Specific Aim 1. Create targeted nanoparticles ca. 50 nm diameter for the effective MR imaging of
lymphoma that have sizes and surface properties (including surface presentation of protein targeting
ligands) that are analogous to those used with the targeted therapeutics so that the imaging and
therapeutic agents have the same biodistribution in mammals.
Assembly of Targeted Iron Oxide Nanoparticles
We have used transferring receptor (TfR) as a target for delivering nucleic acid therapeutics to
tumors (Section B.4.c). Here, we will create new targeted MRI agents that will rely on tumor cell
surface receptor binding for localization within the animal. Transferrin receptor has previously been
identified as a potential target for molecular imaging of human cancer (Hogemann-Savellano et al.,
2003). Weissleder and co-workers have shown that MR imaging of tumors can be achieved with
transferrin-containing, dextran coated iron oxide nanoparticles (Weissleder et al., 2000; Moore et al.,
1998). Dextran coated iron oxide particles of ca. 60 nm diameter (Feridex IV) are FDA approved for
liver imaging (Varallyay et al., 2002). Dextran coated iron oxide particles of ca. 40 nm diameter have
been conjugated with Tf binding to partially oxidized dextran with subsequent reduction of the formed
Schiff’s bases (Moore et al., 1998).
These particles denoted Tf-MION (holo Tf (iron loaded Tf)
conjugated with monocrystalline iron oxide nanoparticle), were able to bind TfR, although with low
affinity.
To overcome the low affinity, TfR mutants were employed that gave higher affinities.
Successful whole animal (mice) imaging of tumors was achieved with Tf-MION when high affinity TfR
were expressed on the tumor cells (Weissleder et al., 2000). When creating nanoparticles with
protein targeting ligands, it is known that the protein should not be attached directly to the particle
surface. This configuration yields low binding affinities to cell surface receptors because of the steric
hindrance of the particle. Shaffer and Lauffenburger (1998) have shown with EGF that the protein
must be tethered from the particle by at least 3 nm to have binding affinities unaffected by the
particle. This result has been verified by others. In our work, we tether targeting ligands with PEG for
this purpose and have shown that with Tf, we can achieve “effective” affinities of Tf-containing
particles that exceed the affinity of Tf alone due to multi-Tf binding on the particle (see Fig. 40).
We will prepare iron oxide nanoparticles by the synthetic methodology of Park et al. (2004).
These workers developed large-scale syntheses of monodisperse nanocrystals of iron oxide. For
example, monodisperse crystals of diameters 9 nm, 12 nm, 16 nm and 22 nm have been prepared.
We will follow their procedure to create monodispersed iron oxide nanoparticles. We will confirm the
size and polydispersity by DLS and TEM. Next, we will coat these particles with the cyclodextrincontaining polymer instead of dextran (see
schematic). We will be able to confirm the
coating by measuring the zeta potential of
the particle surface, the increase in particle
size by DLS and TEM and quantitate the
amount of polymer by performing thermal gravimetric measurements that will give weight lost upon
combustion in air of the polymer fractions. All of these methods are available in the Davis lab.
Additionally, we will implement the method of Reynolds et al. (2005) to determine the nanoparticle
iron oxide core weight. The combination of these results will allow us to completely define the CDpolymer coated iron oxide nanoparticles. We will add Tf-PEG-AD (see Fig. 39) to assemble PEGtethered holo Tf onto the iron oxide particles (see schematic above). By fluorescently labeling Tf
(method already developed by us (Bellocq et al., 2003b)), the number of Tf ligands contained on each
particle can be quantitated. The ligand density can be varied using this methodology (Bellocq et al.,
2003b). This synthetic methodology will allow for the investigation of the effectiveness of the MR
imaging as a function of particle diameter, surface charge (see Fig. 33) and ligand density.
Additionally, this methodology will allow for the preparation of iron oxide MRI agents with sizes and
surface properties that are the same as the therapeutic delivery particles.
Other targeting ligands, e.g., anti-CD19 antibodies, will be incorporated into the iron oxide
particles by creating L-PEG-AD conjugates analogous to Tf-PEG-AD. The chemistries necessary for
creating these conjugates are all readily available and proven.
The strategy outline above utilizes known components. Alternatively, the protein targeting
agents can be added to the end groups of the cyclodextrin-containing polymers prior to association
with the iron oxide particles as illustrated in the schematic shown below. When the CD polymer is
synthesized, it is end capped to top the polymerization reactions.
Here, we will stop the
polymerization with a heterobifunctional PEG (obtained from Nektar).
One end of the
heterobifunctional PEG will end cap the polymer while the other end will provide the appropriate
functional group for covalently attaching the protein (analogous to making the protein-PEG-AD
described previously by us). The protein-CD polymer conjugates will then be combined with the iron
oxide nanoparticles and characterized as described above. These nanoparticles will have covalently
attached targeting ligands and will be compared to those with targeting ligand associations by AD-CD
inclusion complex formation.
Specific Aim 2. Create targeted nanoparticles with the same surface properties as those prepared in
Specific Aim 1 that also contain payloads that are able to provide intracellular, molecular-level
readouts of therapeutic targets.
There are numerous examples of molecular imaging in living subjects (Massoud and Gambhir,
2003). Here, we will develop new agents for use with MRI methods. In Specific Aim 1, targeted iron
oxide particles will be prepared.
These MRI agents will provide for the molecular imaging of
lymphoma surface receptors. In Specific Aim 2, we will provide “payloads” that upon endocytosis will
also give molecular imaging of intracellular targets and/or markers of the molecular-level responses of
the cell to the therapeutic agents.
Assembly of Targeted, Cross-Linked Iron Oxide Nanoparticles
Weissleder and co-workers have shown that nanoparticles (3-5 nm) of iron oxides can be
cross-linked with dextrans (Wunderbaldinger et al., 2002) and other biomolecules such as
oligonucleotides and peptides (Perez et al., 2002; Zhao et al., 2003; Perez et al., 2004) to give
assemblies that are 200-300 nm in size.
The key result is that the spin-relaxation time (T1) is
independent of whether the iron oxide particles are assembled or not while the relaxation rate (1/T2)
is proportional to the cross-sectional area of the particle. Thus, T1 can be used to measure the
concentration of the total iron oxide and (1/T2) to test for assembly and disassembly.
Numerous intracellular targets exist in cancer cells.
For example, McIntyre et al. (2004)
developed a fluorogenic proteolytic beacon for the in vivo imaging of tumor-associated matrix
metalloproteinase-7 (MMP-7) activity. This molecular beacon “turns on” when MMP-7 cuts a specific
peptide (RPLALWRS).
Detection of MMP-7 activity was achieved in mice from subcutaneous
xenograft tumors. We choose to develop MRI rather than optical techniques because they can be
readily translated from animals to humans.
As an example of how we will create targeted, crosslinked iron oxide nanoparticles, a specific set of ligands and
peptides will be discussed next. These serve as an illustrative
example and we will not limit our investigation to these specific
biomolecules.
Using very small iron oxide nanoparticles (sub-10 nm)
prepared by the synthetic methodology of Park et al. (2004), two
different strategies of assembly will be investigated (illustrated in
schematic). The small particles can be cross-linked with a peptide (for example RPLALWRS for
monitoring MMP-7 activity), coated with the CD-containing polymer and the targeting ligand added.
Alternatively, the individual sub-10 nm particles could be coated with a CD-containing polymer and
the coated particles cross-linked with the peptide prior to addition of the targeting ligand. Through
MRI experiments, we will test these assemblies and determine if one is preferable. We will explore
the effects of assembly size and other physical properties (assemblies can be characterized by
methods described previously in Specific Aim 1) on the quality of the imaging. One issue that needs
to be carefully investigated is size. Weissleder and co-workers prepared assemblies that are too
large for in vivo use in our opinion (200-300 nm). Here, we will work in the size range of 100 nm or
below.
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Method of Determining
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Magnetic Sensors for Protease