\set{final}

\def\Author{Prow}
\def\author{prow}
\def\vol{12}
\def\year{2006}
\def\anum{67}
\def\pages{606-615}
\def\txt_title{Construction, gene delivery, and expression of DNA tethered nanoparticles}
\def\txt_authors{Tarl Prow, Jacob N. Smith, Rhonda Grebe, Jose H. Salazar, Nan Wang, Nicholas Kotov, Gerard Lutty, James Leary}

\def\rcvd{28 February 2005}
\def\accept{19 April 2006}
\def\publ{26 May 2006}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

\| External links

\| Internal defs


\article{

\title{Construction, gene delivery, and expression of DNA tethered
nanoparticles}

\authors{\mailto{tprow1@jhmi.edu}{Tarl Prow},\sup{1,2,3} Jacob N.
Smith,\sup{2} Rhonda Grebe,\sup{3} Jose H. Salazar,\sup{2} Nan
Wang,\sup{2} Nicholas Kotov,\sup{4} \mailto{glutty@jhmi.edu}{Gerard
Lutty},\sup{3} \mailto{jfleary@purdue.edu}{James Leary}\sup{1,2,5}}

\institutions{Departments of \sup{1}Pathology and \sup{2}Infectious
Diseases, University of Texas Medical Branch, Galveston, TX; \sup{3}The
Wilmer Ophthalmologic Institute, Department of Ophthalmology, The Johns
Hopkins Hospital, Baltimore, MD; \sup{4}Department of Chemical
Engineering, University of Michigan, Ann Arbor, MI; \sup{5}Basic Medical
Sciences and Biomedical Engineering, Purdue University, West Lafayette,
IN}

\correspondence{James Leary, Basic Medical Sciences and Biomedical
Engineering, Purdue University, West Lafayette, IN, 47907; Phone: (765)
494-7280; FAX: (765) 496-6443; email: jfleary@purdue.edu}

\abstract

\abs_purpose{Layered nanoparticles have the potential to deliver any
number of substances to cells both in vitro and in vivo. The purpose of
this study was to develop and test a relatively simple alternative to
custom synthesized nanoparticles for use in multiple biological systems,
with special focus on the eye.}

\abs_methods{The biotin-labeled transcriptionally active PCR products
(TAP) were conjugated to gold, semiconductor nanocrystals, and magnetic
nanoparticles (MNP) coated with streptavidin. The process of
nanoparticle construction was monitored with gel electrophoresis.
Fluorescence microscopy followed by image analysis was used to examine
gene expression levels from DNA alone and tethered MNP in human hepatoma
derived Huh-7 cells. Adult retinal endothelial cells from both dog
(ADREC) and human (HREC) sources were transfected with nanoparticles and
reporter gene expression evaluated with confocal and fluorescent
microscopy. Transmission electron microscopy was used to quantify the
concentration of nanoparticles in a stock solution. Nanoparticles were
evaluated for transfection efficiency, determined by fluorescence
microscopy cell counts. Cells treated with MNP were evaluated for
increased reactive oxygen species (ROS) and necrosis with flow
cytometry.}

\abs_results{Both 5' and 3' biotin-labeled TAP bound equally to MNP and
there were no differences in functionality between the two tethering
orientations. Free DNA was easily removed by the use of magnetic
columns. These particles were also able to deliver genes to a human
hepatoma cell line, Huh-7, but transfection efficiency was greater than
TAP. The semiconductor nanocrystals and MNP had the highest transfection
efficiencies. The MNP did not induce ROS formation or necrosis after 48
h of incubation.}

\abs_conclusions{Once transfected, the MNP had reporter gene expression
levels equivalent to TAP. The nanoparticles, however, had better
transfection efficiencies than TAP. The magnetic nanoparticles were the
most easily purified of all the nanoparticles tested. This strategy for
bioconjugating TAP to nanoparticles is valuable because nanoparticle
composition can be changed and the system optimized quickly. Since
endothelial cells take up MNP, this strategy could be used to target
neovascularization as occurs in proliferative retinopathies. Multiple
cell types were used to test this technology and in each the
nanoparticles were capable of transfection. In adult endothelial cells
the MNP appeared innocuous, even at the highest doses tested with
respect to ROS and necrosis. This technology has the potential to be
used as more than just a vector for gene transfer, because each layer
has the potential to perform its own unique function and then degrade to
expose the next functional layer.}

\introduction

\p{The majority of nanoparticle research, to date, has been carried out
by materials scientists, but recent trends have brought these tools into
the hands of biologists. Nanoparticles have found two broad niches in
biology, detection technologies and payload delivery [1,2]. Since the
late 1970s, nanoparticles have been used to deliver drugs [2,3]. In
fact, the majority of publications concerning biological applications of
nanoparticles are focused on the delivery of chemotherapeutic agents
with nanoparticles ranging from 2 to 3000 nm. Nanoparticle mediated gene
delivery has recently emerged as a promising tool for gene therapy
strategies [4-6]. The main problems with using nanoparticles for gene
delivery are the construction, cost, and quality control of the
nanoparticles themselves. The construction quickly becomes very
complicated when the number of layers increases. This is due to the
interactions between layers and between nanoparticles with incomplete
and complete layering. These factors limit the usefulness of
nanotechnology to laboratories that have chemists capable of
nanoparticle synthesis or to investigators in collaboration with
chemists. This limits the technology, especially for small laboratories.
This study documents the development of a streptavidin nanoparticle
system that is simple and quite flexible from a commercially available
product intended for other uses.}

\p{Magnetic nanoparticles have been primarily applied to three fields:
magnetic resonance imaging, molecular and cell separation technologies,
and drug delivery [7-11]. Many researchers use magnetic particles as
contrast agents [12-16]. Because these agents are used primarily in
diagnostic in vivo imaging, many of the particle formulations are
already approved for use in humans. The magnetic properties of these
particles are quite favorable for layered construction of a nonviral
based gene delivery vector.}

\p{We are currently developing a nanomedicine strategy to prevent
retinopathy of prematurity (ROP) [17], a disease in which it would be
undesirable to use a viral gene delivery system in premature infants.
Our strategy is to deliver a nanoparticle to a cell in the eye that is
capable of detecting and reacting to the initial hyperoxic insult that
is the first stage of ROP, vaso-obliteration [17]. This event will
trigger the expression of a therapeutic gene able to save the cells in
the eye that would normally die and leave the retina with a compromised
vasculature. Before this can be accomplished, however there are many
challenges to overcome.}

\p{One of the most difficult challenges facing researchers constructing
layered nanoparticles is the purification of the particles after each
step. With magnetic particles, the purification is generally simple and
utilizes magnetic columns. The magnetic properties of nanoparticles have
been used to enhance gene transfer for gene therapy applications
[18-22]. In this case, the nanoparticles were used to concentrate the
plasmid to a specific location and thereby increase the likelihood of
transfection [22]. The Plank lab used clusters of plasmid DNA and coated
magnetic nanoparticles were used to target cells using the magnetic
properties of the nanoparticle clusters [18].}

\p{Several nanoparticle cores were investigated in this study, including
gold, semiconductor nanocrystals, and magnetic iron oxide. Gold
nanoparticles have traditionally been used as for immunolabeling in
transmission electron microscopy and are commercially available in a
variety of sizes. We chose to evaluate one of the smaller gold
nanoparticles (5 nm) available with streptavidin already conjugated to
the surface. Semiconductor nanocrystals are about 25 nm in diameter and
have very unique optical properties. These nanoparticles do not
photobleach, fluoresce intensely with UV excitation, and are capable of
having streptavidin bioconjugated to their surface [23].}

\p{Superparamagnetic nanoparticle cores coated with dextran
bioconjugated to streptavidin were chosen for gene transfer because they
were easily obtainable, simple to construct and could be purified from
the unbound layer components using magnetic columns. The core particles
are composed of an iron oxide core coated with dextran and bioconjugated
to streptavidin, with the complete particle measuring approximately 100
nm in diameter. We have developed a simple procedure for DNA
conjugation, purification, and delivery to cells. These particles were
found to have reasonable transfection efficiency, with respect to free
DNA, when coated with lipid. Genes were expressed from magnetic
nanoparticles at levels slightly below that of free DNA. Magnetic
nanoparticles were capable of transfecting several cell types including
an immortalized human hepatoma cell line, Huh-7, and adult retinal
endothelial cells from both dog (ADREC) and human (HREC) sources. This
study demonstrates that the magnetic and semiconductor nanoparticles
were the two largest and most efficient for transfecting ADREC. Finally,
the magnetic nanoparticles alone did not induce oxidative stress or
necrosis as determined by flow cytometry.}

\methods

\subsection{Biotin-labeled DNA fragment preparation}

\p{PCR amplification was used to create biotin-labeled DNA fragments.
Oligonucleotide primers were purchased from Integrated DNA Technologies,
Inc. For initial studies, either the 5' or the 3' oligo was made with a
single biotin tag. The sequences were based on the pEGFP-C1 (BD
Clontech, Inc., Mountain View, CA) template: forward
5'-\color{\red}{T}AG TTA TTA ATA GTA ATC AAT TAC GGG GTC ATT AG-3',
reverse 5'-TAC ATT GAT GAG TTT GGA CAA ACC ACA ACT AGA AT-3' (Integrated
DNA Technologies, Inc., Coralville, IA). The forward primer begins on
the first nucleotide of the CMV promoter (\color{\red}{red} thymine),
whereas the reverse primer is past the last nucleotide of the SV40
polyadenylation signal. Thus all of the components necessary for gene
expression are present in the PCR product. Later studies used only 5'
oligonucleotides labeled with biotin. These oligonucleotides were then
used as PCR primers. A typical reaction would include 25 \mu l Red Taq,
(Sigma, Inc., St. Louis, MO), 1 \mu l 5' biotinylated primer, 1 \mu l 3'
primer, 1 \mu l template, to 50 \mu l with water. The primers were at
200 pM and the template at 50 ng/\mu l. A typical reaction for DNA
tethering to magnetic nanoparticles would include 25 of these reactions
combined. Typical PCR cycles would include about 35 cycles of denaturing
temperature at 94 \deg C for 30 s, annealing temperature at 65 \deg C
for 30 s and extension for 2 min at 72 \deg C.}

\subsection{DNA tethered nanoparticle construction}

\p{Biotin-labeled PCR products were tethered to streptavidin-coated
magnetic nanoparticles (Miltenyi Biotech, Inc., Auburn, CA). This
nanoparticle has an iron oxide core coated with dextran that is
bioconjugated to streptavidin. DNA tethered magnetic nanoparticles were
constructed by incubating the magnetic nanoparticles with the
biotin-labeled PCR fragments at the ratio of 31 ng DNA to 1 \mu l of
nanoparticles. The mixture was incubator at room temperature for 30 min.
During that time, the magnetic column was prepared by washing once with
the 100 \mu l of the nucleic acid buffer and three times with 100 \mu l
of Optimem (Gibco, Inc., Rockville, MD). Once washed, the column was
loaded with the DNA nanoparticle mixture. The column was then washed
three times with 100 \mu l Optimem. The nanoparticles were eluted by
removing the column from the magnet and adding the 100 \mu l of Optimem.
The resulting brownish solution contained DNA tethered nanoparticles.}

\p{The amount of transcriptionally active PCR products (TAP) bound per
\mu l of streptavidin-Cy3 (SA), gold nanoparticles (GNP), nanocrystals
(NC; made by the Kotov lab or purchased from Quantum Dot, Inc., Hayward,
CA), and MNP was determined by gel electrophoresis. An excess of TAP was
added to 10 \mu l of the nanoparticle stock solution and incubated at
room temperature for 20 min. The maximal amount of TAP bound per \mu l
of SA, GNP, NC, and MNP was determined by semi-quantitative gel
electrophoresis to be 37.4, 16.9, 54.4, and 31.1 ng per \mu l,
respectively. Therefore, 100 ng TAP would bind 2.7 \mu l SA, 5.9 \mu l
GNP, 1.8 \mu l NC, and 3.2 \mu l MNP.}

\subsection{Lipid coating of DNA tethered magnetic nanoparticles}

\p{The DNA tethered nanoparticles (400 ng DNA/13 \mu l MNP) were coated
with Lipofectamine 2000. The eluted particles were diluted in the 250
\mu l of Optimem and incubated for 5 min at room temperature. 10 \mu l
of Lipofectamine 2000 was diluted with 250 \mu l of Optimem in a
separate tube and incubated at room temperature for 5 min. After 5 min,
the two tubes were mixed gently and combined. This mixture was allowed
to stand for 20 min before adding to the cell culture. All nanoparticles
had lipid coating except those in \figref{1}, \figref{2}{B}, and
\figref{3} (as noted on the x-axis).}

\subsection{Confocal and fluorescence microscopy}

\p{Cells were examined for reporter gene expression with a Zeiss 510META
confocal microscope. The two types of nanocrystals used for these
studies had 525 and 565 nm emission peaks. After excitation with a 405
nm diode laser the emission light was passed through a 490 nm long pass
filter and finally detected with 520 nm and 560 nm bandpass filters for
the 525 and 565 nm nanocrystals, respectively. EGFP was excited with a
488 nm argon ion laser and the emission light was passed through a 490
nm long pass filter and finally detected with a 520 nm bandpass filter.
DsRed was excited with a 543 nm HeNe laser and the emission light was
passed through a 490 nm long pass filter and finally detected with a 560
nm bandpass filter. Differential interference contrast (DIC) microscopy
was also used to image all cells using the 488 nm argon ion line.}

\p{Fluorescence microscopy was performed using a Nikon Eclipse TE2000-U
inverted microscope. Photomicrographs were taken with a SPOT RT SE
digital camera, Diagnostic Instruments, Inc. (Sterling Heights, MI).
Nanocrystals were imaged using a Nikon Fluorogold filter set, which
excites with UV and contains a 505 nm long pass emission filter. EGFP
was imaged with a standard Nikon FITC filter set, while DsRed was imaged
using a standard Nikon Cy3 filter set.}

\subsection{Image analysis for gene expression levels}

\p{A standard wave-propagation algorithm was used to segment the images
over a singular threshold. Upper and lower boundaries were chosen for
subsegmentation. Segments which fell below the lower area bound were
removed. Segments which were above the upper boundary were re-segmented
with a higher threshold and reexamined. The threshold level was computed
as the average of the intensity of the pixels within the segment minus
the standard deviation of the intensity of the pixels bounded below by
zero. Threshold levels are computed individually for each subsegment.
The output is a list of segments associated with a bitmap representing
the segment, the total intensity, area, and standard deviation of
intensity for that segment [24].}

\subsection{Cell culture}

\p{Cells were incubated at 37 \deg C in 5% CO\sub{2}. The Huh-7 cell
line, derived from a human hepatoma (gift from Rene Rijnbrand,
University of Texas Medical Branch, Galveston, TX), was cultured in DMEM
supplemented with 10% FBS (Sigma, Inc.) and penicillin/streptomycin
(Sigma). Each experiment was done at least in triplicate and positive
and negative controls were present in all experiments.}

\p{Primary cell lines of ADREC were established as reported by Lutty et
al. [25]. Adult beagles were euthanized by an intraperitoneal overdose
of pentobarbital sodium. Animals were treated in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
The eyes were enucleated and placed in cold, sterile PBS and excess
tissue was cleared away. The eyes were then soaked in cold Betadine for
15 min. The retina was washed thoroughly and homogenized in PBS, with a
Dounce homogenizer. The homogenate was filtered with a 105 \mu m Nitex
nylon mesh in a porcelain funnel under a gentle vacuum. The filtrate was
then passed through a 58 \mu m Nitex mesh. The vessel retentate was
digested in 0.375% collagenase and 0.25% bovine serum albumin in PBS for
45-60 min at 37 \deg C. The digestion was then stopped by the addition
of DMEM/F12 media supplemented with 10% fetal bovine serum, 1%
penicillin/streptomycin/fungizone (Gibco, Inc.). The cells were then
incubated at 37 \deg C in 5% CO\sub{2} for 24 h in a T-25 flask. After
24 h, the unattached cells and debris were washed away and the remaining
cells were fed fresh media. All studies were done with cells at less
than passage 10. The endothelial cells used in these studies were vWF
positive and took up Acetylated LDL.}

\p{Human retinal endothelial cells were purchased from Cell Systems,
Inc. (Kirkland, WA). These cells were maintained on CSC medium from Cell
Systems, Inc. Later, however, the cells were successfully grown and
split in the same fashion and with the same medium as the ADREC above.}

\subsection{Cytotoxicity of magnetic nanoparticles}

\p{Cytotoxicity was evaluated by morphological analysis of ADREC
incubated with MNP for 24 h at several concentrations including, 0, 200,
400, 4000, and \gt 15,000 ng of DNA. After incubation, the cells were
photographed under phase illumination as described above. Reactive
oxygen species formation after nanoparticle treatment was determined by
treating AREC with 0, 200, 400, and 1000 ng DNA equivalents of MNP
(nanoparticles without DNA or lipid coating) for 48 h. After incubation
with the nanoparticles, the cells were washed three times in PBS and
trypsinized to attain a cell suspension and stained with 50 \mu m/ml
propidium iodide (Sigma) for 10 min in DMEM/F12 at 37 \deg C in 5%
CO\sub{2}. After staining the cells were washed three times with PBS.
Next, the cells were stained with 5-(and
6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester
(CM-H\sub{2}DCFDA) in DMEM/F12 at 5 \mu M for 30 min under culture
conditions. After CM-H\sub{2}DCFDA staining, the cells were washed three
times in PBS and trypsinized to attain a cell suspension. The cells were
then washed three times in complete media without phenol red. As a
positive control some cells were treated with 100 \mu M tert-butyl
hydrogen peroxide for 30 min prior to staining. The cells were analyzed
on a FACScalibur flow cytometer, and data were analyzed by CellQuest
software (both from Becton Dickinson Immunocytometry Systems, San Jose,
CA).}

\subsection{Magnetic nanoparticle quantification with transmission
electron microscopy (TEM)}

\p{The stock solution of MNP was purchased from Miltenyi Biotech, Inc.
MNP stock solution (10 \mu l) was diluted 1:100,000 with PBS. The
diluted MNP were then incubated with ten \mu l of biotin coated
polystyrene beads (1.75 million beads, 3.27 \mu m diameter from
Spherotech, Inc., Libertyville, IL) in 990 \mu l of PBS for 30 min at
room temperature. After 30 min, the beads were washed three times in PBS
by centrifugation at 1500 RPM in a Beckman TJ-6 centrifuge with a TH-4
1-88 rotor for 5 min. After washing, the beads were fixed in fresh, 2%
paraformaldehyde for 10 min. The beads were then dried overnight in a
Savant SpeedVac concentrator SVC100H. The beads were then mixed with
LX112 polymer (Ladd Research Industries, Inc., Burlington, VT) and the
solution placed in a cylindrical block mold in a heated block form
(Pelco International, Inc., Redding, CA) and polymerized at 60 \deg C
for 36 h. Thin sections (95 nm) were cut on a Leica Ultracut UTC
ultramicrotome and collected on 150 mesh uncoated copper grids. Samples
were viewed and photographed with a JEOL 100CX TEM (JEOL USA, Inc.,
Peabody, MA). NIH Image was used to determine the area of the bead
(n=90). The area and section thickness of the beads were used to
calculate the surface area of the cylinder (2\pi r\sup{2}) exposed to
nanoparticles. Nanoparticles were also quantified and the data used to
calculate the number of nanoparticles per micrometer squared. This was
then used to determine the number of nanoparticles per \mu l (40
million).}

\results

\subsection{Conjugation of DNA to magnetic nanoparticles}

\p{Biotin-labeled PCR primers were used to generate CMV-EGFP-pA (CMV
promoter, EGFP reporter gene, and poly A signal) containing DNA
fragments (1.5 kb) with 5' biotin-labeled, 3' biotin-labeled, or
unlabeled. Streptavidin-coated magnetic nanoparticles were incubated
with each of the DNA fragments, and no Lipofectamine, and analyzed by
agarose gel electrophoresis (\figref{1}). \figref{1} lanes A to C
contained only the PCR product. \figref{1} lanes D and F contained
magnetic nanoparticles incubated with the PCR fragments. DNA in
\figref{1} lanes C and F contained no biotin tag and were, therefore,
used as negative controls. The black square indicates increased
molecular weight DNA. The dark staining seen at the top of \figref{1}
lanes D and E indicate that the DNA was able to bind to magnetic
nanoparticles and was now trapped at the top of the gel due to its large
size. This gel also shows that there is a significant amount of unbound
DNA present. Because of this, the magnetic nanoparticles need to be
purified from the contaminating free DNA fragments as described in the
next section.}

\p{The MNP's were the only nanoparticles capable of being easily
separated from unbound DNA. Therefore, the other streptavidin-tagged
nanoparticles used in this study had to be incubated with the correct
amount of DNA so as to avoid nanoparticles without DNA and free DNA.
This binding ratio of nanoparticle to DNA was determined for each
nanoparticle and streptavidin by semiquantitative gel electrophoresis
(data not shown). Nanoparticles and streptavidin were individually
incubated with a known excess of 5' biotin labeled TAP. This mixture was
then electrophoresed on an agarose gel similar to that shown in
\figref{1}. The amount of unbound TAP was determined in a
semiquantitative nature. The maximal amount of TAP bound per \mu l of
SA, GNP, NC, and MNP was determined by semi-quantitative gel
electrophoresis to be 37.4, 16.9, 54.4, and 31.1 ng per \mu l,
respectively. Therefore, 100 ng TAP would bind 2.7 \mu l SA, 5.9 \mu l
GNP, 1.8 \mu l NC, and 3.2 \mu l MNP.}

\subsection{Removal of free DNA from magnetic nanoparticle/DNA
solutions}

\p{In these experiments, the mixtures of DNA and magnetic nanoparticles
were washed four times to remove unbound DNA using a magnetic column. It
was found that the magnetic properties of these particles enabled the
rapid purification of the magnetic nanoparticles from the DNA solution.
These samples were then run on an agarose gel (\figref{1}). \figref{1}
lanes A and C represent only DNA fragments. \figref{1} lanes D and F
contain the magnetic nanoparticle/DNA mixture. After washing, a portion
of the magnetic nanoparticles were run onto this gel (\figref{1} lanes G
and I). If carefully examined (inset) dark staining can be seen only in
\figref{1} lanes G and H near the loading well. This suggests that the
free DNA has been removed and only the DNA tethered magnetic
nanoparticles remain in solution.}

\subsection{Cytotoxicity of uncoated magnetic nanoparticles in adult dog
retinal endothelial cells}

\p{Adult dog retinal endothelial cells (ADREC) were treated with
increasing concentrations of non-lipid-coated MNP and examined for signs
of cytotoxicity. As seen in \figref{2}, there are no differences in
cells exposed to MNP versus the untreated control. There are no apparent
signs of cytotoxicity, including pycnosis, blebbing, or detachment. All
of these cells appeared normal from the beginning of the experiment to
the end and were treated with several concentrations of MNP ranging from
0 to the stock concentration (40 million MNP per \mu l), which was dark
brown. Uncoated magnetic nanoparticles with no lipid coating were tested
for their ability to induce ROS formation or necrosis in ADREC after
incubation for 48 h. One hundred \mu M tert-butyl hydrogen peroxide was
added to the cells for 30 min prior to flow cytometric analysis and was
used as a positive control. The results from this experiment are
summarized in the graph in \figref{2}{C}. The three groups of cells
treated with nanoparticles were equivalent to the untreated control (0)
with respect to both ROS and necrosis. The positive control values were
substantially greater than any of the treatment groups. In fact in all
of the treatment groups, the number of positive cells was approximately
5%.}

\subsection{Expression levels of cells transfected with lipid-coated and
non-lipid-coated DNA tethered magnetic nanoparticles}

\p{This experiment was designed to assess the ability of the cellular
machinery to properly express a protein from a nanoparticle tethered
gene. This experiment reveals the level of gene expression as measured
by fluorescence microscopy, not to be confused with transfection
efficiency, which is discussed later. Different combinations of DNA,
biotin labeling, MNP, and lipid coating were tested for their effects on
gene expression. The lipid coating has previously been shown to
dramatically increase transfection efficiency with naked DNA [26]. The
MNP-DNA-Lipid complex was then delivered to Huh-7 cells cultured in
chamber slides, incubated for 48 h. The cells were then photographed and
the images obtained were then analyzed with an in house slide based
cytometry software program (written by JNS, UTMB) and the resulting data
presented in \figref{3}. All of the values were normalized to the
samples treated with the non-biotinylated GFP fragment transfected with
Lipofectamine 2000. The labeling of the DNA did not affect the
expression of EGFP, as shown in \figref{3} groups B and D. When the DNA
was bound to the nanoparticles (\figref{3} groups E and F), there was a
decrease in expression levels when compared to naked DNA alone
(\figref{3} groups B and C). \figref{3} groups G and J were controls for
the construction of the nanoparticles, in that, without a biotin tag the
TAP cannot bind the nanoparticle and should be washed away during
construction. Indeed, we found that the gene expression levels of
\figref{3} groups G and J were equivalent to the true negative control,
\figref{3} group A. The intact pEGFP-C1 plasmid was found to have about
two times greater expression level than the TAP fragment. This is
possibly due to the degradation of the TAP due to the free ends and more
substantial transcriptional machinery-binding site.}

\subsection{Transfection efficiencies of lipid-coated magnetic
nanoparticles in adult dog retinal endothelial cells}

\p{Transfection efficiencies were also determined for different amounts
of DNA so that comparisons to naked DNA could be made. The MNP with
lipid coating, were evaluated for dose responsiveness and compared to
TAP only transfections (\figref{4}). The chosen dosages were nanograms
of DNA (100, 200, and 400 ng) transfected over 1 h. The majority (\gt
90%) of the transfection occurs in less than 1 h after exposure to MNP
(data not shown). The percentage of DsRed positive cells was determined
24 h after transfection. Both the TAP and MNP groups showed a dose
dependent increase in transfection efficiency. The MNP showed greater
average transfection efficiency than TAP treated cells, in the 200 and
400 ng groups. The MNP groups were less variable than the TAP treated
cells.}

\subsection{Transfection efficiencies of different lipid-coated
nanoparticles}

\p{Transfection efficiency in ADREC was used to determine the optimal
size and core material for future nanoparticle experiments. Growing
cells were treated with SA, GNP, NC, and MNP all tethered to DNA
encoding DsRed and all coated with lipid. As a control, a DNA only group
was also run. Forty eight h after exposure, the cells exposed to naked
DNA, nanocrystals, and MNP had the highest transfection efficiency
(\figref{5}). MNP clearly had the best transfection efficiency, at 2
times the naked DNA control in the highest dose (400 ng DNA). All
transfection efficiencies were normalized to DNA alone (400 ng DNA, at
10% of the total cells), because this is the most appropriate
comparison.}

\subsection{Lipid-coated semiconductor nanocrystal transfection of adult
dog retinal endothelial cells}

\p{The ability of lipid-coated semiconductor based nanocrystals as
potential gene carriers was tested first in ADREC and later in HREC.
Confocal imaging of these transfected cells (\figref{6}) demonstrated
that this nanoparticle was capable of delivering genes to human cells.
One interesting observation was that gene expression from nanocrystals
transfected cells appeared to be very high in the first 24 h when
compared to the DNA alone transfected cells. This was observed in both
ADREC and HREC. The downside of using nanocrystals in this way is that
purification between construction steps is difficult at best. One of the
problems that has plagued the use of nanocrystals is their tendency to
form aggregates (\figref{6}{B}, arrowhead). When we attempted to purify
the nanocrystals by centrifugation, there was an increase in aggregate
formation, so for these studies, the nanocrystals were not purified away
from free DNA.}

\subsection{Quantification of magnetic nanoparticles with TEM}

\p{Transmission electron microscopy was used to determine the number of
MNP in 10 \mu l of MNP stock solution. The MNP were bound to
biotin-coated polystyrene beads and then embedded for analysis. Ninety
bead sections were analyzed for the presence of bound nanoparticles and
bead area. The bead area was found to have a normal distribution.
Because the thickness of the bead section, the amount of nanoparticles
and beads added to the mixture were known, we were able to determine the
number of nanoparticles per \mu l. Additionally, it was determined that
3.2 \mu l of MNP to bind 100 ng of TAP, therefore, it is possible to
enumerate the approximate number of TAPS bound per MNP, 47. Finally, 128
million nanoparticles can bind approximately 100 ng of TAP.}

\discussion

\p{These studies have demonstrated that lipid-coated nanocrystals and
MNP can be used to transfect a variety of cell types including retinal
vascular endothelial cells; however, MNP are easier to purify.
Construction of these nanoparticles using the streptavidin-biotin
conjugation can also be monitored with gel electrophoresis. Magnetic
nanoparticles were the most efficient gene delivery vectors tested.
Cells incubated with these particles showed no visible signs of toxicity
(blebbing, apoptosis, etc.), even though the particles are made of iron.
The MNP offer the most promise of the nanoparticles evaluated.}

\p{DNA fragments with 5' or 3' biotin tags were attached to streptavidin
coated magnetic nanoparticles. Free DNA fragments were successfully
removed by washing the particles using a magnetic column. These data
indicated that without lipid coating, there were slightly decreased
levels of EGFP reporter expression when compared to those MNP with
lipid. This may be a result of the number of nanoparticles entering the
nucleus. The addition of lipid to transfection solutions is a widely
recognized method to increase the amount of DNA that reaches the
nucleus. This is also likely to be true for the MNP.}

\p{One important finding is that the DNA tethered magnetic nanoparticles
not coated with lipid were able to successfully transfect cells in
vitro. This result shows that the size range of these particles is
appropriate. Another interesting result is that cells treated with the
DNA tethered magnetic nanoparticles coated with lipid had expression
levels well within range of those transfected with only labeled DNA
fragments. These data show that we can effectively express gene products
from DNA tethered to magnetic nanoparticles in either the 5' or 3'
configuration. Finally, the unlabeled DNA exposed magnetic nanoparticles
that were subsequently washed with a magnetic column, did not show any
appreciable expression of GFP. Therefore, the expression we observed
with the labeled DNA and magnetic nanoparticles was from DNA tethered to
magnetic nanoparticles.}

\p{One way to improve the transfection efficiency is to eliminate
defective or incomplete nanoparticles prior to delivery. This scenario
would explain the differences in transfection seen in this study. This
further underscores the importance of nanoparticle purification during
construction, one of the main benefits of using MNP. Secondly, although
the cellular mechanisms at work are not known, our results suggest that
while the nanoparticles get close to the nucleus, most never get in
(data not shown). This problem could be solved or at least ameliorated
by the addition of targeting molecules to the nanoparticle. However,
with the addition of another layer comes the problem of how to purify
only the nanoparticle with all of the layers.}

\p{Another benefit of this technology is the ability to transfect cells
with low concentrations of DNA. The lower limits of this technology are
often overlooked and researchers tend to examine maximal doses rather
than minimal doses in order to show the best case scenario. These
studies often utilized conditions that can never be achieved in live
animals. We chose to examine the lower limits of MNP compared to DNA
alone to directly compare the two methods of gene transfer. These data
demonstrate a distinct advantage by the MNP at the 200 and 400 ng doses
(\figref{4}). This argument is further strengthened by the fact that
there are about 40 times more DNA particles in the TAP treated cells.
There are thought to be approximately 47 biotin-binding sites per
nanoparticle. If each copy of DNA in the TAP group is considered a
particle capable of transfection, then, on a per particle basis, the
particles of DNA in the TAP alone group out number the MNP particles 40
to 1. This means that the MNP are much more efficient at transfection
than TAP. Even so, the critical factor is the total volume that will be
delivered to the eye. Given the size of the premature infant eye or
neonatal dog eye, our model of ROP, it is possible to inject 50 \mu l
into the vitreous without having to remove any of the vitreous body. In
these studies we have kept the nanoparticle solutions very dilute. This
was primarily due to the costs of the reagents. Therefore, the 50 \mu l
target volume should be capable of delivering more than enough
nanoparticles, 20 billion MNP or almost 1 trillion copies of the gene,
to an individual eye. Reduction of the nanoparticle size would allow for
more nanoparticles and more efficient construction could yield an
increase in the number of usable DNA strands per nanoparticle.}

\p{Another point of contention in the field of nanoparticle based gene
delivery is the optimal size and material of the nanoparticles. Our
personal experience has guided us to nanoparticles less than 200 nm in
diameter. The current study included nanoparticles from 0 (naked DNA) to
100 nm. Although the materials were different, there seemed to be a
clear correlation between size and transfection efficiency: the larger
the particle the better the transfection efficiency. This is most likely
a size and mass issue. If the particle is larger and heavier, there
might be a greater chance that the cell and nanoparticle will be in
contact with each other. On the other hand there will be more particles
present as the core size gets smaller, with the greatest number of
particles present in the DNA only group. With nanoparticles, the smaller
the TAP containing nanoparticles, the less streptavidin that will be
present and consequently fewer DNA particles will be bound. So, by this
formula, increasing the size would decrease the likelihood of
transfection because there would be less nanoparticles per given volume.
We found the opposite to be the case. One possible scenario is that the
addition of multiple TAP copies (47 in the case of MNP) increases the
mass of the nanoparticle so much, that the nanoparticles settle to the
bottom. More experiments need to be done in order to clarify this issue
of size versus transfection efficiency.}

\p{The issue of nanoparticle toxicity is an important area of research.
One of the more frequent criticisms of MNP in culture and in animals is
the toxicity of the nanoparticle. Specifically, the major concern
revolves around the hypothesis that the iron in the core of the MNP
could induce the formation of ROS, via Fenton cycling or other
mechanisms. We have tested this hypothesis with CM-H\sub{2}DCFDA and
flow cytometry. This experiment demonstrated that the levels of ROS, in
MNP-treated cells, were equivalent to those in untreated controls. This
experiment confirms our observation that the cells do not seem to be
harmed by the penetrating MNP. This is likely due to the fact that the
nanoparticles do not readily break down within the cells. Rather, they
appear quite stable during the course of these experiments. Although
this is very promising, the next step is to evaluate their performance
and toxicity in animal models of ROP.}

\p{In summary, multilayered nanoparticles can be constructed with
reasonable ease in a molecular biology laboratory. These particles have
the potential to transfect a multitude of cells, including those
isolated from human sources. Tethered nanoparticle transfection has the
potential to decrease the possibility of the delivered gene integrating
into the host genome. Additionally this technology can increase the
stability of the DNA in the cellular milieu. These particles also have
benefits over virally delivered genes, like decreased inflammation and
immune response, and the MNP do not induce ROS. One limitation of the
MNP reported here is that expression is short-term and transfection
efficiency is low, when compared to other means of gene delivery (i.e.,
viral). These shortcomings may be remedied with better cell entry and
nuclear localization molecules attached to the surface of the
nanoparticle. The nanoparticles investigated offer the possiblity of
adding multiple ordered layers. Perhaps the need to better filter out
defective nanoparticles in order to increase functionality is also
important. If these efforts are successful, the potential reward is
huge. The nanoparticle platform is capable of delivering molecular
programming to a single cell and dictating its actions toward insults.
This behavior modification at a molecular level can be used to prevent
disease as it happens. To this end, we are developing a MNP based system
to deliver biosensors to the eye which can detect, react and thereby
prevent retinopathy of prematurity [17]. The next step is to deliver
nanoparticles to relevant cells that can detect and respond to the
initial high oxygen insult that initiates ROP.}

\acknowledgements

\p{This work was supported by the Biomolecular, Physics and Chemistry
Program under NASA grant NAS2-02059 (TP, JS, NW, MM, JL), National Eye
Institute grant R03EY013744 (GL), R01EY09357 (GL), EY01765 (Wilmer), and
the Johns Hopkins Hematology Training grant T32HL007525 (TP).}

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}

\beginfigures

\figfile{1}{
\figtitle{1}{Construction and purification of DNA tethered magnetic
nanoparticles}

\p{Gel electrophoresis was used to examine the successful construction
of the DNA tethered magnetic nanoparticles and to verify that the
purification removed the unbound DNA from the nanoparticles. Lanes A-C
represent only DNA fragments. Lanes D-F contain the MNP/DNA mixture.
After washing with a magnetic column, a portion of the purified MNP were
also run on this gel (Lanes G-I). On the right is a magnified image of
the box around Lanes F-I. This gel shows three critical stages of
nanoparticle construction, DNA alone, DNA tethered nanoparticle in
excess DNA, and finally DNA tethered nanoparticles free of unbound DNA.}

\ctr{\jpgimage{1}{800}{253}{40}}

}

\figfile{2}{
\figtitle{2}{Adult dog retinal epithelium cells cytotoxicity from
uncoated magnetic nanoparticles}

\p{The images are representative phase contrast photomicrographs of
ADREC treated with 0 ng (\panel{A}) or 400 ng (\panel{B}) DNA tethered
MNP without lipid-coating for 24 h. \panel{C}: The effects of uncoated
magnetic nanoparticles (0, 200, 400, 1000 ng DNA equivalents) on
reactive oxygen species (ROS; white bars) and necrosis (red bars) after
48 h incubation. 100 \mu M tert-butyl hydrogen peroxide was used as a
positive control. The error bars represent standard deviation. One
potential cause for concern when using iron derived nanoparticles is
iron induced oxidative stress, while another concern may be membrane
integrity. Therefore, an oxidative stress sensitive fluorescent dye was
used to confirm the absence of nanoparticle induced oxidative stress.
Likewise, propidium iodide was used to confirm the absence of ruptured
membrane. Flow cytometric analysis of these cells confirmed that there
was no increase in oxidative stress or ruptured membranes (Necrosis).}

\ctr{\jpgimage{2a}{800}{302}{75}}

\ctr{\gifimage{2b}{800}{623}{21}}

}

\figfile{3}{
\figtitle{3}{Expression levels of enhanced green fluorescent protein
from lipid-coated and uncoated DNA tethered magnetic nanoparticles}

\p{In vitro gene expression levels are shown as the percentage of
lipofectamine-transfected enhanced green fluorescent protein DNA. Biotin
tagged DNA was also transfected into cells. All samples were incubated
with or without lipofectamine 2000.}

\ctr{\gifimage{3}{600}{509}{30}}

}

\figfile{4}{
\figtitle{4}{Transfection efficiencies of lipid-coated magnetic
nanoparticles in adult dog retinal endothelial cells}

\p{Three different concentrations of transcriptionally active PCR
products (TAP; white bars) were used (100, 200, and 400 ng per well of
24 well plates) either alone or tethered to lipid-coated nanoparticles
(MNP; red bars). Approximately 1000 cells were analyzed for each data
point. The bars represent the mean of 3 wells; the error bars represent
the standard error of the mean. These data show that TAP tethered
magnetic nanoparticles are better at transfecting cells than free TAP in
the 200 and 400 ng groups.}

\ctr{\gifimage{4}{550}{430}{18}}

}

\figfile{5}{
\figtitle{5}{Transfection efficiencies of multiple lipid-coated
nanoparticle sizes and cores}

\p{Five transfection groups, including DNA alone, streptavin-Cy3 (SA), 5
nm gold nanoparticles (GNP), 25 nm semiconductor nanocrystals (NC), and
100 nm magnetic nanoparticles (MNP), were tested at three different DNA
concentrations (100, 200, and 400 ng). All values were normalized to the
400 ng DNA alone group and all groups were coated with lipid. The 400 ng
MNP group had statistically significant greater transfection (p\lt
0.01), when compared to the 400 ng DNA alone, SA, GNP, or the NC groups
using the Student's t-test.}

\ctr{\gifimage{5}{600}{334}{29}}

}

\figfile{6}{
\figtitle{6}{Lipid-coated nanocrystal transfected human retinal
epithelium cells}

\p{Cells were cultured with lipid-coated nanocrystals tethered to either
EGFP (green in \panel{B}) or DsRed (red in \panel{D}) for 48 h or 10
days, respectively. Confocal (\panel{A},\panel{B}) and fluorescence
(\panel{C},\panel{D}) microscopy were used to simultaneously visualize
nanocrystals and tethered fluorescent gene expression. The nanocrystals
are marked by white arrows and nanocrystal aggregate is marked with a
white arrowhead. \panel{A}: DIC microscopy. \panel{C}: Phase contrast
microscopy.}

\ctr{\jpgimage{6}{500}{497}{32}}

}