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Experiment: Testing different ratios of Lipofectamine-RNPs after 24 hours for determination of positive control

PI:   Ross C. Wilson, PhD  Samira Kiani, PhD  Shaoqin (Sarah) Gong, PhD 

Description: Liver-on-a-chip was used to examine the cellular uptake of CRISPR/Cas9 encapsulated nanoparticles provided from the Gong Lab at the University of Wisconsin-Madison. The Gong lab conducted free radical polymerization of the monomer coating with (PEG)-acrylate to ensure that the RNP-NC be stable and able to conjugate different ligands. Two liver-targeted ligands were provided from the Gong Lab, RNP-NC attached to tri(GalNAc) and RNP-NC containing cell penetrating peptide (TAT). The tri(GalNAc) is known to enhance RNP-NC target to hepatocytes, whereas TAT will enhance target and uptake of RNP-NC in all liver cells such as Kupffer cells. These ligands are tagged with Atto-550 a fluorescent protein reporter for easier detection. The goal was to investigate cellular uptake of Cas9-gRNA nanocapsules using imaging after 24 hours and to determine the appropriate ratio for Lipofectamine-RNP positive control.
Delivery Assays: Calculated transfection efficiency by imaging fluorescent cells (Atto 550) compared to DAPI stained cells, and quantified using Image J.
Download: Submitted files
Filters ..
Editors
 sNLS-SpCas9-sNLS
Guides
 Negative control gRNA
  Delivery Systems
 RNP-NC-CPP
 RNP-NC-no ligand with Lipofectamine 2000
 RNP-NC-no ligand with Lipofectamine 2001
 RNP-NC-no ligand with Lipofectamine 2002
 RNP-NC-tri(GalNAc)
Models
 Liver On Chip (LoC)
Select experimental variable to highlight records on the chart: 
 

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Results
Testing_different_ratios_of_Lipofectamine-RNPs_after_24_hours_for_determination_of_positive_control
Delivery Efficiency
Record Id Condition Editor Model Delivery Guide Dosage Delivery Efficiency Corrected total fluorescence (mean gray value) Image
15000003497 RNP-NC Lipo 1:1 sNLS-SpCas9-sNLS Liver On Chip (LoC) RNP-NC-no ligand with Lipofectamine 2000 Negative control gRNA 2.4µg RNP
15000003498 RNP-NC Lipo 2:1 sNLS-SpCas9-sNLS Liver On Chip (LoC) RNP-NC-no ligand with Lipofectamine 2001 Negative control gRNA 2.4µg RNP
15000003499 RNP-NC Lipo 5:1 sNLS-SpCas9-sNLS Liver On Chip (LoC) RNP-NC-no ligand with Lipofectamine 2002 Negative control gRNA 2.4µg RNP
15000003500 RNP-NC-CPP TAT sNLS-SpCas9-sNLS Liver On Chip (LoC) RNP-NC-CPP Negative control gRNA 2.4µg RNP
15000003501 RNP-NC-tri(GalNAc) sNLS-SpCas9-sNLS Liver On Chip (LoC) RNP-NC-tri(GalNAc) Negative control gRNA 2.4µg RNP
Synthesis scheme for acrylate-PEG-ligand [CPP and tri(GalNAc)].

Shows the synthesis schemes for acrylate PEG-ligand (i.e., CPP or tri(GalNAc)). Acrylate-PEG-CPP was synthesized via a Michael addition reaction. Trimeric GalNAc (i.e., tri(GalNAc)) was conjugated to the terminal end of acrylate-PEG via an amine-succinimidyl ester reaction. 
A schematic illustration for the synthesis of RNP-NC

NLS-Cas9-NLS protein was mixed with the desired sgRNA at a 1:1 molar ratio and allowed to complex for 5 min. The Cas9 RNP complex (2.4ug) was diluted in 0.2 mL sodium bicarbonate buffer (10 mM, pH=9.0). The monomers, crosslinker and initiators were accurately weighed and dissolved in degassed sodium bicarbonate buffer (2 mg/ml). Monomers were added into the above solution under stirring in the order of acrylic acid (AA), N-(3-aminopropyl)methacrylamide hydrochloride (APMA), and 1-vinylimidazole (VI) at 5 min intervals
Transfection of RNP NC in Primary Human Hepatocytes (PHH) shows little but detectable uptake (Part 1)

A) Shows transfection of 2.4ug RNP-NC tagged with no ligand but labeled with Atto 550 in PHH when used in a 1:1 ratio with lipofectamine. B) Shows transfection of 2.4ug RNP-NC tagged with no ligand but labeled with Atto 550 in PHH when used in a 2:1 ratio with lipofectamine. C) Shows transfection of 2.4ug RNP-NC tagged with no ligand but labeled with Atto 550 in PHH when used in a 5:1 ratio with lipofectamine.
Transfection of RNP NC in Primary Human Hepatocytes (PHH) shows little but detectable uptake (Part 2)

D) Shows transfection of 2.4ug RNP-NC tagged with TAT ligand and labeled with Atto 550 in PHH. E) Shows transfection of 2.4ug RNP-NC tagged with TrigalNac ligand and labeled with Atto 550 in PHH