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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

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Page 1: Proposal for Magnetic Labeling of Stem Cells for Subsequent …web.ics.purdue.edu/~jfleary/nanomedicine_course_2011... · 2010-11-30 · BME 695-Engineering Nanomedical Systems-Final

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

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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,

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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

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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

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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

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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

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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

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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.

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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

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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).

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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.