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.