Download Cell-penetrating nanocarrier

Jin Lab: Research Update 2.24.09
Stabilized Immunoliposomes for
Targeted Drug Delivery
Nwanyinma Nnodum
Background
Potential of Liposomes as Pharmaceutical Nanocarriers
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.
Background
Potential of Liposomes as Pharmaceutical Nanocarriers
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.
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
Methods
FITC Encapsuation
• FITC encapsulated during lipid hydration step (0.48 mg FITC/mL HBS)
• Floatation assays using sucrose
• Mix 500 µL sample + 500 µL 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
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
Results
FITC Encapsulation
• Less than 5%
encapsulated
FITC Encapsulation
Absorbtion
Original Abs
average
Free FITC Abs
average
FITC+Liposome Abs
average
495nm
Sample 1
Sample 2
0.168
0.168
0.176
0.176
0.182
0.182
0.175333333
0.17533333
0.166
0.128
0.155
0.136
0.1605
0.132
0.023
0.041
0.03
0.043
0.0265
0.042
Efficiency (%Encapsulated) 15.11406844
23.9543726
Efficiency (%Free)
91.53992395
75.2851711
0.5
0.48
0.5
0.48
volume (mL)
Original Conc (mg/mL)
FITC encapsulated (mg)
0.036273764
0.24mg
0.05749049 0.046882129
Results (cont.)
Flow Cytometry (FACS)
HeLA
NIH-3T3
Results (cont.)
Flow Cytometry (FACS)
MDA-MB231
•
•
•
•
Noise!
Minimal shift
Incubation at 37°C
More cells
MCF-7
Future Plans
• FACS
• With proteins
• With proteins plus liposomes
• Confocal microscopy
• Drug encapsulation
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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