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Systems for Intracellular Delivery of Drugs

#3175


Novel Self-Organized Macromolecular Systems for Delivery of Drugs and Genetic Material into Living Cells

Tech Area / Field

  • MED-DRG/Drug Discovery/Medicine
  • CHE-POL/Polymer Chemistry/Chemistry
  • MED-OTH/Other/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology

Status
8 Project completed

Registration date
15.12.2004

Completion date
16.06.2009

Senior Project Manager
Visser H

Leading Institute
Mendeleev Chemical Technological University, Russia, Moscow

Supporting institutes

  • Moscow State University / Department of Chemistry, Russia, Moscow

Collaborators

  • University of Lund / Center for Chemistry and Chemical Engineering, Sweden, Lund\nUniversity of Crete, Greece, Heraklione

Project summary

Design of new approaches for drug and gene delivery has been a key point for biology and medicine for the last thirty years. However, in spite of impressing achievements, a number of principal questions remain insoluble. This concerns, in particular, lyyophilization of drugs and control for their circulation time in organism as well as protection of drugs from environmental, e.g. because of their biodegradation or uptake by cells.

Use of polymers, capable of drug/gene immobilization, is a promising way to overcome these difficulties. Nanoscale containers: polymer micelles and complexes, with incorporated bioactive additives can be thus prepared. Physico-chemical and biomedical parameters of such supramolecular constructs can be varied within wide limits. At the same time, ionizable polymers are often characterized by a high level of cytotoxicity, meanwhile non-charged polymers, e.g. ethylene oxide/propylene oxide block-copolymers (Pluronics) demonstrate a low affinity to biological membranes. This requires the search of new polymeric carriers for drugs and genes.

The specific goals of the Project are:


1) to synthesize a series of novel amphiphilic water soluble polymers for biomedical applications,
2) to clarify the factors controlling their self-organization in aqueous solutions,
3) to quantify the binding of an antitumor drug Doxorubicin (Dox) and DNA with polymeric micelles, and
4) to study the interaction of Dox and DNA loaded micelles with cell and model lipid membranes.

Hydrophobic fragments of such polymers will be represented by aliphatic radicals, hydrophilic by water-soluble biocompatible oligomers with controllable degree of polymerization. The latter will contain a few amount of functional monomers. for covalent immobilization of vector molecules to address the whole constructs to target cells. Cationic units in hydrophilic corona is expected to favor complexation of negative DNA with polymeric micelles and binding of the resulting constructs to biological membranes. Anions groups will stabilize the micelles in biological environment.

The project will be performed by the group of Prof. Shtilman from Mendeleyev Russian University of Chemistry and Technology (RMUCTR) and the group of Prof. Yaroslavov from Lomonosov Moscow State University (MSU). The interest of the first concerns mainly the synthesis of water-soluble polymers. The RMUCTR group participants have experience in the defense area. Second group is active in the study of polymer solutions as well s polymer interaction with biological membranes. Publications relevant to the project are listed below:


V.P. Torchilin, M.I. Shtilman, V.S. Trubetskoy, K. Whiteman, A.M. Milshtein, Amphyiphyilic vinyl polymers effectively prolong liposome circulation time in vivo, Biochim. Biophys. Acta, 1195 (1994)), 181-184.
T.K. Bronich, S.V. Solomatin, A.A. Yaroslavov, A. Eisenberg, V.A. Kabanov, A.V. Kabanov, Steric stabilizatuion of negatively charged luiposomes by cationic graft copolymers, Langmuir, 16 (2000), 4877-4881.
V.P. Torchilin, T.S. Levchenko, K.R. Whiteman, A.A. Yaroslavov, A.M. Tsatsakis, A.K. Rizos, E.V. Michailova, M.I. Shtilman Amphiphilic poly-N-vinylpyrrolidones: Synthesis, properties and liposome surface modification, Biomaterials, 22 (2001), 3035-3044.
V.A. Kabanov, A.A. Yaroslavov, What happens to negatively charged lipid vesicles upon interacting with polycation species?, J. Controlled Release, 78 (2002), 267-271.
A.A. Yaroslavov, A.A. Efimova, V.I. Lobyshev, V.A. Kabanov, Reversibility of structural rearrangements in the negative vesicular membrane upon electrostatic adsorption/desorption of the polycation, Biochim. Biophys. Acta, 1560 (2002),, 14-24.
A.A. Yaroslavov, O.Ye. Kuchenkova, I.B. Okuneva, N.S. Melik-Nubarov, N.O. Kozlova, V.I. Lobyshev, V.A. Kabanov, F.M. Menger, Effect of polylysine on structure and permeability of negative vesicular membrane, Biochim. Biophys. Acta, 1611 (2003), 44-54.
O.O. Krylova, N.S. , Melik-Nubarov, G.A. Badun, A.L. Ksenofontov, F.M. Menger, A.A. Yaroslavov, Pluronic L61 accelerates flip-flop and transbilayer doxorubicin permeation, Chem. Eur. J., 9 (2003), 3930-3936.

Synthesis of polymers will be performed using the procedures designed by the project participants. Physico-chemical characteristics of polymers will be controlled by element and functional analysis as well as by UV/Vis, IR, NMR and EPR spectroscopy.

Micelle formation in water solutions of the amphiphilic polymers will be studied with a fluorescent probe method, size of micelles with quasi-elastic light scattering, and their surface echarge with laser microelectrophoresis. The fluorescence approach will be also use for the study of solubilization activity of polymer micelles towards Dox and model dyes. An ability of polymer micelles to complex with two-strained DNA molecules will be examined, DNA modified with fluorescent ethydium bromide being used.

The central part of the project is to examine binding of the prepared polymer micelles with incorporated drugs, or complexed with DNA, to biological membranes. For this, spherical bilayer lipid vesicles (liposomes), lipid monolayers at the water/air interface and native cells will be used.

It is expected that the micelle/liposome interaction will be accompanied by incorporation of hydrophobic polymer radicals into the vesicular membrane. This can result in structural rearrangements in the membrane and influence its permeability. The lateral segregation in mixed lipid/polymer membranes will be studied by a differential scanning calorimetry. The membrane permeability will be controlled by measuring conductivity and fluorescence of solutions (towards simple salts and doxorubicin, respectively). Size of particles in the system will be determined by quasi-elastic light scattering. The latter will be also used to estimate a thickness of the hydrophilic corona formed by interacting polymers. An additional important information about structural organization of lipid membranes, including those with incorporated polymers, will be obtained from the behavior of lipid and mixed lipid/polymer monolayers.

Cytotoxicity of polymers will be controlled with a methyl-tetrazolium blue assay, intracellular localization of penetrated doxorubicin and model fluorescent probes by fluorescent microscopy. Human erythroleucemia K562 and human breast carcinoma MCF-7 cells will be used.

Interaction of DNA/polymer complexes with cells will be studied using SR1 cells. DNA delivery will be monitored using fluorescent microscopy. For this, DNA labeled with an intercalator dye with a bright green fluorescence will be used to follow uptake of DNA molecules. To study expression of genes, plasmid DNA containing gene of green fluorescent protein (GFP) will be used, and appearance of green fluorescence in cytoplasm of the transfected cells will be studied.

Thus, a series of novel functionalized amphiphilic polymers will be synthesized, and a correlation between structure and properties of polymer micelles with their membrane activities and biological effects will be disclosed.

The project will allow a group of qualified Russian researchers from polymer chemistry, membranology and cell biology, involved earlier in the defense-related programs, to use their potential for design of new polymers for biomedical applications. In the course of project realization, the Russian participants will contact with leading European researchers, visit foremost laboratories and present the results at international conferences. The hope is to synthesize industry-oriented polymers, thus developing the principles of market economy in Russia.


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