Executive Summary
In the past few years, gene therapy as a treatment for diseases and disorders has been a topic of
great interest and research. Research into gene therapy utilizing non-viral vectors has greatly increased
due to the lack of immunogenic effects. However, non-viral vectors tend to have high cytotoxicity and
low transfection efficiency. Therefore, new peptides are being researched in order to rectify the obstacles
of non-viral vectors present. This study analyzed the effectiveness of newly designed, non-viral peptides
(Arginine)10(Serine)10 (R10S10), (Arginine)10(Lysine-Glutamate)10 (R-KE), and
(Arginine)10(Lysine-
Glutamate)10 –RGD (R-KE-RGD). The newly designed peptides were compared to currently available
standards Poly(L-lysine) (PLL) and Polyethylenimine (PEI). PEI was primarily analyzed to try and
reproduce previously reported results in order to validate the experimental procedure. The results of PEI
testing confirmed that the experimental procedure was adequate.
The effectiveness of a peptide is based on its transfection efficiency and toxicity. For a particle to
successfully transfect a cell, it must have a particle size that will allow it to be transfected into the cell,
and a zeta potential that will promote the neutral or positively charged peptide/DNA complexes to enter
negatively charged cells. In this work, particle sizes and zeta potentials for each peptide were measured
with a Malvern Zetasizer. Size and zeta potential measurements for R 10S10 were determined to be less
accurate since it was out of the measurement range of the Zetasizer. The “Expert Advice” feature
indicated there was sediment/large particles/or dust present in the sample that would have affected size
and zeta potential and most likely skewed the results. The sizes and zeta potentials for R10S10 in different
buffers ranged from 150-2000 nm and -43-25 respectively. For R-KE sizes ranged from 120-5500 nm for
the sodium acetate (NaAc) buffer, and 150-1250 nm for tris. Zeta potentials were mostly negative for
both NaAc and tris. The sizes at lower N/P ratios were around 170 nm, an adequate size. The zeta
potentials were all negative.
Different buffers were tested to determine which buffer/complex
combinations resulted in the desired size and zeta potential. Tested buffers included tris, sodium acetate,
and nano-water. Tris and NaAc were determined to be the best buffers for R 10S10 and R-KE/R-KE-RGD
respectively.
Prior to transfection testing, the relative light units (RLU) were normalized to the protein content
measured using the Pierce® BCA Protein Assay Kit, and transfection was measured via the Bright-GloTM
Luciferase Assay System. A certain kind of DNA, pCMV-Luc DNA, that carries the luciferase gene was
used in luciferase testing. Transfection was tested for both R-KE, and R-KE-RGD because of reasonable
size and zeta measurements. For comparison, PEI was also tested and resulted in relative light units per
well (RLU/well) of just under 1.0E+7. The highest transfection reported for the R-KE/DNA complex was
just under 6.0E+5 RLU/well which occurred at an N/P ratio of 3. The highest transfection reported for
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the R-KE-RGD/DNA complex was just under 1.6E+6 RLU/well which occurred at an N/P ratio of 10.
The transfection of R-KE-RGD was closest to that of PEI but was still significantly lower. Toxicity was
measured for R-KE-RGD via the Vybrant® MTT Cell Proliferation Assay Kit. The toxicity was found to
be very low, with the lowest percentage of live cells being only 94%.
This study determined that R-KE-RGD is non-toxic and has the best transfection efficiency out of
the newly designed peptides. The results from this study have already been put to use in other research.
In the current research, an N/P value of 10 is being used based on the R-KE-RGD transfection results.
Also, the R-KE-RGD peptide design has been modified to include the amino acids histidine and valine to
improve transfection.
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Introduction
In the past few years, gene therapy has been a topic of great interest and research. “Gene therapy
is defined as the transfer of genetic information to specific cells to direct the synthesis of a specific
protein.” [1] People in the fields of medicine, pharmaceuticals, and biotechnology have been looking to
gene therapy as a treatment for chronic diseases and genetic disorders such as cancers and cystic fibrosis.
Non-viral DNA vectors, such as plasmids, have many advantages over viral vectors, such as low
immunogenicity and low production cost that make them more desirable than viral vectors. Because of
the lack of possibly dangerous side effects of a viral vector, research on non-viral vectors has increased
[2].
However, non-viral vectors also have their drawbacks.
Non-viral vectors tend to have high
cytotoxicity and poor delivery efficiency. Therefore, research has been put into developing non-viral
vectors with increased delivery efficiency [1].
Despite the significant research put into gene therapy and the progress that has been made, gene
therapy is still a long way off from being an acceptable cure for diseases and disorders. Therefore,
methods and carriers are still being researched to find a delivery system that is non-viral, non-toxic, and
has high delivery efficiency, or transfection. One category of non-viral gene delivery systems is polymerbased. Disadvantages and advantages of polymers are that they “generally do not have the capacity for
cell-specific targeting, but provide flexible chemistry for the attachment of targeting moieties that allow
both increased cell uptake and often, cell specificity,” [3]. To overcome the main barriers in the field of
gene delivery, it is desired to design integrated peptide materials that carry all of required functions
(efficient drug encapsulation, long blood circulation, and effective targeting) in one material for efficient
targeted gene delivery.
Background
Important Properties of Gene Delivery Materials
For a material to efficiently deliver a gene for mammalian cells, certain physical criteria must be
met. The DNA used in the project was a plasmid that encodes the luciferase enzyme. The size of
material after it has been allowed to complex with DNA (DNA condenses in the presence of polymerbased vectors) must be within a certain range if the drug carrier is to be transfected into the desired cells
because of the size required to enter through the endosomal pathway. The ideal size is less than 300 nm.
Secondly, the carrier needs to have a certain surface change if it is to be attracted to the surface of the
target cells. The surface charge can be determined by measuring zeta potential. The surfaces of the cells
are slightly negative, so it is ideal for the carrier to have a neutral or slightly positive charge. The size and
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zeta potential can be measured with a Malvern Zetasizer. For the carrier to be successful, it must not be
toxic to the cells it is trying to transfect, known as cytotoxicity. It must also have a high transfection
efficiency. The luciferase enzyme encoded by the DNA is desired because it catalyzes a reaction used to
test for transfection. Transfection is measured by relative light units (RLU) which is a measure of the
number of cells that emit light when using luciferase testing. Transfection has occurred in cells that emit
light.
Current Non-Viral Vectors
So far several polymer-based delivery systems have been extensively studied. Two such systems
that were tested in this study are Poly(L-lysine) (PLL) and Polyethylenimine (PEI). PLLs with high
molecular weight have been found to possess properties suitable for a gene carrier. However, with
condensed DNA/PLL, complexes have high cytotoxicity [2]. A PLL with a molecular weight of 10,000
was used in this study. In order to rectify some of the issues with cytotoxicity, a PLL-Poly(ethylene
glycol) (PEG) block copolymer can be synthesized [2]. This form of PLL reduces zeta-potential and cell
cytotoxicity [2]. PLL-PEG was not analyzed in this study. The chemical structures of PLL and PLLPEG can be found in Figures 1 and 2 respectively.
Figure 1: Poly(L-lysine) (PLL) chemical structure
Figure 2: PLL-PEG chemical structure.
PEI can be in one of two forms: linear or branched. The respective chemical structures are shown
in Figure 3 and 4. The branched form of PEI is the standard form used for gene delivery because that
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form has yielded significantly greater success in terms of cell transfection [4]. Godbey et al. measured
both the size and zeta potential of branched PEI. When using an analysis method similar to the Zetasizer,
sizes of PEI/DNA complexes have been found to be approximately 150 nm. The zeta potentials of PEI by
itself, when allowed to complex with DNA, and the complex after centrifugation were found to be 37 mV,
31.5 mV, and 29.2 mV respectively [4]. Godbey et al. also measured transfection efficiencies of PEI with
different molecular weights.
It must be noted that the transfection efficiency and cell toxicity are
dependent on the ratio of nitrogens in PEI to phosphates in DNA (N/P). Godbey et al found the optimum
ratio to be between 9 and 13.5 N/P. The highest transfection efficiency reported by Godbey et al was
25.5% ± 9.7 [4]. The results of Godbey et al.’s research was used to validate the procedure used for this
project and was also used as a comparison for the newly designed carriers.
Figure 3: Linear PEI chemical structure.
Figure 4: Branch PEI chemical structure.
Designed Vectors
The focus of this project was to analyze newly designed vectors based on their size, zeta
potential, toxicity, and transfection. Vectors tested in this project include R10S10, R-KE, and R-KE-RGD.
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R10S10 has 10 arginines (R) followed by 10 serines (S). R-KE has 10 arginines followed by 10 each of
alternating lysines and glutamates (K and E). R-KE-RGD has the same number of arginines, lysines, and
glutamates as R-KE, but has additional amino acids including alanine (A), tyrosine (Y), cysteine (C),
glycine (G), asparagine (D), and phenylalanine (F). The sequence of the additional amino acids after the
arginines, lysines, and glutamic acids is ARYCRGDCFDG.
The most important component of R-KE-RGD peptide is the RGD amino acid sequence. The
cysteines on both sides of RGD form a disulfide bond making it circular and giving it a higher affinity
towards ligands. The purpose of the RGD is to target specific proteins, α vβ3 and αvβ5, in cancer cells.
Those specific proteins, αvβ3 and αvβ 5, are common in tumor vasculature and are also found in metastic
melanoma [5].
Experimental Procedure
Size and Zeta Potential Measurement
A Malvern Zetasizer was used to determine size and zeta potential of peptide/DNA complexes.
To prepare samples for the Zetasizer, the volume of peptide to be added to each sample was based on the
ratio of nitrogens in the peptide to the phosphates in DNA (N/P). N is equal to the moles of peptide
multiplied by the number of nitrogens in the peptide. The moles of peptide can be found using the ratio
of the mass of peptide to the molecular weight of the mer of the peptide. P is equal to the moles of DNA
multiplied by the number of phosphates in the DNA. The moles of DNA can be found using the ratio of
the mass of DNA to the molecular weight of DNA. The molecular weight of DNA was taken to be 660
and the DNA contains 2 phospates. A mass of 1 µg for the DNA and a concentration of 1 µg/ 1µL for the
peptide were chosen in order to make the calculation of the required volume of peptide simple. The
following is an example of calculating the volume of PLL for N/P = 1.
MWpep = 208.08
MWDNA = 660
#Nitrogens = 1
massDNA = 1 µg
concentrationpep = 1 µg/1 µL
#Phosphates = 2
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After the volume of peptide was calculated for N/P = 1, the volume for other N/P values were
calculated by multiplying the N/P = 1 volume by the N/P value itself. For example, for N/P = 2, the
volume of the peptide for N/P = 1 was multiplied by 2. In this project, N/P values ranging from 0 to 20
were used. Volumes for different peptides were calculated in the same manner.
Once the volume of peptide was calculated for each N/P value, a specified volume of a buffer
solution was added to the desired volume of peptide and mixed well with a pipette. The same volume of
buffer was then added to the 6 µL of DNA in a separate vial and mixed well with a pipette. The peptide
solutions were then slowly added dropwise to the DNA solutions with adequate mixing and some heat.
The complexes were allowed to incubate for 20 minutes to allow the DNA to fully condense. Adequate
mixing of the DNA and peptide solutions was achieved with the use of a stir bar and a hot plate was used
to warm the solutions. The volumes of buffer added to both the peptide and DNA ranged from 50 µL to
500 µL. Depending on the volume of the complexes after the incubation time, more buffer solution was
added to give each solution a total volume of 900 µL or 1000 µL. Different buffers used include nanowater, sodium acetate, and tris (tris(hydroxymethyl)aminomethane). The size and zeta potential for each
N/P solution was then analyzed using the Zetasizer and the results were recorded.
Cell Culture
To test both toxicity and transfection efficiency of complexes, fetal bovine cells first had to be
grown and cultured. To grow cells, fetal bovine serum (FBS) was used as the serum-supplement for the
cell culture. The culture medium used was a mixture of DMEM (Dulbecco's Modified Eagle Medium),
serum, penicillin streptomycin, non-essential amino acids solution, and sodium pyruvater solution. The
fetal bovine serum was incubated with the culture medium for 2 days before the original culture medium
was replaced. After another 3-4 days, the culture medium was removed and the cells were rinsed with
PBS (phosphate buffered saline). Trypsin-EDTA was mixed with the culture medium and was added to
the cells. Once the cells became round, the trypsin-EDTA was removed and additional culture medium
was added. The cells were allowed to incubate again for another week. After the second week of
incubation, the steps of adding and removing the trypsin-EDTA were repeated once more [6].
To trypsinize the cells, the culture medium was removed and the cells were rinsed with a PBS
buffer. Trypsin-EDTA was added and then removed once the cells rounded up. The flask was patted to
detach the cells and buffer was used to titrate. The suspension was then transported to a tube. The
suspension was centrifuged. After centrifugation, the solution was removed, leaving the cells, and
DMEM with no phenol red was added to titrate. To determine the cell density, the cell solution was
added into trypan blue and mixed well. The cell density was then measured using a hemocytometer.
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After the density has been counted, the cell solution was diluted with DMEM. The buffer was removed
from the well-plate and the diluted cell solution was added to each well. The cells were then allowed to
incubate at 37°C for 2 hours [6].
In vitro Transfection
For transfection experiments, COS-7 cells were seeded into each well of a 96-well plate. The
cells were allowed to attach overnight in a growth medium of 90% phenol red-free DMEM and 10% fetal
bovine serum. A stock solution of the peptide was prepared in DMSO solvent. Working dilutions of
each peptide were prepared in sodium acetate buffer. The diluted peptide solution was added to pCMVLuc DNA in each well of a 96 well-plate. The mixtures were incubated for to allow the DNA to
condense. Then the peptide/DNA solutions were added to Opti-MEM with sodium bicarbonate in 96well plates. Growth medium was removed from the cells and the previously prepared solution was
immediately added. The complexes were allowed to incubate, removed, and replaced with the growth
medium. Cells were allowed to grow for 3 days at 37°C, 5% CO2 after which the cells were ready for
transfection analysis [6]. Transfection testing was done with a Bright-GloTM Luciferase Assay System.
The test for transfection was simple. After the Bright-GloTM Reagent warmed to room temperature, a
volume of reagent equal to that of the culture medium in each well was added and mixed. After a period
of two minutes elapsed to allow for complete cell lysis, luminescence was measured in a luminometer [8].
The relative light units (RLU) were normalized to the protein content measured using the Pierce® BCA
Protein Assay Kit [9].
Cytotoxicity Measurement
To determine the toxicity of the delivery system a Vybrant MTT Cell Proliferation Assay Kit was
used. After cells have been cultured in the well-plate, medium was added to each well in addition to an
MTT stock solution. Each N/P value used has 5 repeats. The MTT solution was also added to 5 wells
that contained just medium as a control. The samples were incubated at 37°C for 4 hours. After
incubation, the medium was removed and DMSO was added to each well and mixed thoroughly with a
pipette. The samples were incubated at and then the absorbance of each well was read at 540 nm [7].
Results
Peptides analyzed in the project include both known standards and newly designed peptides. The
purpose of analyzing the standard PEI was to determine if the experimental procedure used was accurate
and would be able to reproduce previously reported results. Tests were not replicated so no averages or
standard deviations are reported. The following figures depict the results for each peptide analyzed.
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Size, Zeta Potential and Transfection efficiency of PEI
Figure 5: The size of PEI/DNA complex at different N/P ratios in Tris and water buffers.
Figure 5 depicts the size measurements of PEI. The normal buffer used for PEI is tris. When tris
was used as the buffer in this study, a size similar to that of literature was recorded (between 75 nm and
160 nm). Using tris with the Opti-MEM increased the size of the complex as was expected due to the
larger size of particles in the Opti-MEM.
Figure 6: The zeta potential of PEI/DNA complex at different N/P ratios in Tris and water
buffers.
The zeta potential of PEI in tris was slightly lower than the reported results (between 10 and 30)
as seen in Figure 6. Using Opti-MEM in addition to tris for the buffer decreased the zeta potential, but
still kept it positive which is desired for transfection. The zeta of the complexes in the nano-water buffer
differed due to the different ionic strength of the buffer.
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Figure 7: Transfection efficiency of PEI/DNA complex at different N/P ratios in Tris buffer.
In Figure 7, transfection was most successful at an N/P ratio of 10 which resulted in almost
1.0E+7 RLU/well. The measurement of RLU/well for PEI, a known standard, was used as a comparison
for other peptides.
Size and Zeta Potential of PLL
Figure 8: The size of PLL/DNA complex at different N/P ratios in water buffer.
The sizes of the complexes in samples that contained the PLL were approximately between 90
nm and 150 nm as seen in Figure 8. This is close to the desired size of the peptide/DNA complex.
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Figure 9: The zeta potential of PLL/DNA complex at different N/P ratios in water buffer.
In Figure 9, it can be seen that the zeta potential of PLL in nano-water was positive, the desired
charge on peptide/DNA complexes.
Size and Zeta Potential of R10S10
Figure 8: The size of R10S10/DNA complex at different N/P ratios in PBS, water, and Tris
buffers.
The results from the Zetasizer indicated that there were multiple peaks for each N/P ratio ranging
from very high to low sizes which most likely skewed the results. Multiple tests were performed on
R10S10 with different buffers and at different N/P values due to the wide range of sizes within one test.
Sizes at lower N/P ratios were smaller and closer to the desired size as seen in Figure 10.
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Figure 9: The zeta potential of R10S10/DNA complex at different N/P ratios in PBS, water, and
Tris buffers.
The “Expert Advice” feature of the Zetasizer indicated that there was sediment/large particles/or
dust present in the sample that would have affected zeta potential and most likely skewed the results. In
Figure 11, zeta potentials at lower N/P ratios were negative and did not become positive until N/P ratios
of 4 and higher depending on the buffer. However, with multiple peaks reported for each N/P ratio for
each sample, the large sizes are most likely less accurate measurements.
Size, Zeta Potential and Transfection Efficiency of R-KE
Figure 10: The size of R-KE/DNA complex at different N/P ratios in Tris and NaAc buffers.
The results from the Zetasizer indicated that there were multiple peaks for some N/P ratios
ranging from very high to low sizes which most likely skewed the results. Sizes measured ranged from
120 nm to almost 5500 nm for the NaAc buffer, and 150 nm to 1250 for tris as see in Figure 12.
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Figure 13: The zeta potential of R-KE/DNA complex at different N/P ratios in Tris and NaAc
buffers.
The “Expert Advice” feature of the Zetasizer indicated that there was sediment/large particles/or
dust present in some samples that would have affected and zeta potential and most likely skewed the
results. In Figure 13, in can be seen that the zeta potential was negative for the majority of samples in
NaAc, however, the positive potential seemed out of place. The zeta potential of the complex in Tris
increased from negative to positive as the N/P ratio increased.
Figure 11: Transfection efficiency of R-KE/DNA complex at different N/P ratios in NaAc buffer.
As shown in Figure 14, the RLU/well for R-KE was very low and didn’t even reach 1.0E+6. As
seen in Figure 7, PEI reached a RLU/well of 1.0E+7 so the transfection of R-KE was poor in comparison.
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Size, Zeta Potential, Transfection Efficiency, and Toxicity of R-KE-RGD
Figure 12: The sizes of R-KE-RGD/DNA complex at different N/P ratios in NaAc buffer.
The results from the Zetasizer indicated that there were multiple peaks for each N/P ratio ranging
from very high to low sizes which most likely skewed the results. Figure 15 shows the sizes for N/P
ratios less than 2.5 were very close to the desired size. However, with multiple peaks reported for each
N/P ratio for each sample, the large sizes are most likely not accurate measurements.
Figure 13: The zeta potential of R-KE-RGD/DNA complex at different N/P ratios in NaAc
buffer.
The “Expert Advice” feature of the Zetasizer indicated that there was sediment/large particles/or
dust present in the sample that would have affected the zeta potential and most likely skewed the results.
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All N/P ratios reported negative zeta potentials as shown in Figure 16. However, the data does not follow
a pattern and with the error from the Zetasizer, it is possible the measurements are not accurate.
Figure 17: Transfection efficiency of R-KE-RGD/DNA complex at different N/P ratios in NaAc
buffer.
Transfection for R-KE-RGD at an N/P ratio of 10 had fairly high transfection. It was still an
order of magnitude less than the standard PEI, but was a significant improvement over R-KE.
Figure 14: Cytotoxicity of R-KE-RGD/DNA complex at different N/P ratios in NaAc buffer.
Positive cells indicate cells that were still alive. A control of just cells with no peptide was used
as a comparison for results. Therefore, the higher the number of positive cells, the less toxic the peptide.
If no cells were killed, the number of positive cells would be 100. As is shown in the graph, it is possible
to have more than 100 due to error in the measurements. The majority of N/P ratios resulted in at least
100 positive cells indicating the peptide has very low cytotoxicity.
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Discussion
PEI and PLL
The results of sizes and zeta potentials of PEI in the tris buffer were close enough to that of the
reported size and zeta potential, that the experimental procedure for testing size and zeta potential was
validated. The measured sizes and zeta potentials for PLL were close to the desired values. Transfection
testing of PEI was used as point of comparison for the newly designed peptides. The results of PEI and
PLL testing support the use of PEI and PLL as standards.
Newly Designed Peptides
Due to some property of the designed peptides R 10S10, R-KE, and R-KE-RGD, the measurements
of sizes and zeta potentials seemed to possibly be less accurate as indicated by the “Expert Advice”
feature of the Zetasizer. Size and zeta potential measurements for R10S10 can be found in Figures 10 and
11. Sizes and zeta potentials seemed to vary significantly within one set of samples of various N/P ratios
made at one time. The data did not show a correlation between N/P ratio and size/zeta potential as would
be expected. Runs were not replicated so no averages or standard deviations were available. Replication
of testing would have indicated if the variations were due to regular fluctuations of measurements or due
to a property of the samples that didn’t allow for accurate measurements by the Zetasizer. However,
since tests were not replicated, R 10S10, was eliminated as a viable option due to the inconsistency in size
and zeta potential measurements.
The size and zeta potential results for R-KE seemed to follow more of a pattern and had a size
desirable for transfection, although the zeta potential was negative. NaAc was chosen as the most
reasonable buffer for R-KE because the sample with the most acceptable sizes used NaAc. Despite the
negative zeta potential, transfection testing was performed for R-KE with NaAc buffer based on the size
measurements. As can be seen in Figures 14 and 17, in the transfection testing the RLU/well didn’t even
reach 1.0E+6 when the RLU/well was 1.0E+7 for PEI. This indicated the transfection efficiency for RKE is not as good as PEI.
The NaAc buffer was used for R-KE-RGD based on the results of R-KE. The results for R-KERGD, which can be found in Figure 15, indicated that for N/P ratios below 2.5 the peptide/DNA complex
had a size within the desired range for transfection. Above an N/P ratio of 2.5, the Zetasizer reported
multiple peaks ranging from very high to low sizes which most likely skewed the results. Similar to the
R10S10, the “Expert Advice” feature on the Zetasizer indicated some sediment/large particles/or dust
present in the sample that would explain the larger peak present in the size measurements. The zeta
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potentials for all N/P ratios were measured to be negative as seen in Figure 16. Despite the negative zeta
potential, transfection testing was performed for R-KE-RGD with NaAc buffer based on the size
measurements. The transfection results shown in Figure 17 indicated that the transfection for an N/P ratio
of 10 was significantly better than for R-KE, but still a magnitude of 10 less than PEI. Since the
transfection testing was successful compared to R-KE, toxicity testing was performed on R-KE-RGD.
Figure 18 shows that R-KE-RGD has low cytotoxicity. Cytotoxicity is typically a disadvantage of nonviral vectors, but R-KE-RGD has shown that non-viral vectors have the potential to have low toxicity.
Based on the results of this study, R-KE-RGD has the most potential as an effective non-viral
vector for gene therapy. However, its transfection is not as efficient as that of PEI. A cause for the lower
transfection could be that the peptide is unable to escape from the endosome or lysosome after
endocytosis. This capability is an important factor in the success of non-viral vectors. The addition of the
amino acid histidine helps peptides with this capability and has been shown to increase transfection [6].
Since R-KE-RGD had moderately high transfection, there are currently studies being completed on a
modified version of R-KE-RGD that includes histidine and valine. Current and future studies will take
the information gained from the study of R 10S10, R-KE, and R-KE-RGD and improve upon the designs to
design a peptide that is a non-viral vector with high transfection, low cytotoxicity.
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References
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3. Pack, Daniel W., Allan S. Hoffman, Suzie Pun, and Patrick S. Stayton. "Design and Development
of Polymers for Gene Delivery." Nature Reviews Drug Discovery 4.7 (2005): 581-93.
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Gene Delivery." Journal of Controlled Release 60 (1999): 149-60.
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Melanoma Cells." Protein Engineering Design and Selection 17.5 (2004): 433-41.
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