed them to perforate the membrane of different cell lines and investigated nanoparticle mediated laser
cell perforation as an alternative optical transfection method. Therefore, the gold nanoparticles (GNP) of different shapes
were applied. Furthermore, we varied the methods for attaching GNP to the membrane, i.e. co-incubation of pure gold
nanoparticles and bioconjugation of the surface of GNP. The optimal incubation time and the location of the GNP at the
cell membrane were evaluated by multiphoton microscopy. If these GNP loaded cells are irradiated with a fs laser beam,
small areas of the membrane can be perforated. Following, extra cellular molecules such as membrane impermeable dyes
or foreign DNA (GFP vectors) are able to diffuse through the perforated area into the treated cells. We studied the
dependence of the laser fluence, GNP concentration, GNP size and shape for successful nanoparticle mediated laser cell
perforation. Due to a weak focusing of the laser beam a gentle cell treatment with high cell viabilities and high
perforation efficiencies can be achieved. A further advantage of this perforation technique is the high number of cells
that can be treated simultaneously. Additionally, we show applications of this method to primary and stem cells.
We present our results for nanoparticle mediated laser
poration as an alternative transfection technique. As a fundamental part for the perforation of the cell membrane the interactions of gold nanoparticles and living cells were studied.
Investigation on the interaction of small particles, e.g. gold nanoparticles with light is a current field of high interest. As
light can be absorbed, enhanced or scattered by the nanoparticles a wide variety of possible applications become
possible. If the electrons of such a nanoparticles oscillate with the incident light, plasmon resonances occur. Provided
that these particles are brought very close to a cell, the cell membrane gets perforated due to the laser induced effect. We
investigate nanoparticle mediated laser perforation as an alternative technique for cell transfection. By using weakly
focussed femtosecond laser pulses, 150 nm gold particles were stimulated to perforate the cell membrane. Through the
perforated area of the membrane macromolecules e.g. DNA are able to enter the cell. By this technique GFSHR-17 rat
cells were successfully transfected with GFP vector and the dependence on laser parameters and concentration were
studied. Even after 48 hours after manipulation the transfected cells show no indications of apoptosis or necrosis. This
technique allows the transfection of cells by opto-perforation without the need of tight focusing conditions and single
cell targeting- opening the way for a wide field of applications.
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