Judith Krawinkel

Mechanisms of gold nanoparticle mediated ultrashort laser cell membrane perforation

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.

Gold nanoparticle-mediated delivery of molecules into primary human gingival fibroblasts using ns-laser pulses: A pilot study

Interaction of gold nanoparticles (AuNPs) in the vicinity of cells’ membrane with a pulsed laser (λ = 532 nm, τ = 1 ns) leads to perforation of the cell membrane, thereby allowing extracellular molecules to diffuse into the cell. The objective of this study was to develop an experimental setting to deliver molecules into primary human gingival fibroblasts (pHFIB-G) by using ns-laser pulses interacting with AuNPs (study group). To compare the parameters required for manipulation of pHFIB-G with those needed for cell lines, a canine pleomorphic adenoma cell line (ZMTH3) was used (control group). Non-laser-treated cells incubated with AuNPs and the delivery molecules served as negative control. Laser irradiation (up to 35 mJ/cm2) resulted in a significant proportion of manipulated fibroblasts (up to 85%, compared to non-irradiated cells: p < 0.05), while cell viability (97%) was not reduced significantly. pHFIB-G were perforated as efficiently as ZMTH3. No significant decrease of metabolic cell activity was observed up to 72 h after laser treatment. The fibroblasts took up dextrans with molecular weights up to 500 kDa. Interaction of AuNPs and a pulsed laser beam yields a spatially selective technique for manipulation of even primary cells such as pHFIB-G in high throughput.

Intrazelluläre Manipulation mit peptidkonjugierten Goldnanopartikelagglomeraten

Neben spharischen Partikeln konnen auch Agglomerate spharischer Partikel zur Manipulation von Zellen verwendet werden. Mehrere spharische AuNP in unmittelbarer Nahe zueinander fuhren zu einer verstarkten Intensitat der Nahfeldstreuung und so zu den sogenannten „hot spots“ zwischen den Partikeln [36]. Uberdies ist der Warmeeintrag bei gleicher Laserleistung im Vergleich zu einzelnen AuNP erhoht.

Alternative zu Partikeln: Zellmanipulation mit goldbeschichteten Nanostrukturen

Neben spharischen AuNP und Goldnanopartikelagglomeraten konnen auch an goldbeschichteten (strukturierten) Oberflachen durch Interaktion mit Laserstrahlung entstehende plasmoneninduzierte Effekte fur die Zellmanipulation verwendet werden. Solche Oberflachen lassen erwarten, dass bei der Manipulation keine NP in die Zellen gelangen. So kann bei klinischen Anwendungen eine Anlagerung im menschlichen Korper verhindert werden.

Zellmanipulation mit Hilfe sphärischer Partikel und Laserstrahlung

Membranadharente spharische NP in Kombination mit Laserstrahlen, die uber die Probe gescannt werden, konnen Zellen schonend und mit hohem Durchsatz manipulieren [9, 160, 162, 180]. Bei Lasereinstrahlung fungieren AuNP als „Nanolinsen“ bzw. „Nanoheizungen“. Durch die dabei entstehenden Effekte (s. 2.1) wird die Zellmembran lokal in der Nahe der Partikel perforiert, so dass Molekule vom extra- in den intrazellularen Raum gelangen konnen (s. 2.2.3).

Grundlagen laserbasierter Manipulation von Zellen mittels plasmoneninduzierter Effekte

Der Mechanismus der laserbasierten Zellmanipulation mittels plasmoneninduzierter Effekte nutzt die Auswirkungen der Interaktion von Laserstrahlung mit Goldnanostrukturen auf biologische Materialien. Da die Interaktion von den Laserparametern sowie den Eigenschaften der Goldnanostrukturen abhangt, ist das Verstandnis ihrer optischen Eigenschaften, also ihr Verhalten bei Interaktion mit einer EMWelle, essentiell. Deswegen werden in diesem Kapitel zunachst die optischen Eigenschaften von AuNP und die durch Laserstrahlung an ihnen induzierten Effekte beschrieben.

Gold nanoparticle mediated cell manipulation using fs and ps laser pulses for cell perforation and transfection

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.