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Research Papers: Regular Papers

Transient Low-Temperature Effects on Propidium Iodide Uptake in Lance Array Nanoinjected HeLa Cells

[+] Author and Article Information
John W. Sessions

Department of Mechanical Engineering,
Brigham Young University,
Provo, UT 84602
e-mail: sessions.john84@gmail.com

Brad W. Hanks

Department of Mechanical Engineering,
Brigham Young University,
Provo, UT 84602
e-mail: hanksbr90@gmail.com

Dallin L. Lindstrom

Department of Exercise Science,
Brigham Young University,
Provo, UT 84602
e-mail: dallin.lindstrom@gmail.com

Sandra Hope

Department of Microbiology and
Molecular Biology,
Brigham Young University,
Provo, UT 84602
e-mail: sandrahope2016@gmail.edu

Brian D. Jensen

Department of Mechanical Engineering,
Brigham Young University,
Provo, UT 84602
e-mail: bdjensen@byu.edu

1Corresponding author.

Manuscript received September 11, 2015; final manuscript received March 25, 2016; published online May 10, 2016. Assoc. Editor: Feng Xu.

J. Nanotechnol. Eng. Med 6(4), 041005 (May 10, 2016) (9 pages) Paper No: NANO-15-1077; doi: 10.1115/1.4033323 History: Received September 11, 2015; Revised March 25, 2016

Understanding environmental factors relative to transfection protocols is key for improving genetic engineering outcomes. In the following work, the effects of temperature on a nonviral transfection procedure previously described as lance array nanoinjection are examined in context of molecular delivery of propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to HeLa 229 cells. For treatment samples, variables include varying the temperature of the injection solution (3C and 23C) and the magnitude of the pulsed voltage used during lance insertion into the cells (+5 V and +7 V). Results indicate that PI is delivered at levels significantly higher for samples injected at 3C as opposed to 23C at four different postinjection intervals (t = 0, 3, 6, 9 mins; p-value ≤ 0.005), reaching a maximum value of 8.3 times the positive control for 3 C/7 V pulsed samples. Suggested in this work is that between 3 and 6 mins postinjection, a large number of induced pores from the injection event close. While residual levels of PI still continue to enter the treatment samples after 6 mins, it occurs at decreased levels, suggesting from a physiological perspective that many lance array nanoinjection (LAN) induced pores have closed, some are still present.

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Figures

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Fig. 1

Scanning electron microscope image of lance array silicon chip. Lances measure 10 μm in height and 1–2.5 μm in diameter, with a 10 μm spacing.

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Fig. 2

Lance array nanoinjection stepwise process. (a) Staging lance array into injection solution maintained at either 3 C or 23 C. (b) Injection of cell culture, consisting of both physical penetration of the cell membrane and electrical treatment. (c) Staining of cell culture by introduction of PI into the extracellular solution at 0, 3, 6, and 9 mins postinjection. (d) Induced pores from injection event eventually close and PI molecules in the intracellular space of injected cells interact with nucleic acids, increasing PI fluorescence.

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Fig. 3

Diagram of nanoinjection device. Components shown (top to bottom) include: stepper motor mounted to ABS orthoplanar spring, threaded screw acting to actuate silicon lance array mounted above three-dimensional (3D) printed cell culture platform, and associated electrical connections (ground connected to cell culture platform).

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Fig. 4

Cell culture platform assembly. Glass slide containing cell culture secured between the bottom PLA base and top PLA clip to provide proper alignment during injection.

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Fig. 5

Experimental setup illustrating injections performed for 3 C treatment samples. A thermocouple was used to verify surrounding fluid temperature of ice/water bath of 3 C prior to treatment. labview program was used to verify proper electrical signals delivered to nanoinjection device. Six-well plate containing cell culture platforms and associated cell cultures were aligned to nanoinjection device.

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Fig. 6

Flow cytometry sample of six different sample types. (a) Demonstrates how cells were gated for living cells and dead cells from forward and side-scatter signals. (b)–(g) Represent gating of living cells based on signal intensity of samples from blue-laser 2 sensor (BL-2). Note: The threshold for PI+ signals was determined from nontreated control samples and is used globally to determine PI+ cells in treatment samples. (a) foward scatter/side scatter with gating, (b) NTC, (c) BC for PI, (d) 5 V pulsed, 23 C treatment, (e) 5 V pulsed, 3 C treatment, (f) 7 V pulsed, 23 C treatment, and (g) 7 V pulsed, 3 C treatment.

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Fig. 7

Box plots of normalized propidium iodide uptake for all sample types, grouped according to postinjection time when PI was added: (a) normalized PI uptake, t = 0 min postinjection, (b) normalized PI uptake, t = 3 min postinjection, (c) normalized PI uptake, t = 6 min postinjection, and (d) normalized PI uptake, t = 9 min postinjection. Statistically significant relationships based on the permutation testing are indicated with a star.

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Fig. 8

Mean normalized PI uptake for all sample types through time

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Fig. 9

Mean normalized cell viability for all sample types through time

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