Molecular nanosyringe for environment-selective delivery of drugs and imaging probes into tumor

(In collaboration with Dr. Reshetnyak, URI, and Dr. Engelman, Yale University).

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, University of Copenhagen, Denmark. This work is supported by NIH R21 grant (PI Dr. Andreev).

b)      phalloidin cellimpermeable 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, Aachen, Germany.