Optimal cell therapies require efficient, selective and rapid delivery of molecular cargo into target cells without compromising their viability. Achieving these goals ex vivo in bulk heterogeneous multi-cell systems such as human grafts is impeded by low selectivity and speed of cargo delivery and by significant damage to target and non-target cells. We have developed a cell level approach for selective and guided transmembrane injection of extracellular cargo into specific target cells using transient plasmonic nanobubbles (PNB) as cell-specific nano-injectors. As a technical platform for this method we developed a laser flow cell processing system. The PNB injection method and flow system were tested in heterogeneous cell suspensions of target and non-target cells for delivery of Dextran-FITC dye into squamous cell carcinoma HN31 cells and transfection of human T-cells with a green fluorescent protein-encoding plasmid. In both models the method demonstrated single cell type selectivity, high efficacy of delivery (96% both for HN31 cells T-cells), speed of delivery (nanoseconds) and viability of treated target cells (96% for HN31 cells and 75% for T-cells). The PNB injection method may therefore be beneficial for real time processing of human grafts without removal of physiologically important cells.
"Viral vector is currently the method mostly used to introduce foreign material into cells but safety and immunogenicity concerns [6,7] favored the development of alternative techniques to accomplish this task [2,8]. The main physical methods that have been developed include electroporation [9,10], direct injection  and laser methods such as laser induced stress wave [12,13], direct optoporation [14,15] and selective cell optoporation using light absorbing particles [16–19]. These methods however face major drawbacks. "
[Show abstract][Hide abstract] ABSTRACT: In this paper, we report a light driven, non-invasive cell membrane perforation technique based on the localized field amplification by a nanosecond pulsed laser near gold nanoparticles (AuNPs). The optoporation phenomena is investigated with pulses generated by a Nd:YAG laser for two wavelengths that are either in the visible (532 nm) or near infrared (NIR) (1064 nm). Here, the main objective is to compare on and off localized surface plasmonic resonance (LSPR) to introduce foreign material through the cell membrane using nanosecond laser pulses. The membrane permeability of human melanoma cells (MW278) has been successfully increased as shown by the intake of a fluorescent dye upon irradiation. The viability of this laser driven perforation method is evaluated by propidium iodide exclusion as well as MTT assay. Our results show that up to 25% of the cells are perforated with 532 nm pulses at 50 mJ/cm(2) and around 30% of the cells are perforated with 1064 nm pulses at 1 J/cm(2). With 532 nm pulses, the viability 2 h after treatment is 64% but it increases to 88% 72 h later. On the other hand, the irradiation with 1064 nm pulses leads to an improved 2 h viability of 81% and reaches 98% after 72 h. Scanning electron microscopy images show that the 5 pulses delivered during treatment induce changes in the AuNPs size distribution when irradiated by a 532 nm beam, while this distribution is barely affected when 1064 nm is used.
"A PNB is a laser pulse-induced nanoscale explosive event that develops only if the energy (fluence) of laser pulse exceeds specific threshold of the evaporation of NP environment, a vapor nanobubble and not a NP, although it employs a gold NPs or their cluster to convert optical energy into heat for the evaporation of the liquid surrounding super-heated NP. The major difference that distinguishes PNBs from existing nano-agents is their on-demand threshold and transient nature, dynamic tunability and mechanical, not thermal, therapeutic mechanisms 37-40. PNBs offer several unique oportunities that are not addressed by current methods (both traditional and those using various NPs and external energies). "
[Show abstract][Hide abstract] ABSTRACT: The resistance of residual cancer cells after oncological resection to adjuvant chemoradiotherapies results in both high recurrence rates and high non-specific tissue toxicity, thus preventing the successful treatment of such cancers as head and neck squamous cell carcinoma (HNSCC). The patients' survival rate and quality of life therefore depend upon the efficacy, selectivity and low non-specific toxicity of the adjuvant treatment. We report a novel, theranostic in vivo technology that unites both the acoustic diagnostics and guided intracellular delivery of anti-tumor drug (liposome-encapsulated doxorubicin, Doxil) in one rapid process, namely a pulsed laser-activated plasmonic nanobubble (PNB). HNSCC-bearing mice were treated with gold nanoparticle conjugates, Doxil, and single near-infrared laser pulses of low energy. Tumor-specific clusters of gold nanoparticles (solid gold spheres) converted the optical pulses into localized PNBs. The acoustic signals of the PNB detected the tumor with high specificity and sensitivity. The mechanical impact of the PNB, co-localized with Doxil liposomes, selectively ejected the drug into the cytoplasm of cancer cells. Cancer cell-specific generation of PNBs and their intracellular co-localization with Doxil improved the in vivo therapeutic efficacy from 5-7% for administration of only Doxil or PNBs alone to 90% thus demonstrating the synergistic therapeutic effect of the PNB-based intracellular drug release. This mechanism also reduced the non-specific toxicity of Doxil below a detectable level and the treatment time to less than one minute. Thus PNBs combine highly sensitive diagnosis, overcome drug resistance and minimize non-specific toxicity in a single rapid theranostic procedure for intra-operative treatment.
[Show abstract][Hide abstract] ABSTRACT: A novel transient nanoprobe was developed whose biomedical functions can
be dynamically and selectively tuned inside living cells. This probe is
not a nanoparticle, but a transient event, a vapor nanobubble generated
with a laser pulse around gold (plasmonic) nanoparticles. This is a
phenomenon that we recently discovered and named plasmonic nanobubbles
(PNBs). PNBs represent an entirely new class of probes due to their
dynamic tunability in situ, including single cells. We show that PNB
provides highly sensitive diagnosis and immediate follow-up and guided
treatment with true nanoscale precision at the level of single cells.
Proceedings of SPIE - The International Society for Optical Engineering 02/2012; DOI:10.1117/12.910639 · 0.20 Impact Factor
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