Fluorescent image of cells after BSA delivery with a nanoneedle array
A Highly Dense Nanoneedle Array for Intracellular Gene Delivery
Investigation into cellular functions requires the ability to
apply specific and controlled treatment to cells. Such treatment
includes the delivery of biological effectors across cell membranes
using various approaches such as chemical, mechanical or electrical
perturbations to cells. In addition to investigation, gene
correction represents an active treatment in which genetic diseases
and cancer may be treated by correcting the causative mutations in
the genetic code. Sickle cell disease is the most common inherited
blood disorder caused by a mutation of a single nucleotide of a gene.
The disease causes hemoglobin-containing red blood cells to tend to
deform, clump and break apart, resulting in clogged blood vessels
and causing severe pain, serious infection and organ damage. Bone
marrow transplantation, the only permanent cure for the disease, has
the undesirable limitation of donor compatibility. Research has
shown that transforming defective blood-forming cells into normal
ones by inserting corrective genes can alleviate the disease.
Several techniques currently exist for gene correction,
including biological methods or physical injection of therapeutic
agents into cells. Physical methods such as conventional
microinjection typically use glass microcapillaries; however, thesetechniques have relatively low throughput due to the
time-consuming operation of targeting individual cell nuclei. A
micron-scale microcapillary array, made of SiO2, was previously
demonstrated for fluorescent dye injection and DNA injection
to plant cells. In this paper we propose a silicon nanoneedle
array with needle tip diameters in tens of nanometers, enabling
pinpoint injection of individual cells in a high throughput manner
without causing cell death by puncture.
By exploiting conventional isotropic etching and a thermal-oxidation-based sharpening process, high-density nanoneedle arrays were produced. The nanoneedles possessed sharp tips as small as 16nm in radius and a high density as large as 1000000 per square centimeters regardless of shapes of mask patterns as long as opening gaps between array elements were consistent. The silicon-micromachined nanoneedles were able to deliver small molecules as well as macromolecules into cells by mechanically penetrating cell membranes. The punctured cells showed uptake of calcein and BSA, respectively, with no significant cell death caused by membrane penetration. The demonstration of intracellular delivery with nanoneedles shows promise for high throughput gene transfer. Additional study on correlation of intracellular uptake of macromolecules with mechanical parameters of nanoneedle treatment for cell membrane penetration, and subsequent demonstration of gene incorporation and expression, is required to further validate this technique.