Molecular nanosyringe for environment-selective
delivery of drugs and imaging probes into tumor
(In collaboration with Dr.
Reshetnyak, URI, and Dr. Engelman,
Many diseases, such as cancer, atherosclerosis,
inflammation, heart infarction or stroke create an acidic extracellular
environment. We have engineered a molecular syringe that enables to inject drug
or imaging probe into cell at acidic pH. The molecular syringe consists of the
water-soluble peptide that inserts itself into membrane at pH < 7.0 with its
C-terminus going into the cell, a disulfide link, cleavable only in cytoplasm,
and therapeutic or imaging agent. We called this peptide – pHLIP (pH Low
Insertion Peptide). We demonstrated that pHLIP was able to translocate the
fluorescent dyes and cell-impermeable toxin, phalloidin, through the membrane
of cancer cells and released them in cytoplasm. Phalloidin induced
stabilization of actin cytoskeleton and inhibition of cell contractility. The
molecular syringe is of nanoscale size and can be easily delivered by blood
flow to the diseased tissue, where low pH will trigger the injection of drug or
probe into the cells. Since we know the mechanism of translocation of
cargo-molecules across the cell membrane by the peptide we can tune its
properties by varying the sequence. The discovered peptide can be used not only
for injection of molecules into the cells but also for specific labeling of
their surface with imaging probes or with molecules (like tissue factor,
mannose and others) that can induce the immune attack of these cells.
1) pHLIP –
new delivery agent specifically targeting diseased tissue with acidic
environment
pHLIP offers a new way to deliver molecules inside the
cell – it is insertion of membrane peptides in lipids bilayer. In collaboration
with Don Engelman Lab we propose to elucidate the general principles underline
the insertion process. We know that there is a restriction in size and polarity
of cargo molecules to be translocated through the membrane (for example, pHLIP
can not translocate 20-mer ODN). The major question we would like to answer:
what are the properties of molecules, which could be translocated by pHLIP.
In parallel to the in
vitro investigation of interaction of pHLIP with liposomes we propose to
study the ability of pHLIP to translocate functional moieties in cultured live
cells and in vivo. There are 3 major
classes of functional cargo molecules, which especially attract my attention:
a) PNA – as a gene regulation agent. PNA itself has poor
membrane permeability, however pHLIP significantly enhances its translocation
and antisense activity. We are collaborating with the PNA inventor, Professor Peter E. Nielsen,
b) phalloidin – cell–impermeable toxin.
pHLIP-S-S-phalloidin could be considered as a potential new anticancer drug
that can prevent cell migration and metastasis (phalloidin was not considered
for therapeutic purposes, since it is cell impermeable, and only being attached
to pHLIP it can be delivered into cell and stabilize actin cytoskeleton). The
work is supported by 3-years DoD research grant (PI - Dr. Reshetnyak ).
c) cell permeable drugs, such as doxorubicin or taxol. We
know that pHLIP does not insert in cell membrane at normal pH, so I assume that
it can prevent the entry of drug into cell in healthy tissue with normal
extracellular pH, however it will promote drug delivery in diseased tissue with
low extracellular pH. The technology can offer the enhancement of drug efficacy
and reduction of side effects of anti-cancer therapy.
2) The
specific tethering or assembling of nanoparticles at surface of cancer cells in
vivo.
We show that modification of the N-terminus of pHLIP
does not affect the insertion process; therefore many kinds of nanoparticles
containing therapeutic, diagnostic and/or cell regulation agents could be
specifically delivered to tumor upon their conjugation to N-terminus of pHLIP.
The list of extracellular cargo molecules includes (but not limited): imaging
probes; immunogenic agents; radiation, thermo- and photo- sensitizers; radio-
and chemo- therapeutic agents; boron for neutron-capture therapy; quantum dots,
carbon nanotubes, nanospheres, magnetic particles, dendrimers and liposomes.
Currently, we are exploring the possibility to deliver
carbon nanotubes to the cancer cells via pHLIP. Carbon nanotubes can serve as
nano-heaters as a result of their illumination at 800 nm wavelength. The work
is supported by DoD grant 2007-2010 (PI
– Dr. Andreev).
3) pHLIP for
neutron-capture therapy
pHLIP can deliver variety of molecules to the diseased
tissue. The advantage of pHLIP use is its ability to concentrate the
therapeutic or diagnostic agents in site of disease, where it can stay for
several days. The delivery of radiopharmaceuticals to the site of disease in
concentration appropriate to produce desirable effect is one of the main issues
in neutron-capture therapy. Recently, we started a new project for the use of
pHLIP in delivery of boron or Gd to the cancer tissue in vivo. The experiments will be carried out on the RI reactor,
which generates thermal neutron that are used for the irradiation of boron or
Gd. Also some experiments is planned to do in collaboration with our colleagues
from the MIT Nuclear Reactor Lab. Joined URI, Yale
and MIT grant has been submitted to Depertment of Defense (I am Co-PI, PI –
Dr.Reshetnyak)
4) pHLIP for
the diagnostic of early pathology
We show that pHLIP can map acidity in tumors and site
of inflammation in vivo. The signal
is very stable, since pHLIP being inserted in membrane is protected from the
proteases attack and stays in membrane for several days (it was possible to
image tumor for 8 days by fluorescence and 24 hours by PET methods, the later
depends mostly on a short half-life time of isotopes used). Moreover pHLIP
consisting of D-amino acids exhibits an excellent performance. The major goal
of the project is to explore the possibility of pHLIP use for cancer
diagnostics and particularly for the early detection of pathology. We are working
on various tumor models using whole-body fluorescence imaging techniques. We
are developing pHLIP constructs conjugated with MRI and PET probes, some of
them are currently tested in collaboration with specialists in MRI (UCSF, San
Francisco) and PET imaging (Washington University, St. Lois). PET imaging results
were presented on 17th International Symposium on Radiopharmaceutical Sciences,