Umanzor-Alvarez J et al. (2011), Near-infrared laser delivery of nanoparticles t...

XB-ART-42888

Biotechnol J 2011 May 01;65:519-24. doi: 10.1002/biot.201000205.

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Near-infrared laser delivery of nanoparticles to developing embryos: a study of efficacy and viability.

Umanzor-Alvarez J , Wade EC , Gifford A , Nontapot K , Cruz-Reese A , Gotoh T , Sible JC , Khodaparast GA .

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Targeted delivery of materials to individual cells remains a challenge in nanoscience and nanomedicine. Near infrared (NIR) laser injection may be a promising alternative to manual injection (where the micropipet diameter limits targeting to small cells) or other laser techniques (such as picosecond green and UV lasers, which can be damaging to cells). However, the efficiency with which NIR pulses can deliver nanoparticles and any adverse effects on living cells needs thorough testing. Toward this end, we have determined the efficacy and toxicity of delivering quantum dots (QDs) into cells of Xenopus laevis embryos by NIR laser injection. Because this model system provides not only living cells but also a developing organism, we were able to assess relatively long-term effects of NIR pulses on embryonic development (through the tadpole stage). We developed parameters for NIR pulses that did not affect embryonic viability or morphology and delivered QDs as effectively as manual injection. Higher intensities of NIR pulses caused permanent damage to the targeted cells, and thus NIR pulses may also prove useful for ablation of specific cells within tissues.

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	Figure 1. Low-power images of X. laevis embryos. (A) Four-cell embryo, 2 h pf. Embryo is being microinjected into one cell, a dorsal blastomere. (B) 32-cell embryo, 4 h pf. (C) Late blastula stage embryo, 9 h pf. (D) Gastrulating embryos, vegetal view (left), animal view (right), âˆ¼14â€“15 h pf; (E) tailbud stage embryo prior to hatching, âˆ¼24 h pf; (F) high-power view of embryo immediately after laser targeting for 1 s. Arrow indicates targeted region. In an experiment with 10 embryos, 80% of embryos targeted with similar settings as the measurement presented in Fig. 1 F survived through the swimming tadpole stage with no observable abnormalities. Scale bars = 1 mm (Aâ€“E), 100 Î¼m (F).
	Figure 2. Delivery of QDs at 1 s exposure and subsequent embryonic development. Control embryos (A,C,E) or embryos targeted with QD solution (B,D,F) for 1 s at 700 nm with an ROI of 100 Î¼m were photographed at 3 h (A,B), 6 h (C,D), and 24 h (E,F). QDs fluoresce green and autofluorescence of embryos is blue. (G) A control embryo and four targeted embryos were photographed at 72 h using a stereoscope the tadpoles are alive and swimming, where the views are from different angles. The embryo indicated by an asterisk (*) is the same embryo shown in (B,D,F). Scale bar = 100 Î¼m in AâF. A fifth targeted embryo died after 24 h. On three different experimental trials involving 40 embryos, a survival rate of more than 60% was observed over a time span of 50 h.
	Figure 3. Delivery of QDs X. laevis embryos via NIR. (A) Schematic of laser targeting approach. Embryos in a dish containing a solution of QDs were first scanned at low power (700 nm; 10â12% intensity) to target a single cell in the vegetal pole since this side faces down and the incident laser comes up through the bottom of the dish. A ROI of 100 Î¼m was then targeted for 1 s at 700 nm. (B) Low-power scan of an embryo showing the ROI. A 660 nm reflector, which only reflects wavelengths >660 nm, were used.
	Figure 4. Low and high power views of embryos after laser injection with QDs. (A) An uninjected (left) and laser-injected (right) embryo right after injection. (B) Control- and (C) laser-injected embryos photographed at the early gastrula stage, âˆ¼8 h after injection. (D) Control- and (E) laser-injected embryos photographed at the tadpole stage, âˆ¼3 d after injection. Note some nonspecific autofluorescence in the gut (arrows) of the control embryo but a higher level of fluorescence in the injected embryo. (F) Control- and (G) laser-injected embryos âˆ¼2 days pf photographed in the gut region using the Nikon confocal microscope. Scale bars = 100 Î¼m in A, F, and G and âˆ¼1 mm in B â€“E. Under similar experimental conditions, four laser and one manually injected embryos with QDs were monitored and photographed, suggesting a higher level of fluorescence in the guts.
	Figure 5. Z-stack series to visualize QDs in laser-targeted embryos. QDs were delivered to embryos at the 32â64-cell stage, and embryos were imaged using the Zeiss LSM at the 32â64 cell stage. Uninjected (A) and laser-targeted (B) embryos were imaged at 5 Î¼m intervals through a 100 Î¼m section in the center of the embryo. The Z-Stack images presented here demonstrate an example of six embryos injected by QDs, using the NIR pulses, under similar experimental conditions.

References [+] :

Beumer, Whole-mount immunohistochemistry on Xenopus embryos using far-red fluorescent dyes. 1995, Pubmed, Xenbase