Virus-like particle (VLP) nanotechnology for the generation of drug and diagnostics delivery tools
Project is being carried out within the ERDF 220.127.116.11. activity „Support for Science and Research”
Project agreement Nr. 2010/0314/2DP/18.104.22.168.0/10/APIA/VIAA/052
Project duration: 36 months
Project funding: 266419 LVL
Project leader: Dr. biol. Kaspars Tārs
Collaboration partners - „LabochemLV Ltd.” and „Rīgas Austrumu klīniskā universitātes slimnīca Ltd.”
Specific and efficient delivery of therapeutic and diagnostic materials to the appropriate organs, tissues, and cell types is one of the most urgent problems of medicinal nanotechnologies. For example, the anti-cancer chemotherapeutic drugs are highly toxic also for normal organ cells. Therefore, dosage of such drugs is limited, but side effects are ineradicable. The problem could be resolved theoretically by packaging of the appropriate drugs into nanocontainers and selective delivery of the latter directly to the cancer cells. Besides anti-cancer drugs, magnetic, diamagnetic, and paramagnetic nanoparticles, contrast reagents, DNA and RNA genes and regulatory elements, antibiotics, therapeutic proteins and peptides could be packaged into such nanoconteiners.
Viruses are obliged to enter host cells and to transport their genomes into the latter during viral life cycle. Therefore, viruses can be considered to be natural nanocontainers, which are adjustable for the use in the form of delivery vehicles, as it is realised now in gene therapy. However, application of whole viruses for therapeutic and diagnostic purposes is connected with high risks and heavy practical problems of viral production. Indeed, it is not always possible to provide viral genome with mutations, which would be able to ensure packaging of active substances and decoration of the virions with appropriate addresses, from one side, and normal viral replication and self-assembly, from other side. Moreover, viral production needs special safety precautions, in order to avoid potential health care (human and animal viruses) and pollution of the environment (plant and microorganism viruses) risks.
Virus-like particles (VLPs) are the most reliable basis for the specific delivery of a broad spectrum of the nanomaterials. VLPs are artificially constructed viral envelopes or capsids, which bear structural and morphological resemblance to the appropriate viruses, but do not contain viral genomes and/or their fragments. Therefore, VLPs are not infectious and much safer for medicinal application than initial viruses. Since VLPs can be cultivated in recombinant bacterial and yeast strains with high level expression of the target genes, production of VLPs is profitable economically. Packaging of VLPs with immunostimulatory DNA oligonucleotides (so-called CpG elements) is used now in the generation of vaccine candidates undergoing clinical trials. For some time now, viral particles (e.g., some plant viruses) with packed materials are used in laboratory practice for generation of nanoparticles for non-biological applications, including construction of semi-conductor elements.
VLPs are easily subjected to modifications (stabilisation, gene fusion, chemical coupling of biomolecules) in order to construct so-called chimeric VLPs supplied with specific addresses for the recognition of definite cell types.
The project tasks: elaboration of (1) technologies for VLP packaging with the appropriate materials and their transportation to leukocytes, hepatocytes, and cancer cells, (2) product prototypes, namely, VLPs packaged with different materials for diagnostic and therapeutic purposes.
VLPs are chosen for packaging of following therapeutic substances: low molecular weight drugs (antibiotics, first of all, anti-cancer drug doxorubicin) and thermotherapy agents (gold un iron oxide nanoparticles). Different contrast reagents, irone oxide, nanogold, nanolantanoides, fluorescent markers, marker genes un marker proteins are intended for usage as diagnostic agents to be packaged in VLPs.
Hepatocytes, leukocytes, T-cell lymphomas, as well as HER2 antigen exposing breast cancer cells are selected as specific target cells. VLP will be supplied with specific addressing biomolecules, in order to ensure their recognition and uptake by target cells.
To address VLPs to hepatocytes, HBV preS1 peptide will be exposed on the VLP surface. Appearance of the preS1 peptide on the VLP surface will be assured by either genetic fusion, or chemical coupling
To address VLPs to leukocytes, HBc VLPs will be used, since they are able to recognize leukocytes without other addressing agents.
To address VLPs to T-lymphoma cells, oligonucleotide sc8 will be coupled chemically to the VLP surface.
To address VLPs to HER2 antigen presenting cancer cells, previously described affibody ZHER2:342 will be exposed on the VLP surface by either genetic fusion, or chemical coupling.
To wide range addressing of the VLPs, fragments of HIV Tat protein and its synthetic analogues, so-called PTD4, viral inflammation antagonist vMIP II, and stromal cell-derived factor SDF1 will be used.
VLPs, which are available at the BMC, possess diverse properties, which may influence markedly VLP ability to pack definite materials and accept addition of different cell addresses. For this reason, broad spectrum of VLP platforms will be involved into evaluation of VLP packaging and addressing capacities during the project time. First, HBc is the most studied and well characterised VLP model. A method for packaging of oligonucleotides, GFP, and magnetic nanoparticles into the HBc is elaborated at the BMC. Ability of the HBc to be recognised by lymphocytes without any additional addresses is documented. Small RNA phages AP205, GA, and phiCB5 are structurally well characterized. Methods for dissociation and re-association of RNA phage VLPs in vitro are elaborated, as well as a method for oligonucleotide and oligonucleotide/doxorubicin complex packaging into GA VLPs. Dissociation and in vitro re-association protocols are developed for icosahedral plant viruses ApMV un RYMV VLPs. First successful attempts have been performed at the BMC for the packaging of gold nanoparticles into icosahedral plant virus VLPs. Polyoma virus VLPs are able to packaging and efficient delivery of DNA and proteins to numerous cell types.
Constructed VLPs with exposed addresses and packaged material will be tested in (1) commercially available cell cultures and (2) cell and biopsy specimens of real patients, which will be aquired in cooperation with the RAKUS partner. During testing, the following features will be estimated: (1) toxicity of VLPs to target and control cells; (2) binding efficiency of VLPs to target and control cells; (3) capacity of packaged substances to perform therapeutic and/or diagnostic functions.
All activities listed above will be realised in the frame of industrial study. As a result of the study, the most prospective product will be chosen as lead products for the experimental development and the latter will be performed.
In the frame of experimental development, scaling of the selected prototypes, namely, elaboration and approval of protocols for large-scale production, characterisation, and quality control of the lead products, will be put into practice.
Before implementation of the project, it is not possible to judge, which VLP candidate from the possible VLP prototypes will be forced to the experimental development. From the current point of view, VLPs packaged with doxorubicin and provided with the ZHER2:342 address seem to be the most prospective candidate, which would be able to recognise and destroy selectively breast cancer cells with exposed HER2 antigen.