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Research Papers

Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array OPEN ACCESS

[+] Author and Article Information
Zachary K. Lindstrom, Steven J. Brewer, Melanie A. Ferguson

Department of Mechanical Engineering,
Brigham Young University,
Provo, UT 84602

Sandra H. Burnett

Department of Microbiology
and Molecular Biology,
Brigham Young University,
Provo, UT 84602

Brian D. Jensen

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

1Corresponding author.

Manuscript received April 4, 2014; final manuscript received September 15, 2014; published online October 3, 2014. Assoc. Editor: Malisa Sarntinoranont.

J. Nanotechnol. Eng. Med 5(2), 021008 (Oct 03, 2014) (7 pages) Paper No: NANO-14-1028; doi: 10.1115/1.4028603 History: Received April 04, 2014; Revised September 15, 2014

Delivering foreign molecules into human cells is a wide and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via nonviral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. We present an electromechanical method for delivering foreign molecules into human cells. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. This paper describes an array of solid silicon microlances that was tested to determine efficiency and viability when nanoinjecting tens of thousands of HeLa cells simultaneously. Propidium iodide (PI), a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers PI into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78–91% viability. With these results, we submit the nanoinjection method using a silicon lance array as another promising particle delivery method for mammalian culture cells.

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Many methods to deliver foreign material into living animal cells are common in research and clinical studies. By delivering certain particles into cells, diseases can be treated or even cured. Gene therapy is possibly the most common field of foreign material delivery into cells and has great implications for curing human genetic diseases. Certain nucleic acids, when delivered into cell nuclei, can suppress undesirable cellular processes or supplement certain processes that have been limited due to genetic disease. For example, two monkeys colorblind from birth were given gene therapy treatment in a recent experiment. As a result of gene therapy, both animals had their color vision restored with no apparent side effects [1].

Traditionally, gene therapy has been divided into two categories: biological (viral) vectors and chemical or physical (nonviral) approaches [2-4]. Although viral vectors are currently the most effective approach to delivering deoxyribose nucleic acid (DNA) into cells, they have certain limitations, including immunogenicity, toxicity, and limited capacity to carry DNA [2,5-9].

Microinjection, or injecting DNA solution through a micropipette into a cell, is a highly efficient delivery method. However, microinjection is limited to in vitro applications and injecting a single cell at a time, which can be time consuming [3,7,10]. Electroporation techniques use an electric field to increase permeability of the plasma membrane, allowing foreign particles to enter the cells through membrane openings. Although electroporation can be an efficient gene delivery method, the viability of electroporated cells can be limited [3,6,7,11,12]. Other studies have used chemicals to deliver foreign particles into cells [6,11,13,14,15]. Chemical reagents can be efficient at delivering DNA into cells, but studies have found that these chemicals can also be toxic to cells [16]. Numerous other methods exist and can be reviewed in Refs. [2-4].

Genetic-based therapy is a promising area of drug delivery, although there are currently many limitations and advantages to all gene therapy methods in practice. In this paper, we discuss a new mechanical approach to deliver foreign particles into cells. Nanoinjection is a new gene delivery technique that uses a solid silicon lance to penetrate the cell membrane and deliver foreign material. Nanoinjection techniques have the potential for high material delivery efficiency while maintaining high viability. Previous nanoinjection studies have designed and used a single polysilicon lance mechanism to inject mouse zygotes with DNA. These mouse zygote injections have proven more efficient and less damaging to mouse zygotes when compared to parallel studies using a microinjection technique [17-19].

A fluorescent dye, PI, is frequently used as an injection particle when performing preliminary experiments with new gene therapy methods. In one study, optical injection (photoporation with a laser) was used to deliver PI molecules into living HEK293 cells in a microfluidic device. At a PI concentration of 1 mg/mL solution, an average PI delivery efficiency and viability of 42% and 67% were obtained, respectively [20]. Another study used highly localized electroporation with nanostraws to deliver PI into small amounts of HEK293 cells. With PI concentrations at 0.1 mg/mL solution, >95% PI uptake and >98% viability were achieved [21].

We have previously developed a nanoinjection lance array for injecting thousands of culture cells simultaneously [22]. We have used PI as the injection particle to measure particle uptake efficiency and cell viability when using the nanoinjection lance array. The nanoinjection lance array penetrates cell membranes in an entire cell culture, opening up pores in the membranes where injection particles can enter cells. The lances are small enough, relative to the size of the cells, that damage to the cell is minimal. This paper presents the proposed nanoinjection method and discusses results from testing the lance array on HeLa culture cells. This method could have tremendous implications as an injection technique in academic or clinical research, including the gene therapy field.

Lance Array Fabrication and Injection Device.

We fabricate lance arrays using standard photolithography and etching methods for silicon wafers outlined in Ref. [22]. First, we pattern a bare silicon wafer with a grid of photoresist dots. The locations of the photoresist dots determine where lances will be located on the wafer by the end of the fabrication process. A combination of plasma etches at specific etch times results in an array of high-aspect ratio lances on the surface of the wafer. The lances are 1–1.5 μm in diameter and 8–10 μm in length (see Fig. 1). We dice the silicon wafer into square pieces 2 cm × 2 cm. Each 2 × 2 cm silicon chip contains approximately 4 × 106 lances on the surface, with the space between lances being 10 μm. We optimized lance geometry and spacing for testing on a culture of HeLa 229 cells [22].

For injection experiments, we mounted the lance array on an injection device. The injection device design fulfills several purposes: (1) provide the motion necessary to plunge the lances through cell membranes and withdraw them during the injection process, (2) constrain lance movement to the vertical direction only, (3) fit snugly into each well of the culture dish, and (4) contain conductive sections of material that allow voltage to be applied to the lance array chip [23]. We fabricated the injection device from ABS plastic with rapid prototyping technology (Fig. 2). Two parallel orthoplanar springs ensure injection motion is strictly vertical [24]. Voltage is required for some of our tests to determine if an electric charge on lances increases PI uptake into cells. A combination of stainless steel wire, conductive epoxy, and gold contacts makes up the conductive section of the device necessary for voltage application [23].

PI.

PI (Sigma) was used in this experiment as an injection particle to determine the efficiency and viability of the lance array nanoinjection method. When PI comes into contact with genetic material, PI molecules intercalate between base pairs of DNA and ribonucleic acid (RNA) [25-31]. PI molecules attached to nucleic acids emit enhanced (20×–30×) fluorescent light waves when excited by certain lamps, making PI positive cells detectable with flow cytometry. PI does not penetrate the membrane of viable cells [26,30,31,32], so PI positive cells in this experiment must be either successfully injected, dead, or both. In previous experiments, no cytotoxicity has been seen in cells for at least 1 h after the addition of PI to islet cells [33]. In theory, the double positive charge on a PI molecule [34] allows PI to be manipulated in the presence of an electric field. We performed tests to determine if an electric charge would increase or decrease PI uptake in cells. All the attributes of PI listed in this section make PI molecules good candidates for testing the effectiveness of the nanoinjection lance array system.

Nanoinjection Technique.

The nanoinjection technique uses a solid lance to penetrate the cell membrane and deliver foreign material to the cell [18]. Two methods can be used to disperse particles into the cell: (1) particles can either diffuse into the cell through pores opened by the lance or (2) electric charge, applied to the lance, can assist in particle entry. When including an electric charge on the lance array chip in the nanoinjection method, charged particles are accumulated on the lance through application of voltage. The lance then penetrates the cell membrane, the charge is reversed, and the particles are repelled into the cell. The diffusion method is nearly identical, without the application of an electric charge to the lances.

When using electric charge to assist in particle delivery, the method has some similarities to traditional electroporation with mechanical assistance. However, the maximum voltage used here, 5 V, over a separation distance of 0.15 mm, gives an approximate maximum electric field of 333 V/cm. This is relatively low compared to many electroporation studies (for example, Ref. [6] used an electric field of 750 V/cm). The intention of this paper is to present data on nanoinjection without providing a direct comparison to electroporation.

Testing Methods.

We nanoinjected HeLa 229 cancer cells (Fig. 3) with PI in this experiment. We cultured cells in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum and gentamicin, with incubation at 37 °C with 5.0% CO2. To prepare for the injection process, we cultured cells in six well culture plates, with approximately 2.5 × 105 cells per well. We cultured cells on glass microscope cover slips in each well to eliminate any surface variation in the culture plates. In the experiments performed using voltage, we placed stainless steel plates under glass microscope slips in the base of the wells before adding cells. We spread cells into a circular shape approximately 2 cm in diameter in the center of the well.

Following incubation in six well plates for 24 h, we removed media from each well and rinsed the cells with Hanks balanced salt solution (HBSS). We added HBSS and PI to each well in a predetermined concentration. We used four PI concentrations in these experiments to determine trends in cellular PI uptake and cell viability due to PI concentration in solution: 0.01, 0.02, 0.03, and 0.04 mg PI per 1 mL HBSS. A typical injection experiment consisted of twelve controls (one with no PI and no membrane penetration with lances, and eleven with PI added but no membrane penetration), and twelve injected samples (PI added and cells punctured with lances).

For each injected sample, we lowered the injection device and lance array chip into a well of the six-well culture dish. A side view schematic of the injection device and lance array chip in a single cell culture well is shown in Fig. 4. The stainless steel plate electrode (for voltage injections), lance array chip, and adhered HeLa cells were completely submersed in HBSS/PI solution for these experiments.

At this step in the nanoinjection process, we performed one of six different tests to determine molecule delivery efficiency and cell death due to different variations of the nanoinjection process. These tests consisted of:

  1. PI diffusing through membrane openings created by lances in the nanoinjection process, with no applied voltage
  2. an application of 1.5 VDC to the lances to accumulate and release PI molecules into cells
  3. PI entering cells with the assistance of 1.5 VDC accumulation and a 20 ms release pulse alternating between 1.5 and 5 VDC applied to the lances
  4. an experiment identical to diffusion testing without addition of PI
  5. an experiment identical to diffusion testing, but using a bare silicon chip instead of a lance array
  6. an experiment performed to determine the length of time cell membranes remain permeable following penetration and removal of lances.

Diffusion Protocol.

We applied a manual force of approximately 30 N to the center fixture of the injection device, moving the lances through cell membranes and into the cytoplasm or nucleus of adhered cells. We measured the magnitude of the force using a force plate on a small number of injections to obtain an estimate of the manual force. We arranged the force plate to measure the force actually applied to the injection chip (that is, not including the stiffness of the support spring). After holding the force for 5 s, we released the force and removed the injection device from the well. The diffusion protocol presented PI nanoinjection data for comparison against other variations in the injection process.

DC Voltage Protocol.

In theory, positively charged PI molecules can be accumulated on lances and released in the presence of an electric field [34]. We added DC voltage to the diffusion protocol to test for greater PI uptake efficiencies using an electric field. We used a power supply to apply an electric potential across the stainless steel base electrode and the lances. We applied negative 1.5 VDC to the system for 20 s, allowing lances to accumulate positively charged PI molecules. Using a manually applied 30 N force, we lowered the lances into the cell culture to puncture cell membranes. Immediately following membrane penetration, we switched the negative 1.5 VDC to positive 1.5 VDC for 5 s, releasing PI molecules from the lances into the cytoplasm or nucleus. We withdrew the lances from the cells and moved the entire device to the next injection site. Figure 5 illustrates the steps in the nanoinjection process with a lance array chip and DC voltage.

DC Voltage With Pulse Protocol.

We applied negative 1.5 VDC to the nanoinjection system for 20 s, allowing lances to accumulate positively charged PI molecules. Again, we pushed lances into cultured cells with a force of 30 N. Immediately following membrane penetration, we applied 10 square wave pulses to the electrodes, releasing PI molecules from lances and into the cells. Each pulse was 1 ms long, and acted between positive 1.5 and 5 VDC. Then, we applied positive 1.5 VDC while removing the injection system from the well. We employed pulsed voltage to determine if an increased repulsion voltage would increase PI uptake while mitigating cell death. We applied the 2 ms period square wave pulse with 50% duty cycle for 20 ms, for a total of 10 pulses per injection, since high voltages for an extended period of time can decrease cell viability [35]. We watched the wells during the voltage pulses to see whether bubbles would form, indicating electrolysis. Had electrolysis occurred, it might have further decreased cell viability due to pH changes inside the cell. However, no bubble formation was observed.

Protocol for Injections Without PI.

In order to quantify cell death due to lances puncturing cell membranes, we did not add PI to any samples for these nanoinjections. We applied a manual force of approximately 30 N to the center fixture of the injection device, moving the lances through cell membranes and into the cytoplasm or nucleus of adhered cells. After holding the force for 5 s, we released the force and removed the injection device from the well.

Bare Silicon Chip Injection Protocol.

To determine the difference in PI uptake between cells punctured with lances and cells strained but not punctured, we employed a bare silicon chip without lances. We applied a manual force of approximately 30 N to the center fixture of the injection device, pressing the bare silicon chip against cell membranes of adhered HeLa cells. After holding the force for 5 s, we released the force and removed the injection device from the well.

Cell Membrane Permeability Timing Protocol.

To test for cell membrane permeability over time, we punctured cell cultures with the lance array before adding PI to the solution. PI concentration was constant at 0.04 mg/mL for all samples. After cell cultures were injected, we added PI to samples at intervals of 0, 3, 7, or 10 min.

Collection of Cells for Analysis.

To make sure different PI concentration injections were not mixed, we rinsed the lance array chip before injecting cells at a new PI concentration. Following the injection of each sample, regardless of previous injection protocol, we added 5× Trypsin (Sigma) to each well and incubated the culture dishes for 5 min. After trypsinating the cells, we added DMEM to each well, and the contents of each well were transferred to FACS tubes (BD Biosciences). We centrifuged the tubes at 2000 RPM for 10 min. Following centrifugation, we removed supernatants in each tube and resuspended the pellet of cells in the remaining media. We added 0.5 mL HBSS to each tube, and then analyzed the experimental samples using a flow cytometer (BD Biosciences).

Flow Cytometry and Statistical Analysis.

We employed flow cytometry to quantify PI positive cells and cell death. Using flow analysis software (BD FACSDiva, Dako Summit) and control data, we determined live and dead cell populations on a forward scatter and side scatter basis. For all experiments, we determined cell death using gating techniques in the flow software. For each HeLa cell sample, we analyzed 10,000 events. We also used control data to determine the PI positive population in the PI fluorescence channel.

We repeated each experimental condition in at least 12–15 independently tested wells to produce a data set. We calculated confidence intervals for each data set (α = 0.05) and included them in bar graphs to visualize variation in statistical analysis. We used Student's t-tests for small sample sizes (<30) to determine statistical significance between population means. When comparing population means, we considered p-values less than 0.05 to show statistically significant difference. We considered population means with p-values greater than 0.05 to be statistically the same.

Nanoinjection Efficiency.

Figure 6 compares averages in PI uptake efficiency for controls, diffusion, 1.5 VDC, and pulsed voltage experiments. T-test results indicate that all differences in PI uptake between population means are statistically significant (p < 0.05), except for the difference between 1.5 VDC and diffusion at 0.04 mg/mL PI (p > 0.05). More exact p-values for differences in population means can be viewed in Table 1. The relatively high efficiency (up to about 60% for injections performed without voltage and 80% for injections performed with voltage) suggests that the majority of cells have been punctured by nanoinjection lances during injections.

Increasing PI concentration resulted in similar trends for all controls, diffusion, 1.5 VDC, and pulsed voltage experiments. Greater concentrations of PI resulted in greater PI uptake into cells. As seen in Fig. 6, when comparing controls to diffusion tests, the nanoinjection method penetrates cell membranes and delivers PI molecules to culture cells. Diffusion of PI molecules into culture cells following the insertion and release of nanoinjection lances results in approximately 4.1 times more cells taking in PI than without nanoinjection. The addition of DC voltage, to accumulate PI molecules and release them into the cellular cytoplasm or nucleus, increases efficiency of the nanoinjection process by an average of 11.4% over diffusion. In the case of the highest PI concentration, statistical evidence shows that the application of voltage is no different than diffusion tests for PI uptake. However, due to the evidence that the other three concentrations are statistically different between diffusion and 1.5 V, this discrepancy may be a result of the inherent variation in biological data [36]. We can confidently state that the addition of 1.5 DC volts increases average PI uptake when compared with diffusion alone in the nanoinjection process.

Nanoinjection Viability.

Results of viability calculations are graphed in Fig. 7 along with confidence intervals (α = 0.05) to visualize scatter in the data. The differences in average viability between diffusion testing and all voltage tests are statistically significant (p < 0.05). There is no statistical difference (p > 0.05) in average viability of the 1.5 VDC tests and pulsed voltage tests at all PI concentrations. Average viability for controls and diffusion tests are statistically different for each PI concentration, except for 0.03 mg/mL. Specific p-values for differences in average viability between tests are listed in Table 2.

The viability results show that diffusion nanoinjections induce greater average cell death than that of controls, but only by 2–4%. Both experiments performed with voltage caused greater average cell death than diffusion nanoinjections, by 5–12%. At all PI concentration levels, average viability was higher for 1.5 VDC tests than pulsed voltage tests. However, statistical analysis reveals that there is no statistical difference in average viability between 1.5 VDC and pulsed voltage tests. Therefore, the addition of a 20 ms repulsion pulse does not decrease cell death when compared to 1.5 VDC repulsion tests.

We also compared viability averages between PI concentrations in each experiment for statistical difference. Table 3 lists the p-values obtained from the Student's t-test. For all average viability comparisons in control data and diffusion data, there is no statistical difference between PI concentrations (p > 0.05). When comparing averages between 0.01 and 0.03 mg/mL PI, as well as 0.01 and 0.04 mg/mL PI for both experiments with voltage, statistical difference is apparent (p < 0.05). For all other average viability comparisons in voltage experiments, there is no statistical difference between PI concentrations.

The statistical difference in viability in the electrical experiments between concentrations may be explained by a change in the electrochemistry at higher concentrations. For control and diffusion data, PI uptake into cells is approximately linearly dependent on PI concentration. However, PI uptake does not increase linearly with increasing PI concentration in voltage experiments (see Fig. 6). Instead, there seems to be a decreasing effect of voltage as concentration increases. This data suggest that the application of voltage becomes less effective at accumulating and releasing PI molecules from lances at higher PI concentrations, perhaps due to differences in the electrochemistry of the solution due to the double layer effect. The decrease in efficiency at higher concentrations may also explain the increase in cell viability as PI concentration increases in voltage experiments. If a smaller effective electric field is applied to the cells at higher concentrations due to saturation of the field (the double layer effect), this would explain both the higher viability and the reduced efficiency effect.

Membrane Puncturing Viability Without Injecting PI.

Since PI fluoresces when bound to nucleic acid chains, cells with positive PI expression may have died due to normal passaging procedures, or they may have been successfully injected. We performed an experiment to quantify the difference in cell death occurring as a result of passaging and moving cells and cell death as a result of puncturing membranes with lances. We determined cell death using gating capabilities of flow cytometry software. Figure 8 indicates an average decrease in cell viability of 4.2% due to lances puncturing cell membranes, which agrees with the diffusion versus controls comparison in Fig. 7. From the results of a t-test comparing population means, the difference in average viability between the controls and punctured cells is statistically significant (p < 0.001). We can confidently state that on average the lances induce approximately 4.2% greater cell death than cells not punctured with lances.

Nanoinjections With Bare Silicon.

We performed an experiment to determine if lances were indeed penetrating cell membranes, or if cell membranes were being strained into opening pores for PI delivery. We used pieces of a bare silicon wafer, having the same dimensions as the lance array chips (2 × 2 cm), in place of the lance arrays. Figure 9 compares experimental data from diffusion lance injections with bare silicon “injections” and control groups with the same PI concentration. Bare silicon delivers more PI than control groups, indicating that there is some cell membrane permeability that occurs, allowing more PI to diffuse into cells. The data indicate statistically significant differences in average PI uptake between bare silicon injections and lance penetration experiments (p < 0.01). There is also statistically significant difference in average PI uptake between bare silicon “injections” and control data (p < 0.001). On average, the silicon lance array delivers PI to twice as many cells as a bare silicon chip in similar experiments.

Cell Membrane Permeability Following Nanoinjection.

PI uptake results from the membrane permeability tests are graphed in Fig. 10. Adding PI immediately after removing lances from cell membranes gives nearly identical PI uptake results as in the 0.04 PI concentration in Fig. 6 (60%). Whether PI is added to the cell culture solution before or immediately after membrane penetration with lances makes no difference in PI uptake. PI uptake decreases in nanoinjected cells as time intervals between injections and addition of PI increases. This indicates that cell membrane pores are formed due to nanoinjection, and the pores stay open for an additional amount of time following lance penetration and removal. However, the difference between uptake averages in the 7-min and 10-min tests are not statistically significant (p > 0.05). The similarity between the 7 and 10 min experiments suggests that the closing of membrane pores becomes more gradual after about 7 min. Control data illustrate no difference in PI uptake at any time interval in the experiment.

Figure 11 shows cell viability results from the membrane permeability timing experiments. For all time intervals, there is no statistical difference (p > 0.05) in cell death over time. As discussed in the membrane puncturing section, control viability is an average of 4.2% greater than injected samples. Adding PI at different time intervals after cell membrane penetration makes no difference in cell viability. These results also show that nanoinjection causes the increase in cell death, and not PI exposure.

Particle delivery methods for mammalian cells are numerous, with each one having a unique set of advantages and disadvantages. The nanoinjection process promises high delivery efficiencies while maintaining very low cell death. This paper presents the results of experimental testing of the nanoinjection lance array system on HeLa 229 culture cells. We used PI, a fluorescent dye, to tag nanoinjected cells and quantify nanoinjection efficiency and cell death. We used different concentrations of PI to determine the effect of PI concentration on uptake into cells. We used DC voltage in some experiments to assist in PI delivery, while in others we added a short square wave pulse to enhance delivery further. Delivery efficiencies for these experiments ranged from 25–78%, depending upon PI concentration and application of voltage. Diffusion alone has previously produced efficiencies as high as 98% at much higher PI concentrations [22]. PI delivery efficiency increased with increasing PI concentration. The addition of an electric field also increased PI delivery into cells, as did adding a square wave repulsion pulse. By straining the cell membranes with a silicon chip without lances, we delivered PI to half as many cells as diffusion tests with a lance array.

We also quantified cell death for the lance array nanoinjection process. Depending upon PI concentration and application of voltage, cell viability ranged from 78 to 91%. Nanoinjections without voltage induced 2–4% cell death, while injections with a voltage applied induced 5–15% cell death. Experimental data suggest that higher PI concentrations reduce the effectiveness of voltage in delivery efficiency, which may cause higher viability at higher PI concentrations. Further testing of the lance array without the addition of PI presented an average decrease in cell viability of 4.2%. HeLa cell membranes stay porous for several minutes following penetration and removal of lances. Most membrane pores close quickly within 7 min of nanoinjection, after which the pores close at a slower rate. Future work includes using DNA plasmids and linear gene segments as injection particles. Testing will also be expanded to nanoinject a primary cell line and determine efficiencies in other cell types.

In conclusion, the nanoinjection process using a silicon lance array is a promising physical method for foreign particle delivery into cell cultures. The high delivery efficiencies obtained with PI as well as high viabilities maintained in these experiments make this process a favorable choice for research and clinical applications.

This research was supported by the National Science Foundation under Grant No. ECCS-1055916.

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Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., and Barreda, D. R., 2010, “Conventional Apoptosis Assays Using Propidium Iodide Generate a Significant Number of False Positives That Prevent Accurate Assessment of Cell Death,” Journal of Immunol. Meth., 358(1–2), pp. 81–92. [CrossRef]
Deitch, A. D., Law, H., and deVere White, R., 1982, “A Stable Propidium Iodide Staining Procedure for Flow Cytometry,” J. Histochem. Cytochem., 30(9), pp. 967–972. [CrossRef] [PubMed]
Krishan, A., 1975, “Rapid Flow Cytofluorometric Analysis of Mammalian Cell Cycle by Propidium Iodide Staining,” J. Cell Biol., 66(1), pp. 188–193. [CrossRef] [PubMed]
Darzynkiewicz, Z., Bruno, S., Bino, G. D., Gorczyca, W., Hotz, M., Lassota, P., and Traganos, F., 1992, “Features of Apoptotic Cells Measured by Flow Cytometry,” Cytometry, 13(8), pp. 795–808. [CrossRef] [PubMed]
Vermes, I., Haanen, C., and Reutelingsperger, C., 2000, “Flow Cytometry of Apoptotic Cell Death,” J. Immunol. Meth., 243(1–2), pp. 167–190. [CrossRef]
Crissman, H. A., and Hirons, G. T., 1994, “Flow Cytometry Second Edition, Part A,” Methods in Cell Biology, Vol. 41, Academic Press, Waltham, MA, pp. 195–209. [CrossRef]
Bank, H. L., 1987, “Assessment of Islet Cell Viability Using Fluorescent Dyes,” Diabetologia, 30(10), pp. 812–816. [CrossRef] [PubMed]
Meda, P., 2001, “Connexin Methods and Protocols,” Methods in Cell Biology, Vol. 154, Springer, Berlin, Germany, pp. 201–224.
Tsong, T. Y., 1991, “Electroporation of Cell Membranes,” Biophys. J., 60(2), pp. 297–306. [CrossRef] [PubMed]
Pelkmans, L., 2012, “Using Cell-to-Cell Variability - A New Era in Molecular Biology,” Science, 336(6080), pp. 425–426. [CrossRef] [PubMed]
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References

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Iversen, N., Birkenes, B., Torsdalen, K., and Djurovic, S., 2005, “Electroporation by Nucleofector is the Best Nonviral Transfection Technique in Human Endothelial and Smooth Muscle Cells,” Genet. Vaccines Ther., 3(2), pp. 1–13. [CrossRef] [PubMed]
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Arndt-Jovin, D. J., and Jovin, T. M., 1989, “Fluorescence Microscopy of Living Cells in Culture Part B. Quantitative Fluorescence Microscopy—Imaging and Spectroscopy,” Methods in Cell Biology, Vol. 30, Academic Press, Waltham, MA, pp. 417–448.
Neumeyer, A., Bukowski, M., Veith, M., Lehr, C.-M., and Daum, N., 2011, “Propidium Iodide Labeling of Nanoparticles as a Novel Tool for the Quantification of Cellular Binding and Uptake,” Nanomedicine, 7(4), pp. 410–419. [CrossRef] [PubMed]
Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., and Barreda, D. R., 2010, “Conventional Apoptosis Assays Using Propidium Iodide Generate a Significant Number of False Positives That Prevent Accurate Assessment of Cell Death,” Journal of Immunol. Meth., 358(1–2), pp. 81–92. [CrossRef]
Deitch, A. D., Law, H., and deVere White, R., 1982, “A Stable Propidium Iodide Staining Procedure for Flow Cytometry,” J. Histochem. Cytochem., 30(9), pp. 967–972. [CrossRef] [PubMed]
Krishan, A., 1975, “Rapid Flow Cytofluorometric Analysis of Mammalian Cell Cycle by Propidium Iodide Staining,” J. Cell Biol., 66(1), pp. 188–193. [CrossRef] [PubMed]
Darzynkiewicz, Z., Bruno, S., Bino, G. D., Gorczyca, W., Hotz, M., Lassota, P., and Traganos, F., 1992, “Features of Apoptotic Cells Measured by Flow Cytometry,” Cytometry, 13(8), pp. 795–808. [CrossRef] [PubMed]
Vermes, I., Haanen, C., and Reutelingsperger, C., 2000, “Flow Cytometry of Apoptotic Cell Death,” J. Immunol. Meth., 243(1–2), pp. 167–190. [CrossRef]
Crissman, H. A., and Hirons, G. T., 1994, “Flow Cytometry Second Edition, Part A,” Methods in Cell Biology, Vol. 41, Academic Press, Waltham, MA, pp. 195–209. [CrossRef]
Bank, H. L., 1987, “Assessment of Islet Cell Viability Using Fluorescent Dyes,” Diabetologia, 30(10), pp. 812–816. [CrossRef] [PubMed]
Meda, P., 2001, “Connexin Methods and Protocols,” Methods in Cell Biology, Vol. 154, Springer, Berlin, Germany, pp. 201–224.
Tsong, T. Y., 1991, “Electroporation of Cell Membranes,” Biophys. J., 60(2), pp. 297–306. [CrossRef] [PubMed]
Pelkmans, L., 2012, “Using Cell-to-Cell Variability - A New Era in Molecular Biology,” Science, 336(6080), pp. 425–426. [CrossRef] [PubMed]

Figures

Grahic Jump Location
Fig. 1

Scanning electron micrograph showing the edge of a lance array chip

Grahic Jump Location
Fig. 2

Images of the injection device from (a) above and (b) below

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Fig. 3

Optical micrograph of a HeLa 229 cell culture. The cells are approximately 50% confluent.

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Fig. 4

Schematic of the setup for the nanoinjection process with a lance array, using the application of an electric field (not to scale)

Grahic Jump Location
Fig. 5

Process diagram for the nanoinjection technique using a lance array and voltage (not to scale). The process begins with (a) cultured cells in solution with PI and the lance array, with a negative voltage applied to the lances to accumulate PI molecules. The lances penetrate cell membranes (b), and the voltage is reversed (c), releasing the PI molecules inside the cytoplasm and/or nucleus of cells. The lances are removed from the cells (d), leaving PI molecules inside.

Grahic Jump Location
Fig. 6

Bar graph comparing average PI uptake data for controls, diffusion injections, 1.5 VDC injections, and pulsed voltage injections. The error bars indicate a 95% confidence interval (α = 0.05). *Statistically significant difference in population averages (p < 0.05).

Grahic Jump Location
Fig. 7

Bar graph comparing average viability data for controls, diffusion injections, 1.5 VDC injections, and pulsed voltage injections. The error bars indicate a 95% confidence interval (α = 0.05). *Statistically significant difference in population averages (p < 0.05).

Grahic Jump Location
Fig. 8

Bar graph comparing average viability data for controls and cells punctured with the lance array. The error bars indicate a 95% confidence interval (α = 0.05). *Statistically significant difference in population averages (p < 0.05).

Grahic Jump Location
Fig. 9

Bar graph comparing PI uptake between cells strained with a bare silicon chip, cells punctured with lances, and unpunctured controls. Error bars indicate a 95% confidence interval (α = 0.05). **Statistically significant difference in population averages (p < 0.01).

Grahic Jump Location
Fig. 10

Bar graph comparing PI uptake between controls and cells nanoinjected with PI added at several time intervals. Error bars indicate a 95% confidence interval (α = 0.05). *Statistically significant difference in population averages (p < 0.05).

Grahic Jump Location
Fig. 11

Bar graph comparing viability between controls and cells nanoinjected with PI added at several time intervals. Error bars indicate a 95% confidence interval (α = 0.05).

Tables

Table Grahic Jump Location
Table 1 Table of p-values calculated when comparing the difference in population means for PI uptake
Table Grahic Jump Location
Table 2 Table of p-values calculated when comparing the difference in population means for cell viability
Table Grahic Jump Location
Table 3 Table of p-values calculated when comparing the difference in population means for cell viability, between PI concentrations

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