1. TRICIA LIN M.ENG CANDIDATE CORNELL UNIVERSITY Binding of Streptavidin-Biotin Complex to Liposomes for Potential Tumor Detection

2. Background Rationale:  Past research has focused on in vivo and in vitro methods for drug delivery and for diagnostic protocols  Much interest in the utilization of vesicles as carrier systems with adsorptive endocytosis internalization  Ease of liposomes to encapsulate proteins  Covalent coupling of proteins and liposomes with phosphatidylethanolamine (PE) proved to be time consuming and excessive exposure to toxic reagents (Loughrey, H., et al)  Non-covalent coupling of proteins and liposomes more reliable, specific binding purposed by Loughrey et al.

3. Background  Binding of ligands to liposomal surfaces non-covalently (2-step targeting)  Typically through binding of hydrophobic anchors with functional groups  Anchors incorporated into membrane during formation of liposome itself  Addition of ligands to mixture of phospholipids during preparation of liposome  Percentage of ligand binding to carrier is low (4-40%)  Possible aggregation of liposome [3]  Usage of Streptavidin Protein as ligand  High binding affinity to biotin (10 -15M )  Liposomes coated with biotinated phosphatidylethanolamine (PE) Biotinylated Vesicle Streptavidin Biotinated antibody

4. Questions Will the non-covalent method purposed by Loughrey et al., be sufficient enough to couple biotinated- liposomes to streptavidin? If so, will the SA-liposome complex be functional enough to attach to biotinated antibody or proteins? Can the SA-liposome complex deliver the biotinated antibodies into the cell and not inhibit the function of such antibodies? (Future work)

5. Aim #1 Objective: To synthesize stable liposomes coated with hyaluronan and/or biotinated PE In brief:  Follow protocol done by Loughrey et al. (1987-1993) papers  Synthesis of large unilamellar liposomes via freeze/thaw, extrusion  Biotin combine with DPPE or hyaluronan coat liposome  Possibility of coating liposomes with polyethylenglycol (PEG) to avoid rapid clearance, sterically stabilized liposomes [3]  Side experiment: study the effects of hyaluronon on streptavidin-coated liposomes

6. Aim #1 Experimental Method  Follow similar protocol as Loughrey et al.(1987 and 1993) in non-covalent method  Biotinated phosphatidylethanolamine (PE) incorporated with egg phosphatidylcholine (PC) and DPPE (1:3:1)  Unilamellar vesicles produced through freeze-thaw, cycles extruded through extruder to produce close to 100nm diameter vesicle  Vesicles incubated with streptavidin that was modified with amine reactive reagent SPDP (or NHS) that helps form amide bond  Add DTT for reduction

7. Aim #2 Objective: To couple (hyaluronan) biotinated- liposomes with streptavidin;  Show actual coupling of biotinated PE-liposomes and streptavidin (no coating as negative control)  Measure extent of coupling of SA to liposomes via binding of biotin to streptavidin  Determine optimal concentration of biotinated PE for which SA can bind without liposome aggregation To quantify concentration of SA bound to liposome

8. Aim #2 Experimental Method:  SA prepared in Hepes buffer, modified with SPDP or NHS for amide bond, D-salt for removal of NHS  Addition of DTT to SA, gel exclusion to isolate thiolated product  Usage of microtitre 96-well plate, immobilize with SA, liposomes added  Incubate SA with liposomes at ratio 100 ug SA/umol lipid  Quench with N-ethylmaleimide to aid in cross-linking of cysteine groups Quantification:  Coupling SA and liposomes measured by extent of binding biotin to streptavidin; incubate  Compare with binding of biotin to SA via standard through gel chromatography Sephadex G-50 or size exclusion

9. Aim #3 Objective: Coupling of biotinated antibody to SA- coated liposomes to investigate and mimic in vivo behavior  Research done by Loughrey utilized antibodies in mice (anti- rat erythrocyte IgG) that are bound to biotin  Results: in absence of liposomally bound SA, no detectable antibody binding in vivo; quantified 40 molecules of SA bound per visicle, 40 molecules of antibody bound per vesicle Experimental Method;:  What antibody to use in vitro for this assay?

10. Aim #3 Once the antibody is determined,  In vitro: fluorescently label SA; after binding to liposome and antibody, samples dissolve complex in ethanol  Correlate fluorescent liposomes to known quantity of fluorescein labeled antibody Must demonstrate that targeted liposomes will bind specifically to target cells (in vitro)  Use biotinated antibody with labeled marker  Measure the specific binding

11. Preliminary Data Streptavidin production  Successfully cultured streptomycetes and purified streptavidin after 21 days of culturing  FPLC, nanodrop  Average concentration of purified SA: 0.16 mg/mL per 15 mL of media  Functionality assay  Purified SA successfully bind to biotin (Biacore results)

12. Preliminary Data L Contro l Blank SLMA2- 0 SLMA2-1SLMA2- 2 SLMA2- 3 SLMA2- 4 SLMA2- 5 SLMB 10x LContro l Blank SLMA2- 0 SLMA2-1SLMA2- 2 SLMA2- 3 SLMA2- 4 SLMA2- 5 SLMB 10x Control SA: 1.26mg/mL (3µL) Sample volumes: 20µL

13. Preliminary Research Loughrey et al. 1987  Investigated the binding of streptavidin to biotinated PE liposomes, subsequent attachment of targeting antibodies to streptavidin Pan et al. 2008  Investigated the targeting of tumor necrotic regions (chTNT—3) with biotinated antibody and streptavidin modified liposomes in tumor- bearing mice  Unilamellar liposomes encapsulated DOX to be delivered to tumor site and kill tumor cells  Modified chTNT-3 antibody with biotin  Coupled streptavidin to PEG-coated liposome  Found that SA-delivered DOX at greater concentrations and inhibited tumor growth compared to simple DOX injections

14. Potential Pitfalls Past research indicates increase of vesicle aggregation following attachment of SA to liposomes containing biotin (Loughrey 1990)  Greater vesicle size allowed greater binding of SA to biotinated liposomes, as shown through vesicle size of QELS  But the increase in vesicle size was related to vesicle aggregation  Extrusion must be efficient and the resulting conjugated samples after extrusion must be uniform in size  Reaggregation of vesicles can still occur after extrusion due to DPPE

15. Future Work Hypothesize that biotinated antibodies can be coupled via streptavidin to liposomes containing biotin PE for a targeted vesicle system Advantage of results show technique can be extended to couple any biotinated antibody or protein to liposomal systems for future tumor targeting Streptavidin conjugation with nano-particles for tumor targeting  Quantum dots  SPIO  Usage of MRI as detection