Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(12):e52356.
doi: 10.1371/journal.pone.0052356. Epub 2012 Dec 20.

Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells

Clément Achille  1 , Sowmya Sundaresh, Benjamin Chu, Michael Hadjiargyrou
Affiliations

Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells

Clément Achille et al. PLoS One. 2012.

Abstract

RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL) alone (Control) and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i) and EGFP (EGFPi, also served as a control) shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing) and 0.2-3 µm (Control). While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release) of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Morphology and fiber diameter of electrospun scaffolds.
SEM images of Control scaffolds without DNA (A, B) and those containing plasmid DNA encoding for Cdk2 (Cdk2i) or EGFP (EGFPi) shRNA (D, E and G, H, respectively). C, F, and I indicate the fiber diameter distribution between the three types of scaffolds (using corresponding images in A, C and E). Scale bar in A = 10 µm, B = 2 µm and in D and G = 35 µm, E and H = 5 µm.
Figure 2
Figure 2. Plasmid DNA release from electrospun scaffolds.
Graphs indicates the amount of DNA release per time point (A) and cumulative release (B) over the 21 day study from the two plasmid DNA containing scaffolds, Cdk2i and EGFPi. (C) Agarose gel electrophoresis showing the integrity of the released DNA from both scaffolds. Lane 1, 100 bp MW marker; Lane 2 and 4, EGFP (pKD-EGFP-v1) and Cdk2 (pKD-Cdk2-v5) control (unincorporated) plasmid DNA; Lane 3 and 5, 21 day released EGPF and Cdk2 plasmid DNA, respectively.
Figure 3
Figure 3. Electrospun scaffold degradation.
SEM images of Control scaffolds without DNA (A,C,E,G) and with scaffold containing plasmid DNA encoding for Cdk2 shRNA (Cdk2i, B, D, F, H) at the specified days (0, 8, 14 and 21). Scale bar in A, C, E, G = 2 µm, and in B, D, F, H = 5 µm.
Figure 4
Figure 4. Silencing of Cdk2 and suppression of MCF-7 cell proliferation.
The effectiveness of the plasmid DNA encoding Cdk2 shRNA was tested on MCF-7 cells growing on tissue culture plates, both in terms of suppressing Cdk2 expression, as measured by Q-PCR on culture day 4 (A) and cellular proliferation, as measured by the MTS assay on culture day 1 (B). The effect on proliferation and cell death was also examined via microscopy in the three conditions (C). The plasmid DNA encoding for EGFP shRNA was used as a control in both experiments. Control indicates results from untransfected cells. (A) *p<.03, **p<.05. (B) *p<.001, **p<.001.
Figure 5
Figure 5. Silencing of Cdk2 and suppression of cell proliferation by the Cdk2i scaffold.
The effectiveness of the plasmid DNA containing scaffolds was tested on MCF-7 cells growing directly on them. Control scaffold and those containing plasmid DNA encoding for Cdk2 (Cdk2i) or EGFP (EGFPi) shRNA, both in terms of suppressing Cdk2 mRNA, as measured by Q-PCR on culture day 4 (A) and cellular proliferation, as measured by the MTS assay on culture day 1 (B). The Control and EGFPi scaffolds served as controls in both experiments. (A) *p<.01, **p<.04. (B) *p<.02, **p<.001.
Figure 6
Figure 6. Silencing of Cdk2 and cell death by the Cdk2i scaffold.
LIVE/DEAD assay of MCF-7 cells grown on each of the three scaffolds, Control (A, B, C), and those containing plasmid DNA encoding for Cdk2 (Cdk2i, D, E, F) or EGFP (EGFPi, G, H, I) shRNA. A, D, H show living cells (green); B, E, H show dead cells (red); C, F, I represent the overlay of the two corresponding red/green images. Scale bar = 50 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pecot CV, Calin GA, Coleman RL, Lopez-Berestein G, Sood AK (2011) RNA interference in the clinic: challenges and future directions. Nat Rev Cancer 11: 59–67. - PMC - PubMed
    1. Wu SY, McMillan NA (2009) Lipidic systems for in vivo siRNA delivery. AAPS J 11: 639–652. - PMC - PubMed
    1. Li J, Huang L (2010) Targeted delivery of RNAi therapeutics for cancer therapy. Nanomedicine 5: 1483–1486. - PubMed
    1. Smith MH, Lyon LA (2012) Multifunctional Nanogels for siRNA Delivery. Acc Chem Res 45(7): 985–993. - PubMed
    1. Monaghan M, Pandit A (2011) RNA interference therapy via functionalized scaffolds. Adv Drug Deliv Rev 63: 197–208. - PubMed

Grants and funding

These authors have no support or funding to report.