Supramolecular structure directing unit regulated co-assembly of a protein produces a highly stable fibrillar nanostructure and glutathione responsive release of the protein in its active state.

This manuscript reports the effect of hydrogen-bonding functionality on the supramolecular assembly of naphthalene-diimide (NDI)-derived amphiphilic building blocks in water. All the molecules contain a central NDI chromophore, functionalized with a hydrophilic oligooxyethylene (OE) wedge in one arm and a phenyl group on the opposite arm. They differ by a single H-bonding functionality, which links the NDI chromophore and the phenyl moiety. The H-bonding functionalities are amide, thioamide, urea, and urethane in NDI-A, NDI-TA, NDI-U, and NDI-UT, respectively. All of these molecules exhibit π-stacking in water, as evident from their distinct UV/vis absorption spectra when compared to that of the monomeric dye in THF. However, among these four, only NDI-A and NDI-TA show hydrogelation, while the other two precipitate out of the medium. The NDI-A hydrogel also exhibits transient stability and leads to a crystalline precipitate within ∼5 h. Only NDI-TA produces stable transparent hydrogel with the entangled fibrillar morphology that is typical for gelators. Both NDI-A and NDI-TA showed a thermoresponsive property with a lower critical solution temperature of about 41−42 °C. Powder XRD studies show a parallel orientation for NDI-A and an antiparallel orientation for NDI-TA. Computational studies support this experimental observation and indicate that the NDI-A assembly is highly stabilized by strong H-bonding among the amide groups and π-stacking interaction in the parallel orientation. On the other hand, due to weak H-bonding among the thioamide groups, the binding energy of the parallelly oriented NDI-TA was significantly lower and the optimized structure was disordered. Instead, its antiparallel orientation was more stable, with criss-cross aligned H-bonding interactions and π−π interactions between adjacent aromatic rings. The NDI-TA hydrogel with less ordered OE chains on the surface showed prominent adsorption of serum protein BSA. In sharp contrast, NDI-A did not exhibit any notable interaction with BSA, as evident from the ITC studies.

Cellular uptake is an important event in drug delivery and other biomedical applications. Amphiphilic polymers produce aggregates of different size and shape depending on the intrinsic structural differences and the packing parameter. Although they have been explored for various biomedical applications with immense interest, the relationship between the shape of the aggregate and cellular uptake has been studied only in limited examples. This work reports two polymers (P1 and P2), both of which contain a hydrophobic supramolecular structure-directing unit (SSDU) at the chain-end of a fluorescence dye-labeled hydrophilic polymer. Depending on the difference in the structure of the single H-bonding functional group (hydrazide or amide) of the SSDU, P1 and P2 produce polymersomes (NS1) and spherical micelles (NS2), respectively. An aged solution of P2 produces cylindrical micelles (NS3). Confocal microscopy studies reveal that the uptake of these nanostructures in HeLa cells greatly depends on the shape of the aggregate. Spherical NS1 and NS2 show appreciable uptake at 1 or 4 h of incubation, whereas NS3 shows negligible uptake. Temperature-dependent cellular uptake studies reveal an energy-dependent endocytosis pathway. Kinetic studies show gradual increase in the cellular uptake with time, and at 24 h the relative uptake ratio (NS1:NS2:NS3) is 1.0:0.2:<0.1, implying the polymersome morphology (NS1) is most efficient for cellular uptake compared to the spherical or cylindrical micelles. The same trend was also noticed for MDA-MB 231 cells. Confocal microscopy studies further reveal cellular internalization and intracellular location of NS1, which showed maximum cellular uptake. As the intrinsic difference in the chemical structure of the two polymers is negligible, the observed difference can be explicitly assigned to their difference in shape.