Determining the Effects of Pegylated Epidermal Growth Factor on RPE Cells
Rebecca
1Department
1,2
Drake ,
Corina
2
White ,
Dr. Ronke
2
Olabisi
of Bioengineering, California Lutheran University, Thousand Oaks, CA
2Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ
EGF-Scaffold Conjugation Efficiency
0.3
0.2
Sample Mass Actual Mass Percent
(μg)
(μg)
Efficiency
4.51
18.0
82.0
y = 0.0079x - 0.003
R² = 0.9981
0.1
0
-0.1
0
10
20
30
Mass (μg)
40
50
Sample
Mass (μg)
Figure 2: Standard curve of EGF with varying
dilutions of free EGF in solution.
Actual
Mass (μg)
4.51
2.24
1.74
0.371
200
150
100
y = 866x + 2.55
R² = 0.9966
50
0
Percent
Efficiency
18.0
18.0
27.9
11.9
0
82.0
82.0
72.1
88.1
0.05
0.1
0.15
0.2
Concentration (μg /mL)
A 80
70
60
50
40
30
20
10
0
B
No EGF
Thousands of Cells
Thousands of Cells
B
200
Day 3 PrestoBlue
C
150
100
50
PEG-EGF Free EGF
0
C
C
Day 14 PrestoBlue
Thousands of Cells
B
Day 1 PrestoBlue
500
400
300
200
100
0
No EGF PEG-EGF Free EGF
No EGF PEG-EGF Free EGF
Figure 3: Results of a PrestoBlue assay, used to determine the number of cells grown over a
period of 1 day (A), 3 days (B), or 14 days (C) under treatment with PEG-EGF, Free EGF, or No
EGF.
Viability Staining
No EGF
PEG-EGF
Free EGF
B
C
D
E
F
A
Day 1
No EGF
A
RPE Cell
Morphology
G
H
0.008
0.006
0.004
0.002
0
-0.002
3.4 kDa
20 kDa
MW of PEG in Hydrogel
Figure 7: Amount of PEG-EGF lost
from 3.4 kDa and 20 kDa PEG
hydrogel scaffolds after one day.
Discussion/Conclusion
AMD is a disease caused by a dysfunction that develops in the Bruch’s
membrane and RPE, which leads to photoreceptor death. To reestablish
the failing blood retinal barrier of the Bruch’s membrane and RPE, our
research aimed to develop a scaffold for RPE implantation with an
introduced growth factor, EGF, to promote growth and viability of cells. It
was found that when treated with EGF in both the bound and unbound
forms, the RPE cells experienced a relative increase in growth after three
days as compared to cells untreated with EGF. Similarly, RPE cells treated
with EGF were also found to have morphology different from that of the
untreated cells, signifying differences in cell development. These results
indicate that polymer conjugation of EGF does not result in cytotoxicity.
Conjugation also does not inhibit EGF activity or interaction with RPE
cells. Further experiments demonstrated that when the EGF was bulk
conjugated to a scaffold via UV photopolymerization, it was found that
less than 0.01 μg of EGF was lost after 1 day, which demonstrates a high
efficiency for the conjugation. In the future, further studies can be used to
examine the effect of EGF on RPE cell expression and function in vitro
when cultured on scaffolds, as well as in vivo in animal models.
B
Acknowledgements
REU in Cellular Bioengineering: From Biomaterials to Stem Cells - NSF EEC
1262924
RiSE at Rutgers
C
I
Figure 4: Fluorescent images of cells grown over a
period of 1 day (A, B, C), 3 days (D, E, F), or 14 days (G,
H, I) under treatment with PEG-EGF, Free EGF, or No
EGF. Cells were stained with ethidium and calcein dyes
for live/dead differentiation.
0.25
Figure 6: Standard curve of EGF with varying
dilutions of free EGF in solution.
Quantifying RPE Cells
A
EGF Lost in Scaffold
Concentration (μg)
Absorbance
0.4
EGF Fluorescent Standard
Fluorescence
EGF Ninhydrin Standard
Table 1: Conjugation efficiency of a
100 μg conjugation of EGF to PEG
as measured by a Ninhydrin Assay.
PEG-EGF (100 μg)
PEG-EGF
The overall goal is to develop a scaffold that promotes long-term
viability and functionality in vivo. Based on previous studies4, we will
functionalize a scaffold with EGF to promote these two things. To
complete this goal, the following steps will be taken:
1. Functionalize ACRL-PEG-SVA with EGF.
Covalently link the protein, EGF, to the polymer, ACRL-PEG-SVA.
2. Demonstrate the efficiency of the PEG-EGF conjugation.
Use a Ninhydrin assay to quantify the amount of free EGF in the PEG-EGF
reaction solution.
3. Evaluate the bioactivity of EGF-functionalized PEG.
Determine the effect of PEG bound EGF and unbound EGF on the
proliferation and viability of RPE cells.
4. Incorporate pegylated EGF into scaffolds.
Use in future cell studies to develop an RPE monolayer.
PEG-EGF Conjugation Efficiency
Free EGF
Our Approach
Results
Day 3
Age-related macular degeneration (AMD) is currently the leading
cause of blindness in developed nations1. The disease occurs in two
forms, a wet form and a dry one, where both forms of AMD are
marked by the death of photoreceptors densely populated in the
macula. The Bruch’s membrane, a semipermeable exchange barrier,
separates the retinal pigment epithelium (RPE), from the choroid or
capillary bed2 (Figure 1). These two structures, the Bruch’s membrane
and the RPE, form a blood retinal barrier. The photoreceptor death,
specific to the dry form of AMD, is due to the altered nutrient transport
through the Bruch’s membrane and dysfunction in the RPE, disrupting
the barrier. In previous studies, RPE was derived from stem cells and
was injected into patients in a bolus form3 (Figure 2). The bolus
injection did not address dysfunction in the Bruch’s membrane and
RPE because it does not recreate the failing blood retinal barrier.
Because of this, the bolus injection has been previously shown to only
rescue photoreceptors for a short period in animal models, due to this
lack of repair mechanism since no monolayer was formed. To
overcome this hurdle, several groups have fabricated scaffolds for cell
implantation. However, these scaffolds have also seen challenges such
as
low
viability
and
dklfahsdglaj
Retinal Pigment Epithelium (RPE)
functionality in vivo. The
Photoreceptors Physically supports photoreceptors.
Convert light into
presented work seeks to
Bruch’s Membrane
neural signals.
A semipermeable exchange
address
these
issues
barrier, separates RPE from
through
scaffold
choroid.
functionalization.
Using
Choroid
epidermal growth factor
Blood vessels that
provide nourishment
(EGF),
a
molecule
to eye.
previously
shown
to
stimulate RPE proliferation,
we seek to characterize EGF
conjugation efficiency to
poly(ethylene glycol) and to
understand how polymer Figure 1: Anatomy of the human eye,
conjugation affects EGF including the photoreceptor, RPE, Bruch’s
membrane, and choroid structures.
activity with RPE cells.
Results
Day 14
Introduction
Figure 5: Morphology of
cells differed between those
untreated (A) and those
treated with EGF (B, C).
References
[1] Lim, L. S., Mitchell, P., Seddon, J. M., Wong, T. Y. (2012). Age-related macular degeneration. Lancet,
379: 1728-1738.
[2] Jager, R. D., Mieler, W. F., Miller J. W. (2008). Age-related macular degeneration. N Engl J. Med, 358:
2606-2617.
[3] Treharne, A. J., Thomson, H. A. J., Grossel, M. C., Lotery, A. J. (2011). Developing
methacrylate-based copolymers as an artificial Bruch’s membrane substitute. J. Biomed Mater Res,
100A: 2358-2364.
[4] Steindl-Kuscher, K., Boulton, M. E., Haas, P., Dossenbach-Glaninger, A., Feichtinger, H., Binder, S.
(2011). Epidermal Growth factor: the driving force in initiation of RPE cell proliferation. Graefes Arch
Clin Exp Ophthalmol, 249(8): 1195-1200.
[5] Sonnet, C., Simpson, C. L., Olabisi, R. M., Sullivan, K., Lazard, Z., Gugala, Z., Peroni, J. F., Weh, J.
M., Davis, A. R., West, J. L. (2013). Rapid healing of femoral defects in rats with low dose sustained