Karthikan Rajagopal

Controlling hydrogelation kinetics by peptide design for three-dimensional encapsulation and injectable delivery of cells

A peptide-based hydrogelation strategy has been developed that allows homogenous encapsulation and subsequent delivery of C3H10t1/2 mesenchymal stem cells. Structure-based peptide design afforded MAX8, a 20-residue peptide that folds and self-assembles in response to DMEM resulting in mechanically rigid hydrogels. The folding and self-assembly kinetics of MAX8 have been tuned so that when hydrogelation is triggered in the presence of cells, the cells become homogeneously impregnated within the gel. A unique characteristic of these gel–cell constructs is that when an appropriate shear stress is applied, the hydrogel will shear-thin resulting in a low-viscosity gel. However, after the application of shear has stopped, the gel quickly resets and recovers its initial mechanical rigidity in a near quantitative fashion. This property allows gel/cell constructs to be delivered via syringe with precision to target sites. Homogenous cellular distribution and cell viability are unaffected by the shear thinning process and gel/cell constructs stay fixed at the point of introduction, suggesting that these gels may be useful for the delivery of cells to target biological sites in tissue regeneration efforts.

Spotted vesicles, striped micelles and Janus assemblies induced by ligand binding

Selective binding of multivalent ligands within a mixture of polyvalent amphiphiles provides, in principle, a mechanism to drive domain formation in self-assemblies. Divalent cations are shown here to crossbridge polyanionic amphiphiles that thereby demix from neutral amphiphiles and form spots or rafts within vesicles as well as stripes within cylindrical micelles. Calcium and copper crossbridged domains of synthetic block copolymers or natural lipid (PIP2, phosphatidylinositol-4,5-bisphosphate) possess tunable sizes, shapes, and/or spacings that can last for years. Lateral segregation in these ‘responsive Janus assemblies’ couples weakly to curvature and proves restricted within phase diagrams to narrow regimes of pH and cation concentration that are centered near the characteristic binding constants for polyacid interactions. Remixing at high pH is surprising, but a theory for Strong Lateral Segregation (SLS) shows that counterion entropy dominates electrostatic crossbridges, thus illustrating the insights gained into ligand induced pattern formation within self-assemblies.

Tuning the pH Responsiveness of β-Hairpin Peptide Folding, Self-Assembly, and Hydrogel Material Formation

A design strategy to control the thermally triggered folding, self-assembly, and subsequent hydrogelation of amphiphilic beta-hairpin peptides in a pH-dependent manner is presented. Point substitutions of the lysine residues of the self-assembling peptide MAX1 were made to alter the net charge of the peptide. In turn, the electrostatic nature of the peptide directly influences the solution pH at which thermally triggered hydrogelation is permitted. CD spectroscopy and oscillatory rheology show that peptides of lower net positive charge are capable of folding and assembling into hydrogel material at lower values of pH at a given temperature. The pH sensitive folding and assembling behavior is not only dependent on the net peptide charge, but also on the exact position of substitution within the peptide sequence. TEM shows that these peptides self-assemble into hydrogels that are composed of well-defined fibrils with nonlaminated morphologies. TEM also indicates that fibril morphology is not influenced by making these sequence changes on the hydrophilic face of the hairpins. Rheology shows that the ultimate mechanical rigidity of these peptide hydrogels is dependent on the rate of folding and self-assembly. Peptides that fold and assemble faster afford more rigid gels. Ultimately, this design strategy yielded a peptide MAX1(K15E) that is capable of undergoing thermally triggered hydrogelation at physiological buffer conditions (pH 7.4, 150 NaCl, 37 degrees C).

Probing the importance of lateral hydrophobic association in self-assembling peptide hydrogelators

A class of peptides has been designed whose ability to self-assemble into hydrogel is dependent on their conformationally folded state. Under unfolding conditions aqueous peptide solutions are freely flowing having the viscosity of water. When folding is triggered by external stimuli, peptides adopt a β-hairpin conformation that self-assembles into a highly crosslinked network of fibrils affording mechanically rigid hydrogels. MAX 1, a 20 residue, amphiphilic hairpin self-assembles via a mechanism which entails both lateral and facial self-assembly events to form a network of fibrils whose local structure consists of a bilayer of hairpins hydrogen bonded in the direction of fibril growth. Lateral self-assembly along the long axis of the fibril is mainly facilitated by intermolecular hydrogen bonding between the strands of distinct hairpins and the formation of hydrophobic contacts between residue side chains of laterally associating hairpins. Facial assembly is driven by the hydrophobic collapse of the valine-rich faces of the amphiphilic hairpins affording a bilayer laminate. The importance of forming lateral hydrophobic contacts during hairpin self-assembly and the relative contribution these interactions have towards nano-scale morphology and material rigidity is probed via the study of: MAX1, a hairpin designed to exploit lateral hydrophobic interactions; MAX 4, a peptide with reduced ability to form these interactions; and MAX5, a control peptide. CD spectroscopy and rheological experiments suggest that the formation of lateral hydrophobic interactions aids the kinetics of assembly and contributes to the mechanical rigidity of the hydrogel. Transmission electron microscopy (TEM) shows that these interactions play an essential role in the self-assembly process leading to distinct nano-scale morphologies.

Polymersomes and Wormlike Micelles Made Fluorescent by Direct Modifications of Block Copolymer Amphiphiles

Wormlike micelles and vesicles prepared from diblock copolymers are attracting great interest for a number of technological applications. Although transmission electron microscopy has remained as the method of choice for assessing the morphologies, fluorescence microscopy has a number of advantages. We show here that when commercially available fluorophores are covalently attached to diblock copolymers, a number of their physicochemical characteristics can be investigated. This method becomes particularly useful for visualizing phase separation within polymer assemblies and assessing the dynamics of wormlike micelles in real time. Near-IR fluorophores can be covalently conjugated to polymers and this opens the possibility for deep-tissue fluorescence imaging of polymer assemblies in drug delivery applications.

Microrheology of Responsive Hydrogel Networks

Hydrogels that form via the self‐assembly of β‐hairpin peptides are studied for their potential use in biomedical applications. Multiple particle tracking microrheology and circular dichroism (CD) spectroscopy are used to study the gelation kinetics of four peptides that are engineered to exhibit responsive behavior to changes in environmental conditions. The peptides being compared differ in sequence by a point substitution of a single amino acid near the turn sequence, which predictably alters the energetics of the folding event. The principles of time‐cure superposition are used to rescale the mean‐squared displacement of probe particles onto master curves before and after the gel point. By analyzing the shift factors based on scaling relationships near the liquid‐solid transition, we are able to accurately determine both the gel time and critical exponents of the incipient gel. An empirical relationship is established between the rheologically‐defined gelation time and the onset of beta‐sheet formatio...

Spray stability of self-assembled filaments for delivery

ABSTRACT Filamentous viruses are common in nature and efficiently deliver – sometimes via aerosol – genetic material, viral proteins, and other factors to animals and plants. Aerosolization can be a severe physicochemical test of the stability of any filamentous assembly whether it is made from natural polymers such as viral proteins or synthetic polymers. Here, worm‐like “filomicelles” that self‐assemble in water from amphiphilic block copolymers were investigated as aerosolized delivery vehicles. After spraying and drying, fluorophore‐loaded filomicelles that were originally ˜ 10–20 &mgr;m long could be imaged as 2–5 &mgr;m long fragments that survived rehydration on natural and artificial surfaces (i.e. plant leaves and glass). As a functional test of delivery, the hydrophobic pesticide bifenthrin was loaded into filomicelles (up to 25% w/w) and sprayed onto plants infested with two agricultural pests, beet army worm or two‐spotted spider mites; pesticidal efficacy exceeded that of commercial formulations. Persistent delivery by the filomicelle formulation was especially notable and broadly consistent with previous intravenous delivery of other drugs and dyes with the highly elongated filomicelles. Graphical abstract Figure. No Caption available.

Polymersomes and Filomicelles

Amphiphilic block copolymers represent a major field of research in the design and creation of innovative materials for biomedical applications. Self-directed assemblies of such copolymers have been of great value in the development of novel drug delivery systems. Polymeric vesicles (polymersomes) and worm-like micelles (or filomicelles) are particularly appealing due to their structure and composition that provides them with specific and tunable properties. The work reviewed at an introductory level in this chapter highlights some of the features of such aggregates and reviews the synthesis of their components, their assembly, and characterization. Degradation and drug release kinetics are described as well as their application in therapeutics.