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BME 695-Engineering Nanomedical Systems-Final Project 2010
1
Proposal for Magnetic Labeling of Stem Cells for
Subsequent Reprogramming to a Primitive State Lisa M. Reece
11/29/2010
ABSTRACT
This study presents a proposed novel nanomedical systems based approach to the
reprogramming of mature stem cells (SC) isolated from human peripheral blood, into induced
pluripotent stem cells (iPSC) utilizing the OCT4 gene as a possible gene therapy for cancer.
This oncogene is a known POU family transcription factor expressed in human embryonic stem
cells and tumor cells, but not in normal differentiated tissues (Tai, Chang et al. 2005) and is thus
used as a marker for undifferentiated cells (MacDougall 2008). Peripheral blood-derived iPSC
are comparable to primitive stem cells with respect to morphology, expression of surface
antigens, and activation of endogenouse pluripotency genes (Staerk, Dawlaty et al. 2010).
OCT4 has been shown to be expressed in some human tumors but not normal somatic tissues
(Tai, Chang et al. 2005) leading researchers to believe that this gene may have greater potential
for therapeutic targeting in cancer treatment. In this study, we will be harvesting pooled
samples of freshly collected human peripheral blood in order to identify, target, sort, and
reprogram the adult SC into iPSC for subsequent biodistribution in a nude mouse model. The
delivery vehicles for the OCT4 gene will be superparamagnetic iron oxide nanoparticles
(SPION) constructed to have a CD34 targeting antibody to identify adult SC. The bound SPION
will be taken up by the SC for the delivery of the OCT4 gene sequence into the nucleus for
transcription. Further, because the SPIO NP contain a magnetic core, the bound SPIO NP-SC
complexes will be sorted from the blood via the Quadrupole Magnetic Cell Sorter for molecular
analysis. Proof of the gene transcription and translation of the OCT4 protein will be used to
signify the reprogramming of the sorted adult SC into the more primitive iPSC. To confirm that
OCT4 transgenes are silenced in the blood-derived SC, qRT-PCR via primers specific for
endogenous and total transcripts of the reprogramming factors shall be performed. Once iPSC
reprogramming has been confirmed, these cells will be bound to SPION programmed to target
SKBR3 cancer cells and introduced into a mouse model for a biodistribution study. This new
approach for gene therapy for cancer medicine is cheaper than the current chemotherapeutic
and radiation therapies, and is designed to target specific cells in the body that can regulate the
metastasis of tumors without the debilitating side effects of said cancer treatments.
Keywords: SPION, stem cells, iPSC, OCT4, CD34, nanoparticles, nanomedical systems
1. INTRODUCTION
It is a belief that human induced pluripotent stem cells (iPSC) hold great promise for
modeling human diseases. Successful reprogramming of differentiated human somatic cells
into a pluripotent state would allow for the creation of patient- and disease-specific stem cells
that would be used in regenerative medical techniques. It has been shown that there have been
studies where the derivation of iPSC from peripheral blood mononuclear cells (PBMC) are
similar to human embryonic stem cells when comparing morphology, expressions of surface
BME 695-Engineering Nanomedical Systems-Final Project 2010
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antigens, activation of endogenous pluripotency genes, DNA methylation, and potential for
differentiation (Loh, Hartung et al. 2010). It has further been revealed through immunoglobulin
and T cell receptor gene rearrangement analyses that some PBMC iPSC were derived from T
cells. This means that the derivation of iPSC from terminally differentiated cell types is possible.
It is important to note that PBMC can be isolated in sufficient quantity and with minimal risk to
the donor and can be obtained to enable reprogramming without the need for prolonged
expansion in vitro (i.e. cell culture). Reprogramming from blood cells thus represents a fast,
safe, and efficient way of generating patient-specific iPSCs. Somatic cells can be induced to
the pluripotent state by the enforced expression of several transcription factors including OCT4,
SOX2, KLF4, MYC, NANOG, and LIN28 (Loh, Hartung et al. 2010). For this preliminary study, I
will utilize OCT4 for translation and transcription of this gene after delivery to the SC via a
magnetic nanomedical system construct – superparamagnetic nanoparticles (SPION).
SPION are types of magnetic nanoparticles (MNP) that can be manipulated under the
influence of an external magnetic field. The unique ability to be controlled in this fashion has
been utilized for MRI, targeted drug and gene delivery, tissue engineering, cell tracking, and
bioseparation (cell sorting). When further functionalized with drugs, peptides, ligands and/or
nucleic acids, MNP can penetrate cell and tissue barriers and offer organ-specific therapeutic
and diagnostic modalities (Shubayev, II et al. 2009). The dual ability of MNP to be functionalized
and responsive to a magnetic field has made them a useful tool for theragnostics - the fusion of
therapeutics and diagnostics that targets to individualize medicine. Through multilayered
programmable functionalization, MNP can simultaneously act as diagnostic molecular imaging
agents and drug carriers. Further, SPION have several important advantages over other MNP
(including gadolinium-based MRI contrast agents): lower toxicity, stronger enhancement of
proton relaxation, and lower detection limit. For example, Ferumoxtran-10 (Combidex), a
dextran-coated SPION with a mean diameter of ~30 nm, has a 90.5% sensitivity and 97.8%
specificity for detecting pancreatic cancer (PCa) lymph node disease by passively accumulating
in cancerous nodes. The major shortcoming of Combidex is its inability to detect PCa disease
outside of the lymph nodes (Wang, Bagalkot et al. 2008). A well-designed SPION should be
able to find tumors in vivo as well as other cells that occur in other parts of the body.
For these experiments, I will employ two multilayered programmable MNP approaches.
First, for the sorting of adult stem cells the SPION will contain a payload of the OCT4 gene
sequence to initiate the reprogramming of the adult SC (a DNA-tethered MNP). The outer layer
of these SPION will have CD34 antibodies on the outer layer to target and bind to the SC
residing within isolated human buffy coats (from whole blood samples). The result will be adult
SC that are magnetic and can be easily pulled out from all other non-SC in the buffy coats.
Additionally, the SPION will be taken up by the cells for translation/transcription of the gene to
render them even more primitive than before. This type of approach is necessary to overcome
the disadvantages of other procedures such as isolation of adult SC from dermal fibroblasts
(this type of tissue contains SC) harvested by surgical skin biopsy (Park, Zhao et al. 2008).
Besides being a highly invasive procedure, exposure of the dermis to ultraviolet light during the
procedure increases the risk for chromosomal aberrations (Ikehata, Masuda et al. 2003). This
obviously raises concerns for whether iPSCs will reflect the patient’s constitutional genotype – a
step that must not be corrupted if we are to avoid any inflammatory reaction to the
reprogrammed cells placed back into the patient. The second set of SPION will have two
targeting molecules on the outer surface: one for CD34 receptor on the repgrogrammed iPSC,
BME 695-Engineering Nanomedical Systems-Final Project 2010
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and a peptide that will recognize SKBR3 cells. This approach will yield labeled iPSC magnetic
and will allow for these cells to be attracted to SKBR3 tumors in vivo (nude mouse model). This
is a necessary first step in a regenerative medicine gene therapy. The SPION must reach their
target tumor cells. However, in order to find cells that lie outside of tumor masses, increased
circulation time of our SPION must be addressed. In vivo, macrophages of the
reticuloendothelial system (RES) quickly challenge and internalize MNP, to neutralize their
cytotoxicity. But in order to promote their circulation time, engineering strategies to modify MNP
surface chemistry are used to allow for evasion of macrophages (Shubayev, II et al. 2009). To
overcome attack from the RES and to be biocompatible in the body, MNP must be stable and
monodisperse in water as well as having an outer coating that will render the particles nontoxic.
Therefore, the core of my MNP will be iron oxide (Fe3O4) with the outermost layer being
comprised of poly(maleic anhydride-alt-1-octadecene) bound to poly(ethylene glycol) (PMAO-
PEG) in a water suspension.
2. MATERIALS AND METHODS
2.1. SPION Synthesis
Figure 1 is a schematic of the multlayered process for the SPION used in the identification,
isolation, and reprogramming of adult SC. The figure also shows the layers needed for the
second type of SPION utilized for the conjugation to iPSC and further recognition of in vivo
SKBR3 tumors. The SPION are able to self-assemble due to the attractive forces of each
protein layer. It is necessary, therefore, that the zeta-potential of each layer must be monitored
to ensure correct assembly and particle size increase.
2.2. Synthesis of Iron Oxide Core
Figure 1
Avidin molecules for binding
CD34 antibody or SKBR3
targeting peptide
OCT4 delivery into adult
SC nucleus
OCT4
PMAO-PEG for
biocompatibility
tethered-gene
CD34 for adult SC / CD34 antibody
for iPSC + SKBR3 target peptide
BME 695-Engineering Nanomedical Systems-Final Project 2010
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Monodisperse Fe3O4 NP are synthesized with diameters from 6-30 nm as seen in Figure 2.
Figure 3 shows the structure of the water dispersible iron oxide nanocrystals made by this
strategy. The free –COOH groups in the hydrophobic ligand layer can conjugate the
biomolecules containing –NH2 groups as shown in Fig. 1. It should be noted that the other ends
of the PEG polymers (R in Fig. 3) are functional groups for the binding of other biomedical
molecules needed not already present in the layers over the core. In the actual reaction
process, 1mM FeO(OH) is mixed with oleic acid, octadecene (ODE), and heated at 320°C for a
certain period of time to produce monodisperse iron oxide nanocrystals.
These are precipitated out of the ODE by acetone, and then re-dispersed in chloroform. The
precipitation and redispersal reactions are repeated to obtain pure iron oxide nanocrystals.
Purified MNP and the PMAO-PEG are mixed in chloroform and stirred overnight at room
temperature. Next, the same volume of water is added and the chloroform is slowly removed by
rotary evaporation at room temperature. This last step causes the MNP to be dispersed in
water and results in a clear brown to dark black solution as seen in Figure 4. Excess PMAO-
PEG was removed by ultracentrifugation for 2 h and then the resultant MNP sample was
concentrated via ultracentrifugation as well. The final MNP-water solution was passed through
a 0.2 µ nylon syringe filter. To verify monodispersity, Cryo-TEM images were taken as seen in
Figure 5. The hydrodynamic diameters of the water dispersible MNP are measured through a
size exclusion chromatography as 20–60 nm long, depending on PEG length, PMAO/PEG ratio,
and iron oxide nanocrystal size. These values are generally in good agreement with the results
from dynamic light scattering (Yu, Chang et al. 2006).
Fig. 3
Figure 2
Fig. 4
BME 695-Engineering Nanomedical Systems-Final Project 2010
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2.3. Oct-4 Vector Synthesis and Expression
The primer sequences for human OCT4 used in this study are: 5’-GAC AAC AAT GAG AAC
CTT CAG GAG A-3’ and 5’-CTG GCG CCG GTT ACA GAA CCA-3’ (Integrated DNA
Technologies, Inc., San Diego, CA). Qualitative reverse transcription polymerase chain reaction
(qRT-PCR) is performed using these primers to amplify the OCT4 DNA. The mixture is first
heated at 94°C for 3 min. in a PTC-200 DNA Engine Thermal Cycler (MJ Research, Waltham,
MA). Amplification is performed for 35 cycles at 94°C for 45 sec, 55°C for 30 sec, and 72°C for
90 sec, followed by 72°C for 10 min. The PCR products are separated via gel electrophoresis
on 1.5% agarose gel bed (Tai, Chang et al. 2005). The amplified DNA is subjected to DNA
sequence analysis to confirm the correct sequence.
Once the sequence is confirmed, the DNA is ligated into the expression vector plasmid
pDNR-LIB (The Dana-Farber/Harvard Cancer Center DNA Resource Core, Harvard Medical
School, Boston, MA). Figure 6 shows the scheme for transfection and expression. The OCT4
gene-tethered product consisting of a lentivirus promoter and a GFP reporter gene sequence is
constructed as described in the literature (Loh, Hartung et al. 2010). The Lipofectamine 2000
transfection system (Invitrogen, Inc., Carlsbad, CA) is used to get the OCT4 construct into the
isolated adult SC. To prove that the transfection was successful, the green fluorescent protein
(GFP) expression protocol is used. Basically, the lentivirus (LV) promotes the OCT4
transcription, the GFP protein is expressed and can be easily seen via confocal microscopy
(Figure 6). Once GFP expression occurs, OCT-4 transcription stops.
2.4. CD34 Antibody and LTVSPWY Peptide Binding to SPION Cores
It should be clear that there are two SPION that are being used in this exercise. One type is
for the selection of adult SC and the other is for the magnetic labeling of iPSC and to enable
these cells to target SKBr3 cells in vivo.
2.5. CD34 Antibody Binding for both SPION
CD34-biotin antibody was purchased commercially (eBiosciences, San Diego, CA). These
Figure 5
BME 695-Engineering Nanomedical Systems-Final Project 2010
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antibodies are used to quantify and purify hematopoietic progenitor stem cells for research and
for clinical bone marrow transplantation. The CD34 protein is a member of a family of
transmembrane proteins that show expression on early hematopoietic and vascular-associated
tissue The streptavidin (SAv) molecules bound to the –COOH groups (see Fig. 3) are able to
bind to the biotinylated ends of the CD34 antibodies. This leaves the variable heavy chain
portions of anti-CD34 to bind to the CD34 membrane receptors on adult SC or iPSC.
2.5.1. Binding of LTVSPWY to SPION
The purified SKBr3 targeting peptide is bound to the SPION using passive adsorption according to standardized protocol Technote 204 (Bangs Labs, Fishers, IN). The appropriate amount of purified ligand is dissolved in adsorption buffer. The SPION suspension is added next to the appropriate volume of dissolved protein, and mixed gently for 1-2 hours. The solution is then incubated overnight at 4˚C, with constant mixing. The following day, the suspension is centrifuged, the supernatant removed, and the microsphere pellet is resuspended in storage buffer to desired storage concentration (often10 mg/ml). Absorbance of the suspension and separate adsorption buffer is measured with a UV/VIS spec at 280 nm.
2.6. Generation of PMAO-PEG Outer Layer
For both SPION types, the simplified process for generating the biocompatible iron oxide NP
will be done according to Yu et al (Yu, Chang et al. 2006). The outermost layer rendering the
SPION biocompatible is coated by PMAO-PEG. PMAO has a monomeric unit that can easily
react with PEG. The PMAO is mixed with PEG methyl ether in chloroform at room temperature
overnight (molar ratio of PMAO:PEG is 1:30) as shown in Figure 7.
Figure 8 illustrates how PMAO-PEG polymers form in chloroform through the anhydride
group and the amine (-NH2) group. The reaction leaves COOH groups available for
bioconjugation with biomolecules if needed. The formation of PMAO-PEG is verified using
Fourier Transform Infrared Spectroscopy (FTIR) as seen in Fig. 8.
Figure 6. Schematic illustrating how isolated
adult SC are transfected with the LV-
expression vector plasmid. GFP fluoresces in
cells that contain the plasmid as seen via
confocal microscopy.
Normal Adult SC SC treated with LV
LV
OCT4 Expression
BME 695-Engineering Nanomedical Systems-Final Project 2010
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Figure 8 illustrates how PMAO-PEG polymers form in chloroform through the anhydride
group and the amine (-NH2) group. The reaction leaves COOH groups available for
bioconjugation with biomolecules if needed. The formation of PMAO-PEG is verified using
Fourier Transform Infrared Spectroscopy (FTIR) as seen in Figure 4. The decrease of the 1775
cm−1 peak and the increase of the 1715 cm−1 peak are due to the decomposition of anhydride
and the release of –COOH, respectively. (All the other characteristic vibrations from mPEG-NH2
are seen for PMAO–PEG.)
It is very important to note that this final PEG layer should allow the zeta-potential to become
negative in addition to the R-COOH groups (and the subsequent added DNA) that are already
negatively charged. This results in an overall negative zetapotential of the SPION complexes.
2.7. SKBr3 and Targeting Peptide
In order for the iPSC to target to SKBr3 cells in vitro, the peptide sequence LTVSPWY was
generated and attached to the SPION based on its previous success of induction of oligonu-
cleotide uptake in SkBr3 human breast cancer cell line (ATCC, Manassas, VA) (Haglund, Seale-
Goldsmith et al. 2009).
Figure 7
Figure 8
BME
2.8. Isolation of PBMC
PBMC are isolated from normal volunteers using the standard venipuncture method (~30 cc
per person). The buffy coats are isolated using Ficoll
recommended procedures (GE Healthca
are pooled to form a larger sample volume with which the SPION
incubated to target the SC for magnetic cell sorting.
2.9. Magnetic Labeling of Adult SC
The binding of the SPION to the adult SC within the b
modification of another magnetic bead protocol used in the Leary lab.
conjugating the CD34-biotin to these cells. The pooled buffy coat i
buffered saline (PBS) for 10 min. at room temperature at
is resuspended in 1 ml of PBS an
and mixed gently. Incubation at 4ºC fo
described but done at 4°C.
Secondly, the SPION must be bound to the CD34 labeled adult SC for subsequent magnetic
sorting. After the second wash step, the cell pellet is resuspend
the SPION. Cells are incubated
minutes at 350 x g (or 1000 rpm) at 4°C.
to magnetic cell sorting.
2.10. QMS Sorting
Rapid cell sorting is accommodated through the use of the Quadrupole Magnetic Cell Sorter
(QMS) (Figure 7). This new cell sorting system
Magnetophoretic mobility is the relationship between the speed at
move in a magnetic field and the properties of that field.
only based on whether the cells or particles exhibit magnetophoretic mobility, but also how
much magnetophoretic mobility i
2010). Using this technology, labeled adult SC, are processed through the QMS and as seen in
Figure 8, are attracted toward the quadrupole magnet (sort boundary) and delivered through
the deflected fraction ‘b’. Unlabeled cells are sorted in fraction ‘a’.
Figure 7. QMS showing the QMS sorting
system with user interface.
BME 695-Engineering Nanomedical Systems-Final Project
PBMC are isolated from normal volunteers using the standard venipuncture method (~30 cc
per person). The buffy coats are isolated using Ficoll-Paque according to manufacturer’s
recommended procedures (GE Healthcare, Inc., Piscataway, NJ). The freshly isolated PBMC
are pooled to form a larger sample volume with which the SPION-CD34 complexes are
incubated to target the SC for magnetic cell sorting.
of Adult SC
The binding of the SPION to the adult SC within the buffy coat is a two step process and is a
modification of another magnetic bead protocol used in the Leary lab. The first step
biotin to these cells. The pooled buffy coat is washed with 1X phosphate
buffered saline (PBS) for 10 min. at room temperature at 350 x g (or 1000 rpm)
is resuspended in 1 ml of PBS and 100 µl of biotinylated CD34 antibody is added to the cells
at 4ºC for 15 min is next followed by another wash step as
the SPION must be bound to the CD34 labeled adult SC for subsequent magnetic
sorting. After the second wash step, the cell pellet is resuspend in 300 µl of PBS and
at 4ºC for 15 min, 2 ml more PBS is added and centrifuge for 5
minutes at 350 x g (or 1000 rpm) at 4°C. Cells are resuspended in 1 ml of fresh cold PBS prior
Rapid cell sorting is accommodated through the use of the Quadrupole Magnetic Cell Sorter
This new cell sorting system sorts cells based on magnetophoretic mobility.
Magnetophoretic mobility is the relationship between the speed at which a particle (or cell) will
move in a magnetic field and the properties of that field. The QMS is capable of sorting cells not
only based on whether the cells or particles exhibit magnetophoretic mobility, but also how
magnetophoretic mobility is exhibited by the particles or cells (Reece, Sanders et al.
Using this technology, labeled adult SC, are processed through the QMS and as seen in
, are attracted toward the quadrupole magnet (sort boundary) and delivered through
the deflected fraction ‘b’. Unlabeled cells are sorted in fraction ‘a’.
QMS showing the QMS sorting
Figure 8.
deflected toward the
magnetic field. Cells are
pulled
to form sort boundaries that
are produced by the sample
flow rate.
Final Project 2010
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PBMC are isolated from normal volunteers using the standard venipuncture method (~30 cc
Paque according to manufacturer’s
re, Inc., Piscataway, NJ). The freshly isolated PBMC
CD34 complexes are
uffy coat is a two step process and is a
The first step is
s washed with 1X phosphate
or 1000 rpm). The cell pellet
is added to the cells
is next followed by another wash step as
the SPION must be bound to the CD34 labeled adult SC for subsequent magnetic
PBS and 100 µl of
and centrifuge for 5
in 1 ml of fresh cold PBS prior
Rapid cell sorting is accommodated through the use of the Quadrupole Magnetic Cell Sorter
magnetophoretic mobility.
which a particle (or cell) will
is capable of sorting cells not
only based on whether the cells or particles exhibit magnetophoretic mobility, but also how
Reece, Sanders et al.
Using this technology, labeled adult SC, are processed through the QMS and as seen in
, are attracted toward the quadrupole magnet (sort boundary) and delivered through
Figure 8. How cells are
deflected toward the
magnetic field. Cells are
pulled towards the magnet
to form sort boundaries that
are produced by the sample
flow rate.
BME 695-Engineering Nanomedical Systems-Final Project 2010
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2.11. Reprogramming and Magnetic Labeling of iPSC
After transfection of the adult SC with the OCT4 construct, cells are allowed to grow under
normal cell conditions (37°C with 5% CO2 injection) for several rounds of replication over 35
days (Loh, Hartung et al. 2010). Afterwards, cells were analyzed via immunohistochemistry and
flow cytometry for presence of CD34 positivity. Additionally, DNA sequencing is performed to
verify the presence of OCT4 that will confirm the reprogramming of the adult SC into iPSC.
Once the sequencing yields a positive result, the iPSC are incubated as previously described in
section 2.9 but with the SPION-CD34-LTVSPWY particles. This confers the iPSC to be
magnetic and contain the targeting capability needed for binding to SKBr3 cells.
2.12. In vitro Cytotoxicity Analysis
CometAssay (Trevigen, Inc., Gathersburg, MD) assay was performed according to the
manufacturer’s recommendations on transfected cells to observe any cytotoxic effects of both
types of NP – especially prior to introduction of the SPION-CD34-LTVSPWY complexes.
2.13. In vivo Toxicity Assay
The Leary lab has established protocols for the administration of SKBr3 cells into nude mice.
These same procedures are followed for this study. SKBr3 cells at a volume of 100 µl are
injected into the shoulder pads of nude mice and allowed to metastasize. Tumors are
monitored until they are palpable (typically anywhere from 5 days to 2 weeks post injection).
After the tumors are visible, the SPION-CD34-LTVSPWY particles (100 µl) are intravenously
injected into healthy mice via the tail vein as per previously established protocols performed in
the Leary lab. The mass of each mouse is monitored during 4 weeks after tail vein injection. The
mice are then sacrificed and the kidney, liver, and spleen tissues are stained using
haematoxylin and eosin and examined by a pathologist.
2.14. In vivo Biodistribution Tests
After tumors are visible as described above, iPSC labeled with SPION-CD34- LTVSPWY
particles are injected via tail vein. For in vivo degradation and biodistribution studies, mice are
imaged under anaesthesia before injection and 1h, 2h, 4h, 8h, 24h, 1 week and 4 weeks post
injection using the IVIS 200 Imaging System (Caliper LifeSciences, Mountainview, CA)
(examining signal from Fe3O4). The mice are sacrificed after 4 weeks and the organs (bladder,
brain, heart, kidney, lymph nodes, liver, lung, skin, spleen and bone marrow) are collected and
examined.
3. EXPECTED BIOLOGICAL RESULTS
Figure 8 shows the construction of the two types of SPION that are being used in this study.
The study takes the experiment from the generation of the SPION (including all layers) to the
labeling and sorting of adult SC, to the reprogramming of the adult SC into iPSC and finally the
injection of the iPSC into a live mouse to try to target established breast cancer tumors. The
BME 695-Engineering Nanomedical Systems-Final Project 2010
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feasibility studies and other preliminary studies for cytoxicity of SPION have been conducted in
the Leary lab before (Haglund, Seale-Goldsmith et al. 2009). Further, SPION have a core that
has been tolerated well in humans for many years for MRI diagnostics. However, it is a
necessity to test for cytotoxicity when any molecules are added to nanoparticles. The Comet
assay will be performed on both adult SC and iPSC after uptake of their respective NP.
Preliminary studies with adult SC show that there should not be a problem with apoptosis after
exposure to the SPION complexes as seen in Figure 9d.
Preliminary studies of SPION-CD34 particles show that after tail vein injection of the nude
mice, the MNP lodge in the liver (Figure 10), however, particles are cleared after 4 weeks. The
tissues were examined by a veterinary pathologist and the report came back that they were “not
remarkable” and did not show any signs of inflammatory response.
a.
b.
Ligand
-COOH Groups
PMAO-PEG
LV GFP
Figure 8. a. Construction and anatomy of the DNA-tethered SPION. b. Schematic of the
DNA construct used to assess transfection and LV activity. NOTE THAT THE SPION USED IN
THE IPSC PART OF THE EXPERIMENT DO NOT HAVE THE DNA, BUT INSTEAD HAVE THE
TARGETING PEPTIDE FOR THE IN VIVO SKBR3 TUMORS.
Figure 9. COMET assay performed on sorted adult SC cell samples: (a) untreated adult SC
(negative control), (b) SC treated with 100µM hydrogen peroxide for 18 hours (positive control), (c)
cells exposed to 0.1 mg/mL bare ferric oxide nanoparticles, and (d) cells exposed to 0.1 mg/mL
SPION-CD34 particles.
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In Figure 11, we see that Faure et al (Faure, Dufort et al. 2009) has done preliminary
biodistribution studies on most of the organs of a nude mouse model after subjection to different
PEGylated MNP. We see that PEG250-COOH resides mostly in the kidney, while an aminated
PEG MNP is found in the spleen liver and uterus of female mice. I expect that the SPION
complexes tested here will be similar as far as those particles that do not directly bind to SKBr3
tumor cells. TEM/SEM will be needed to really assess all tissues to see where “loose” SKBr3
tumor cells are residing. It is my hope that the targeting peptide will find these circulating tumor
cells and bind to them so that they may be detected.
For the targeting studies, the SKBr3 tumors have to be palpable and visible (Figure 12)
before the iPSC-SPION-CD34-LTVSPWY particles are injected into the tail vein. In the Leary
lab we have shown that some quantum dots home to the SKBr3 cells (Haglund, Seale-
Goldsmith et al. 2009), so it is very feasible to assume that the SPION-CD34-LTVSPWY will
home to the tumors as well.
There have also been preliminary biodistribution studies involving MNP (Leary lab
unpublished data) in the nude mouse. Images were taken using the IVIS Spectrum device that
uses noninvasive quantitative 3D molecular imaging via transmission fluoresecence and
reflectace fluorescence. In Figure 13 we see MNP that have been homing to bladder cancer
tumors in mice. We also see positive results for MNP localization in ex vivo tissue sections.
With these promising data, I believe that we should be able to successfully image the
programmable NP from this study throughout the mouse.
Figure 10. Preliminary results of organ
sections after exposure of SPION-CD34
particles. Images show tissue sections of
select organs with no morphological
aberrations. Arrows point to places in liver
where SPION are lodged. These MNP seem to
have cleared the body without harming the
animal.
Figure 11. Preliminary PEGylated
MNP biodistribution study
showing where these particles
reside after tail vein injection in a
nude mouse model.
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4. DISCUSSION
This is a very complicated study with many different variables at every level. However, if this
study is to succeed, several steps must take place. I must monitor the assembly of the MNP at
every step using not only DLS that was mentioned in this proposal, but also X-ray Photoelectron
Spectroscopy, in addition to zetapotential. This will confirm that my MNP are indeed what I
think they are in terms of how they are layered and what the cores are composed of exactly.
While there have actually been preliminary data that support my proposal, it would be good to
try another animal model such as a primate instead of a mouse. The nude athymic mouse is a
cross between an in vitro and in vivo model since it has no immune system. It is imperative to
try to find an animal, such as a primate species, that would more closely mimic the human
immune system and that would provide target organs more strongly related to our own.
While this proposal mentions in the Abstract that this type of nanomedical device can
regulate the metastisis of tumors, the data provided herein have not supported this statement.
Nevertheless, OCT4 along with other genes listed previously, do have the ability to not only
reprogram adult SC into iPSC, but to allow for the cessation of tumor growth by causing cells to
enter the primitive state, differentiate, and then apoptose normally. This is a much more
bioenvironmentally safe way to attack a cancer cell than present chemotherapy or radiation
therapy.
It would also be prudent to pursue different shapes of nanoparticles to increase the
circulation time of the MNP. This is extremely important when trying to find that rare tumor cell
disseminating outside of tumor sites (especially in the leaky vasculature surrounding tumors).
Figure 12. Preliminary
PEGylated MNP biodistribution
study showing palpable SKBr3
tumors (left) and the same
tumors prior to excision (right).
Figure 13. Biodistribution of
MNP in live mouse models (left).
MNP has homed and bound to
bladder cancer tumors. Ex vivo
tissue sections confirming the
live imaging (right).
BME 695-Engineering Nanomedical Systems-Final Project 2010
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Nanorods and nanotubes have longer circulation time in other studies and the binding chemistry
is not that different than what has been proposed in this paper. Also, these shapes provide
greater surface area with a very small volume that will allow for binding of several biomolecules.
Finally, this type of approach, once optimized, will allow cancer treatments to become less
invasive, less costly, and provide the patient with a higher quality of life. These nanomedical
devices do not only have the potential to kill tumors or reprogram cells into ones that will
differentiate and apoptose properly, but with the gene thereapy/drug they deliver precisely to
their target, patients will experience far less side effects that can only add to that higher quality
of life and allow their immune system to recover much more quickly. This is the medical
treatment of the future and shows great promise as being a cheaper, cleverer, a more reliable,
and more available method of treatment that now currently exists.
5. LITERATURE CITED
Faure, A.-C., S. Dufort, et al. (2009). "Control of the in vivo Biodistribution of Hybrid Nanoparticles with Different Poly(ethylene glycol) Coatings." Small.
Haglund, E., M.-M. Seale-Goldsmith, et al. (2009). "Design of Multifunctional Nanomedical Systems." Annals of Biomedical Engineering.
Ikehata, H., T. Masuda, et al. (2003). "Analysis of mutation spectra in UVB-exposed mouse skin epidermis and dermis: Frequent occurrence of C→T transition at methylated CpG-associated dipyrimidine sites." Environmental and Molecular Mutagenesis 41(4): 280-292.
Loh, Y.-H., O. Hartung, et al. (2010). "Reprogramming of T Cells from Human Peripheral Blood." Cell Stem Cell 7: 15-19.
MacDougall, M. (2008, 28 October 2010). "OCT-4." from http://en.wikipedia.org/wiki/Oct-4. Park, I. H., R. Zhao, et al. (2008). "Reprogramming of human somatic cells to pluripotency with
defined factors." Nature 451(7175): 141-146. Reece, L. M., L. Sanders, et al. (2010). High-Throughput Magnetic Flow Sorting of Human Cells
Selected on the Basis of Magnetophoretic Mobility. SPIE. Shubayev, V. I., T. R. P. II, et al. (2009). "Magnetic nanoparticles for theragnostics." Adv Drug
Deliv Rev. 61(6): 467-477. Staerk, J., M. M. Dawlaty, et al. (2010). "Reprogramming of Human Peripheral Blood Cells to
Induced Pluripotent Stem Cells." Cell Stem Cell 7: 20-24. Tai, M. H., C.-C. Chang, et al. (2005). "Oct4 expression in adult human stem cells: evidence in
support of the stem cell theory of carcinogenesis." Carcinogenesis 28(2): 495-502. Wang, A. Z., V. Bagalkot, et al. (2008). "Superparamagnetic Iron Oxide Nanoparticle–Aptamer
Bioconjugates for Combined Prostate Cancer Imaging and Therapy." ChemMedChem 3: 1311-1315.
Yu, W., E. Chang, et al. (2006). "Aqueous dispersion of monodisperse magnetic iron oxide nanocrystals through phase transfer." Nanotechnology 17: 4483-4487.