SELF ASSEMBLED TERNARY COMPLEX: non

covalent strategy for Developing gene

delivery carrier

Presented by
Flora Banerjee(19CY90J05)

Under the Supervision of

Dr. SumanKalyan Samanta

Department of Chemistry
Indian Institute of Technology, Kharagpur
2nd August, 2019
• Spontaneously assembled supramolecular complex

• Non covalent self assembly b/w biological

macromolecule mimicked in chemical approach

• Electrostatic attraction b/w cationic polymer and and –

vely charged DNA
• Study of non viral gene delivery carrier
 Formation of Polyplex

 A number of cationic polymers, such as poly- L-lysine (10),

poly(ethylenimine) (PEI) ( 11), PAMAM dendrimer ( 12), and hyperbranched
poly(amino ester) ( 13), were able to compact DNA into submicron-sized water-
soluble par- ticles (polyplexes) and carry DNA into the cell nucleus
 Hydrodynamic Radii by Dynamic light scattering

Figure 1. Size distribution of (A) PPI-DAB/CB/DNA (with 100% CB relative to DAB), (B) PPI-DAB/DNA, (C) PPI-DAB/DNA/CB (10% CB),
and (D) PPI-DAB/DNA/CB (30% CB) complexes by dynamic light scattering. 1 DNA was p EGFPLuc plasmid DNA. 2 All complexes were
formed at a charge ratio ( +/-) of 3. Charge ratios were calculated based on the assumption that only primary and
secondary amines in DAB are protonated.
 Results from DLS Study
The avg. Radii of PPI/DAB-DNA was found to
be smaller(83.5nm) than PPI/DAB/CB-DNA
having 150.8 nm

In the later one all DAB sites are occupied by

CB and complex grew at larger rate

Two intensity population existed at CB conc.

Above 30% in smaller size region
 Agarose gel Electrophoresis

Figure 2. Investigation of polyplex formation by agarose gel band shift assay. Numbers indicate charge
ratio(+/-). (A) PPI/DNA polyplex
(B) PPI-DAB/DNA polyplex. (C) PPI-DAB/CB/DNA polyplex. In PPI-DAB/CB/DNA polyplex, all of the
DAB moieties are occupied by CBThe original positive image was inverted to make it negafor ease of band
discrimination.
 Conclusion from DNA gel electrophoresis

• All the 3rd generation polymer shown at charge ratio

(polymer to DNA) just above 1
• PPI-DAB/DNA few 4th generation had (+/-) ratio
around 2.
• In case of CB bound unit higher the seperation
attributes to generation of polyplex .

• Binding capacity is decreased for polymer as

generation no. Increased.
 CB remains associated or not??
• To demonstrate the validity of this assumption, each of CB,
PPI-DAB (G4)/CB, and PPI-DAB (G4)/CB/DNA were
dialyzed using 3500 M r cutoff tubing against 0.2 m Na 2SO4,
in which CB dissolves verywell . In this system, only CB is
expected to be freely permeable to the membrane.

• The presence of CB in the solution outside the mem- brane

couldeasily be detected by using DAB-tethered acridine (Acr-
DAB) with high sensitivity
Figure 3. Fluorescence emission spectra of (A) a: Acr-DAB; b: Acr-DAB/CB and the solution outside
the dialysis membrane of (B) CB, (C) PPI-DAB/CB, and (D) PPI-DAB/CB/DNA
sates after addition of Acr-DAB. 1: 0.1 µg (1/40 molar equivalent of total CB); 2: 0.3 µg (3/40); 3: 1.0 µg
(10/40) of Acr-DAB were added to the solution.
 As Potential Gene delivery Vector
• Cytotoxicity of polymers and transfection efficiency was
investigated.

• PPI-DAB/CB has relatively lower cytotoxicity even at

very high conc. 50% of the cells are viable.

• TE is basically dependent upon the cell type and for all

the polyplexes nearly same.

• Transfection is higher for PPI-DAB/CB and only 10 times

lower than PEI and PAMAM dendrimer was more
efficient than PAMAM too.
 Conclusion
The complex was formed in a noncovalent way in which DNA
interacts with PPI-DAB electrostatistically and CB with PPI-
DAB through multiple noncovalent interactions.

 DLS data indicated that the diameter and size of polymers

dependent upon sequence of mixing

 CB has no negative effect upon transfection and is also

associated as functionalised moiety.

. Fluorescence studies showed the quantitative binding of CB

to PPI-DAB after ternary complex formation
Thank You