1. Jin Lab: Research Update 2.24.09 Stabilized Immunoliposomes for Targeted Drug Delivery Nwanyinma Nnodum

2. Background 1 – Traditional “plain” nanocarrier (a – drug loaded into carrier) 2 – Targeted nanocarrier or immunocarrier (b – mAb attached to carrier surface) 3 – Magnetic nanocarrier (c – magnetic particles loaded into carrier together with the drug) 4 – Long-circulating nanocarrier (d – surface-attached protecting polymer (usually PEG)) 5 – Contrast imaging nanocarrier (e – heavy metal atom (i.e. 111In) loaded onto the nanocarrier via the carrier-incorporated chelating moiety) 6 – Cell-penetrating nanocarrier (f – cell-penetrating peptide, CPP, attached to the carrier surface) 7 – DNA-carrying nanocarrier (g – DNA complexed by the carrier via the carrier surface positive charge) 8 –Multifunctional pharmaceutical nanocarrier combining the properties of the carriers # 1–7. Potential of Liposomes as Pharmaceutical Nanocarriers

3. Background 1 – Traditional “plain” nanocarrier (a – drug loaded into carrier) 2 – Targeted nanocarrier or immunocarrier (b – mAb attached to carrier surface) 3 – Magnetic nanocarrier (c – magnetic particles loaded into carrier together with the drug) 4 – Long-circulating nanocarrier (d – surface-attached protecting polymer (usually PEG)) 5 – Contrast imaging nanocarrier (e – heavy metal atom (i.e. 111In) loaded onto the nanocarrier via the carrier-incorporated chelating moiety) 6 – Cell-penetrating nanocarrier (f – cell-penetrating peptide, CPP, attached to the carrier surface) 7 – DNA-carrying nanocarrier (g – DNA complexed by the carrier via the carrier surface positive charge) 8 –Multifunctional pharmaceutical nanocarrier combining the properties of the carriers # 1–7. Potential of Liposomes as Pharmaceutical Nanocarriers

4. Overview FITC Encapsulation Floatation assay Cell Culture HeLa: human cervical cancer cells MDA-MB231: human breast cancer cells MCF-7: human breast cancer cells NIH 3T3: mouse fibroblast cells FACS MAP-wGFP-His 9R-wGFP-His RGD HER2 Protein Expression & Purification

5. Methods FITC Encapsuation FITC encapsulated during lipid hydration step (0.48 mg FITC/mL HBS) Floatation assays using sucrose Mix 500 µ L sample + 500 µ L 70% sucrose Gently add 3mL of 20% sucrose to top Ultracentrifuge for 2hrs at 35000 rpm, 8°C Remove band (liposome + FITC) at top with 16g syringe & rest is free FITC Bring fractions to 4mL & check absorbance with Nanodrop Cell Culture alphaMEM+10%FBS+1%PenStrep for MDA-MB231, MCF-7, NIH-3T3 Advanced DMEM +10%FBS+1%Glutamine for HeLa

6. Methods (cont.) Flow Cytometry (FACS) Sample Prep Spin down cells from tissue culture & remove supernatant Resuspend in 1xPBS Aliquot 100µL per sample, spin down, & remove sup. Resuspend in 100µL of PBS+1%BSA+protein sample Incubate for 1hr at 4°C Spin down & remove sup. with free protein Resuspend in 200µL of PBS+1%BSA X-wGFP Synthesis & Purification Large scale production of MAP-, 9R-, wGFP Purification with Ni-NTA-His column, SDS-PAGE, FPLC

7. Results FITC Encapsulation Absorbtion495nm Sample 1Sample 2 Original Abs0.168 0.176 0.182 average0.1753333330.17533333 Free FITC Abs0.1660.128 0.1550.136 average0.16050.132 FITC+Liposome Abs0.0230.041 0.030.043 average0.02650.042 Efficiency (%Encapsulated)15.1140684423.9543726 Efficiency (%Free)91.5399239575.2851711 volume (mL)0.5 Original Conc (mg/mL)0.48 0.24mg FITC encapsulated (mg)0.0362737640.057490490.046882129 Less than 5% encapsulated

8. Results (cont.) Flow Cytometry (FACS) HeLA NIH-3T3

9. Results (cont.) Flow Cytometry (FACS) Noise! Minimal shift Incubation at 37°C More cells MDA-MB231 MCF-7

10. Future Plans FACS With proteins With proteins plus liposomes Confocal microscopy Drug encapsulation

11. References Peer, Park, Morishita, Carman, Shimaoka. “Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target”. Science. 319(2008): 627-630. Ronny Ruger, Dafne Muller, Alfred Fahr, & Roland E. Kontermann. “In Vitro Characterization of Binding and Stability of Single-Chain Fv Ni-NTA-Liposomes”. Journal of Drug Targeting. 14.8 (2006): 576–582.

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