Svarovsky Lab: Research

Intracellular Delivery of Biologically Active Molecules

Gene therapy is defined as the transfer of genetic material to specific cells in order to have a therapeutic effect. One of the issues preventing gene therapy from reaching its clinical potential is the lack of effective gene delivery vehicles. Among many different delivery methods, biolistics (biological ballistics) or pneumatic particle bombardment provides the most direct, rapid and simple procedure for delivering genes into the cells.

Current biolistic technology relies heavily on the particle-mediated delivery protocols where DNA is co-precipitated with the gold microparticles without direct attachment to the particles. The co-precipitated solid of DNA with microparticles, Ca salt and spermidine is not easily controlled, which results in variability in the amount of DNA loaded onto the microparticles and the ratio of the co-precipitates to one another leads to inconsistent and inefficient delivery of DNA into the cells.

In this project we are developing various formulations and uses of novel cationic (positively charged) gold microparticles where DNA is directly attached to the surface of the microparticles by electrostatic forces. For example, in one formulation, the surface of the gold microparticles is chemically modified with positively-charged (cationic) polymer polyethyleneimine (PEI). The negatively-charged (anionic) DNA is then electrostatically (by charge) loaded onto the surface of the positively-charged microparticles. In another formulation, we use DNA-encapsulated gold nanoparticles electrostatically attached to the surface of the aforementioned PEI-modified microparticles. Here, the large gold microparticle serves as a carrier for the smaller agile gold nanoparticles forming a so-called “raspberry” micronanoplexes®, i.e. complexes of DNA with nano- and microconstructs. In yet another formulation, an alternating layer-by-layer (onion-like) assembly of PEI/DNA on the surface of the gold microparticles is employed. The layered assembly not only allows higher loading capacities but also gives flexibility of using different DNAs slowly released over pre-programmed time lapses.

All of these formulations provide clear advantages of (1) simple preparation; (2) fully controllable, consistent and reproducible DNA loading capacity; (3) flexibility, and (3) highly efficient gene delivery. These advantages may open new possibilities for high-throughput gene delivery technologies.

Template Assembled Synthetic Lectins (Synlecs)

The range and importance of glycosylation in biological processes warrants rapid discovery of agents that can modulate or detect glycan-mediated interactions as a promising new avenue towards novel therapeutic and diagnostic agents. To this end, we are developing a novel random peptide sequence microarray approach to rapidly and reliably identify carbohydrate-binding peptides. As a proof of concept we have used lactose-, 3’-sialyllactose-, 6’-sialyllactose-encapsulated quantum dots as multivalent luminescent glycoprobes to probe an addressable random sequence peptide microarray consisting of 4,000 twenty-mer features printed on polylysine glass slides. We demonstrated that despite rather limited chemical diversity, this method allows selection of highly specific non-overlapping sets of peptides having micromolar affinity for their corresponding glycotargets. Validation of the selected peptides by hemagglutination assays, haptenic inhibition, surface plasmon resonance (SPR), and fluorescence assisted cell sorting (FACS) is presented. Furthermore, some of the selected glycan-binding peptides are successfully applied for magnetic cell sorting (MACS) of human versus bacterial cells based solely on the differences in sialylation of the cell membranes.

Combinatorial screening of carbohydrate-protein interactions

Tissue Engineering using Glycopeptide Scaffolds

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the United States. Approximately 1.3 million cases of nonfatal MI are reported each year. The progressive nature of MI often leads to end-stage heart failure, with heart transplantation being the only option. Several revolutionary technologies for the treatment of damaged or missing myocardial tissue have recently evolved as alternatives to heart transplantation. These regenerative technologies include cell-based therapies and scaffold-based therapies. The major technical limitations of these technologies are specific immunological and unspecific inflammation reactions of the body to the implanted cells and biomaterials. Such deleterious reactions are often mediated by the cell-surface carbohydrate antigens.

Self-Assembled artificial ECM nanofibers for cardiac tissue engineering.

The long-term goal of this proposal is to develop localizable carbohydrate-modified immunosorbant scaffolds capable of controlling tissue response after implantation into the body. Specifically, this project describes development of sialyl Lewis X (sLeX) modified scaffolds for local prevention of unspecific inflammatory response. The hypothesis for this investigation is that a mixture of amphiphilic peptides terminated with integrin-binding RGD motif and of amphiphilc glycopeptides terminated with selectin-binding sLeX tetrasaccharide will self-assemble in situ into viscoelastic network of nanofibers exposing variable densities of sLeX on their surface. Such a unique multivalent presentation of sLeX antigen is expected to provide cooperative cell-binding and anti-inflammatory microenvironment at the site of transplantation. The ultimate product resulting from this project will be injectable directly into the site of transplantation or used as a scaffold for cell-based therapies.

Biological Imaging Using Glyco Quantum Dots

The ability to image processes occurring in live organisms is absolutely critical in virtually any area of life sciences. Small organic fluorophores were used for decades for this purpose. Recently, with the advances in nanotechnology, quantum dots (QDs) semiconductor nanocrystals have emerged as a powerful alternative to the organic fluorophores in cell biology, medicine, and biotechnology. QDs have several clear advantages over organic fluorescent dyes, which include (1) broad light absorption allowing their excitation with a single light source, (2) narrow light emission controlled by size and material composition, and (3) exceptional photostability allowing long-term observation. These unique properties make QDs suitable for exploitation in a wide range of established and rapidly emerging commercial applications. It is expected that quantum dots will soon replace organic fluorophores, a multimillion dollar market.

Bioimaging using carbohydrate-encapsulated quantum dots

A wide range of molecules can serve as potential guides for quantum dots in living systems. These include nucleic acids and proteins with affinities for certain receptors, or carbohydrates. The conjugation and use of QDs with the first two classes of molecules have been extensively researched; the current manufacturers of QDs (e.g. Molecular Probes, Inc and Evident Technologies, Inc) are mainly catering to this segment of the biological imaging market. The technologies for encapsulation of QDs with sugars, however, have lagged behind despite rapidly increasing realization of their ubiquitous roles in biological systems. This oversight has occurred mainly for the two reasons: (1) sugars are difficult to synthesize and otherwise manipulate efficiently and (2) glycobiology community, albeit growing exponentially, still represents a disproportionately small niche in the life sciences market.

Sugars (glycans) are of central importance in the development and maintenance of all biological systems as well as in their communication with outside world. The surfaces of most cells are covered with a dense coating of glycoconjugates (glycoproteins, proteoglycans and glycolipids) called the glycocalix. Cellular adhesion and/or recognition are directed by highly selective interactions involving these surface carbohydrates. A myriad of key biological processes such fertilization, pathogen invasion, toxin and hormone mediation, and cell-cell interactions rely on carbohydrate-protein interactions.

Roles of carbohydrates in biological interactions

Many diseases are caused by abnormalities in control mechanisms that affect the normal social behaviors of differentiated cells in multicellular organisms. Here the sugar chains of glycoconjugates play important roles. They have a key function in the control of immuno­logical and cellular functions, and in recognition processes between cells and their environments. Specific carbohydrate-protein interactions play a role in major human diseases like asthma, reperfusion damage (occurring e.g. in stroke and heart attack), rheumatoid arthritis or atherosclerosis and also in metastasis formation in some forms of cancer. For example, inflammation is a universal phenomenon controlled by the carbohydrate-protein interactions directing lymphocytes to the sites of injury. Understanding underlying biomolecular interactions is required for the development of agonists or antagonists that can functionally intervene in the processes that cause disease.

Despite their ubiquitousness in nature, traditionally carbohydrate-protein interactions were difficult to study because the interactions are subtle with multivalent cluster effects being crucial for their real life function. Small molecules or glycoconjugates bearing a single carbohydrate epitope are rarely successful probes or interference tools in this environment. Therefore there is a need for versatile scaffolds that would allow not only multivalent presentation of carbohydrates but also ability to track their interactions within the living systems. GlycoDotsTM are designed to fulfill this need.

Tumor Microenvironment and Glycosylation

Extracellular pH (pHe) is lower in many tumors than in the corresponding normal tissues. Mechanistically, hypoxia and acidity have their roots in poor perfusion and elevated metabolism. This hostile environment is intimately coupled to glycolysis and hence, the "aerobic glycolysis phenotype", first described by Otto Warburg in 1924, may be central to the process of carcinogenesis itself. Although Warburg observed high glycolysis in a few tumors, the universality of this phenomenon was not appreciated until recently, with the wide application of fluorodeoxyglucose (FdG) PET scans. A recent review by Sam Gambhir and Mike Phelps of over 14,000 patient scans has shown that FdG uptake is significantly elevated in over 90% of all metastatic cancers. Further investigations have used both low metastatic (MCF-7) and highly metastatic (MDA-mb-231 and -435) cells. MCF-7 cells do not glycolyze rapidly under aerobic conditions, whereas the metastatic cells do.

Recently the significance of acidic pHe in the development of metastatic disease was investigated. Human melanoma cells (A-07, D-12, and T-22) were cultured in vitro at pHe 6.8 or 7.4 (control) before being inoculated into the tail vein of BALB/c nu/nu mice for formation of experimental pulmonary metastases. Cells cultured at acidic pHe showed increased secretion of proteinases and pro-angiogenic factors, enhanced invasive and angiogenic potential, and enhanced potential to develop experimental metastases.

It is now well recognized that normal cells and their corresponding cancerous cells have altered features reflected in differing physiologies. One example of this is the aberrant_expression of specific glycosyltransferases and glycosidases. As a result, the process of malignant transformation, tumor progression, and metastasis are all accompanied by glycosylation changes. These changes are considered to be the basis for the altered social behavior of cancer cells, such as altered cell adhesion, invasiveness, and metastatic spread. For example, it is well-documented that in cancer cells there is an overall increase in the sialylation of cell-surface and secreted glycoproteins due to upregulated activity of sialyltransferase ST3Gal1. This increased sialic acid content is believed to facilitate migration of cancer cells and their evasion from immune surveillance. On the other hand, another widely recognized cancer-related phenomenon is the overexpression in cell surface O-linked glycoproteins (mucins) which are truncated in cancer to display a variety of desialylated epitopes. Currently there is no generally accepted consensus regarding origins of these somewhat contradictory glycosylation patterns and their role in metastasis. Another related cancer-associated change is the defucosylation of Lewis-type structures (Glycosciences in Cancer meeting, 2006). Incidentally, both alpha-sialyl and alpha-fucosyl linkages are exceptionally acid-labile.

Inter-relationship between tumor microenvironment and cell surface archtecture

In summary, both tumor acidification and altered cell-surface glycosylation patterns are universal phenomena in cancer growth, proliferation and metastasis. Therefore, it is important to explore the interrelationship between microphysiological environment and cell-surface glycosylation of tumor tissues. Not only this will allow us to shed light on some of the most controversial topics of tumor glycobiology but also aid in development of more effective vaccines, targeting, and therapeutic agents.