Drug Encapsulation via Peptide-Based Polyelectrolyte Complexes.

Peptide-based polyelectrolyte complexes are biocompatible materials that can encapsulate molecules with different polarities due to their ability to be precisely designed. Here we use UV-Vis spectroscopy, fluorescence microscopy, and infrared spectroscopy to investigate the encapsulation of model drugs, doxorubicin (DOX) and methylene blue (MB) using a series of rationally designed polypeptides. For both drugs, we find an overall higher encapsulation efficiency with sequences that have higher charge density, highlighting the importance of ionic interactions between the small molecules and the peptides. However, comparing molecules with the same charge density, illustrated that the most hydrophobic sequence pairs had the highest encapsulation of both DOX and MB molecules. The phase behavior and stability of DOX-containing complexes did not change compared to the complexes without drugs. However, MB encapsulation caused changes in the stabilities of the complexes. The sequence pair with the highest charge density and hydrophobicity had the most dramatic increase in stability, which coincided with a phase change from liquid to solid. This study illustrates how multiple types of molecular interactions are required for efficient encapsulation of poorly soluble drugs and provides insights into the molecular design of delivery carriers.

Molecular Dynamics Simulations of Polyelectrolyte Complexes.

Polyelectrolyte complexes (PECs) are currently of great interest due to their applications toward developing new adaptive materials and their relevance in membraneless organelles. These complexes emerge during phase separation when oppositely charged polymers are mixed in aqueous media. Peptide-based PECs are particularly useful toward developing new drug delivery methods due to their inherent biocompatibility. The underlying peptide sequence can be tuned to optimize specific material properties of the complex, such as interfacial tension and viscosity. Given their applicability, it would be advantageous to understand the underlying sequence-dependent phase behavior of oppositely charged peptides. Here, we report microsecond molecular dynamic simulations to characterize the effect of hydrophobicity on the sequence-dependent peptide conformation for model polypeptide sequences that were previously reported by Tabandeh et al. These sequences are designed with alternating chirality of the peptide backbone. We present microsecond simulations of six oppositely charged peptide pairs, characterizing the sequence-dependent effect on peptide size, degree of hydrogen bonding, secondary structure, and conformation. This analysis recapitulates sensible trends in peptide conformation and degree of hydrogen bonding, consistent with experimentally reported results. Ramachandran plots reveal that backbone conformation at the single amino acid level is highly influenced by the neighboring sequence in the chain. These results give insight into how subtle changes in hydrophobic side chain size and chirality influence the strength of hydrogen bonding between the chains and, ultimately, the secondary structure. Furthermore, principal component analysis reveals that the minimum energy structures may be subtly modulated by the underlying sequence.

Targeted polyelectrolyte complex micelles treat vascular complications in vivo.

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium in vitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications in vivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92aEC-TG /Apoe-/- ) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe-/- mice. We tested the therapeutic effectiveness of the VCAM-1-targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling in vivo. VCAM-1-targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells in vitro and in vivo. The therapeutic effectiveness of anti-miR-92a therapy in treating atherosclerosis and stenosis in Apoe-/- mice is markedly enhanced by the VCAM-1-targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications in vivo.

Structure-Property Relationships of Oligonucleotide Polyelectrolyte Complex Micelles.

Polyelectrolyte complex micelles (PCMs), nanoparticles formed by electrostatic self-assembly of charged polymers with charged-neutral hydrophilic block copolymers, offer a potential solution to the challenging problem of delivering therapeutic nucleic acids into cells and organisms. Promising results have been reported in vitro and in animal models but basic structure-property relationships are largely lacking, and some reports have suggested that double-stranded nucleic acids cannot form PCMs due to their high bending rigidity. This letter reports a study of PCMs formed by DNA oligonucleotides of varied length and hybridization state and poly(l)lysine-poly(ethylene glycol) block copolymers with varying block lengths. We employ a multimodal characterization strategy combining small-angle X-ray scattering (SAXS), multiangle light scattering (MALS), and cryo-electron microscopy (cryo-TEM) to simultaneously probe the morphology and internal structure of the micelles. Over a wide range of parameters, we find that nanoparticle shape is controlled primarily by the hybridization state of the oligonucleotides with single-stranded oligonucleotides forming spheroidal micelles and double-stranded oligonucleotides forming wormlike micelles. The length of the charged block controls the radius of the nanoparticle, while oligonucleotide length appears to have little impact on either size or shape. At smaller length scales, we observe parallel packing of DNA helices inside the double-stranded nanoparticles, consistent with results from condensed genomic DNA. We also describe salt- and thermal-annealing protocols for preparing PCMs with high repeatability and low polydispersity. Together, these results provide a capability to rationally design PCMs with desired sizes and shapes that should greatly assist development of this promising delivery technology.

Engineering Peptide-Based Polyelectrolyte Complexes with Increased Hydrophobicity.

Polyelectrolyte complexation is a versatile platform for the design of self-assembled materials. Here we use rational design to create ionic hydrophobically-patterned peptides that allow us to precisely explore the role of hydrophobicity on electrostatic self-assembly. Polycations and polyanions were designed and synthesized with an alternating sequence of d- and l-chiral patterns of lysine or glutamic acid with either glycine, alanine or leucine due to their increasing hydrophobicity index, respectively. Two motifs were considered for the oppositely charged patterned peptides; one with equal residues of charged and uncharged amino acids and the other with increased charge density. Mass spectroscopy, circular dichroism, H- and F-NMR spectroscopy were used to characterize the polypeptides. Polyelectrolyte complexes (PECs) formed using the sequences were characterized using turbidity measurements, optical microscopy and infrared spectroscopy. Our results show that the critical salt concentration, a key measure of PEC stability, increased with both increasing charge density as well as hydrophobicity. Furthermore, by increasing the hydrophobicity, the amount of PEC formed increased with temperature, contrary to purely ionic PECs. Lastly, we assessed the encapsulation behavior of these materials using a hydrophobic dye. Concluding that encapsulation efficiency increased with hydrophobic content of the complexes providing insight for future work on the application of these materials for drug delivery.

Oligonucleotide-Peptide Complexes: Phase Control by Hybridization.

When oppositely charged polymers are mixed, counterion release drives phase separation; understanding this process is a key unsolved problem in polymer science and biophysical chemistry, particularly for nucleic acids, polyanions whose biological functions are intimately related to their high charge density. In the cell, complexation by basic proteins condenses DNA into chromatin, and membraneless organelles formed by liquid-liquid phase separation of RNA and proteins perform vital functions and have been linked to disease. Electrostatic interactions are also the primary method used for assembly of nanoparticles to deliver therapeutic nucleic acids into cells. This work describes complexation experiments with oligonucleotides and cationic peptides spanning a wide range of polymer lengths, concentrations, and structures, including RNA and methylphosphonate backbones. We find that the phase of the complexes is controlled by the hybridization state of the nucleic acid, with double-stranded nucleic acids forming solid precipitates while single-stranded oligonucleotides form liquid coacervates, apparently due to their lower charge density. Adding salt "melts" precipitates into coacervates, and oligonucleotides in coacervates remain competent for sequence-specific hybridization and phase change, suggesting the possibility of environmentally responsive complexes and nanoparticles for therapeutic or sensing applications.

Self-assembly of α-helical polypeptides driven by complex coacervation.

Reported is the ability of α-helical polypeptides to self-assemble with oppositely-charged polypeptides to form liquid complexes while maintaining their α-helical secondary structure. Coupling the α-helical polypeptide to a neutral, hydrophilic polymer and subsequent complexation enables the formation of nanoscale coacervate-core micelles. While previous reports on polypeptide complexation demonstrated a critical dependence of the nature of the complex (liquid versus solid) on chirality, the α-helical structure of the positively charged polypeptide prevents the formation of ß-sheets, which would otherwise drive the assembly into a solid state, thereby, enabling coacervate formation between two chiral components. The higher charge density of the assembly, a result of the folding of the α-helical polypeptide, provides enhanced resistance to salts known to inhibit polypeptide complexation. The unique combination of properties of these materials can enhance the known potential of fluid polypeptide complexes for delivery of biologically relevant molecules.

Deciphering the Role of π-Interactions in Polyelectrolyte Complexes Using Rationally Designed Peptides.

Electrostatic interactions, and specifically π-interactions play a significant role in the liquid-liquid phase separation of proteins and formation of membraneless organelles/or biological condensates. Sequence patterning of peptides allows creating protein-like structures and controlling the chemistry and interactions of the mimetic molecules. A library of oppositely charged polypeptides was designed and synthesized to investigate the role of π-interactions on phase separation and secondary structures of polyelectrolyte complexes. Phenylalanine was chosen as the π-containing residue and was used together with lysine or glutamic acid in the design of positively or negatively charged sequences. The effect of charge density and also the substitution of fluorine on the phenylalanine ring, known to disrupt π-interactions, were investigated. Characterization analysis using MALDI-TOF mass spectroscopy, H NMR, and circular dichroism (CD) confirmed the molecular structure and chiral pattern of peptide sequences. Despite an alternating sequence of chirality previously shown to promote liquid-liquid phase separation, complexes appeared as solid precipitates, suggesting strong interactions between the sequence pairs. The secondary structures of sequence pairs showed the formation of hydrogen-bonded structures with a ß-sheet signal in FTIR spectroscopy. The presence of fluorine decreased hydrogen bonding due to its inhibitory effect on π-interactions. π-interactions resulted in enhanced stability of complexes against salt, and higher critical salt concentrations for complexes with more π-containing amino acids. Furthermore, UV-vis spectroscopy showed that sequences containing π-interactions and increased charge density encapsulated a small charged molecule with π-bonds with high efficiency. These findings highlight the interplay between ionic, hydrophobic, hydrogen bonding, and π-interactions in polyelectrolyte complex formation and enhance our understanding of phase separation phenomena in protein-like structures.

Self-assembly of rationally designed peptides under two-dimensional confinement.

The rational design of interfacially confined biomolecules offers a unique opportunity to explore the cooperative relationship among self-assembly, nucleation, and growth processes. This article highlights the role of electrostatics in the self-assembly of ß-sheet-forming peptides at the air-water interface. We characterize the phase behavior of a periodically sequenced sheet-forming peptide by using Langmuir techniques, Brewster angle microscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. We find that peptides with an alternating binary sequence transition at high pressures from discrete circular domains to fibrous domains. The qualitative behavior is independent of surface pressure but dependent on molecular areas. In addition, thermodynamic models are employed to specifically quantify differences in electrostatics by obtaining parameters for the critical aggregation area, the limiting molecular area, and the dimensionless ratio of line tension/dipole density. Using these parameters, we are able to relate localized charge distribution to phase transitions, which will allow us to apply these molecules to examine how the dynamics of self-assembly can be directly coupled to the formation of composite nanostructures in biology.

Structural transitions and encapsulation selectivity of thermoresponsive polyelectrolyte complex micelles.

Polyelectrolyte complex (PEC) micelles are formed by mixing a block copolymer composed of a neutral block and a charged block, with an oppositely charged polymer. The micelles formed have a PEC core, capable of encapsulating charged molecules like nucleic acids or proteins, while the neutral block(s) forms the corona that offers protection that can prevent cargo from being degraded under physiological conditions. This work explores using a thermosensitive polymer, poly(N-isopropyl acrylamide) (pNIPAM), as the primary corona-forming block and how that can be leveraged in the context of drug delivery. pNIPAM has a lower critical solubility temperature (LCST), above which a hydrophilic to hydrophobic transition occurs. We are characterizing micelles formed using a diblock copolymer of pNIPAM-b-poly(acrylic acid) with (1) poly(lysine) and (2) poly(ethylene glycol)-b-poly(lysine) using dynamic light scattering, small angle X-ray scattering, absorbance and fluorescence spectroscopy, and transmission electron microscopy. Our results indicate that mixing pNIPAM-b-poly(acrylic acid) and poly(lysine) creates worm-like micelles, while mixing the same diblock with poly(ethylene glycol)-b-poly(lysine) forms spherical micelles. At temperatures above 35 °C, the transition temperature of pNIPAM-b-poly(acrylic acid), the worm-like micelles lose their structure, while the spherical micelles retain their structure, but both aggregate into larger assemblies. Lastly, we evaluate the ability of these micelles to encapsulate and release model charged therapeutics, using a cationic monoion, methylene blue and a cationic polyion, polylysine conjugated rhodamine. We find that methylene blue is not encapsulated by the micelle, and instead exhibits counterion-like behaviour upon polyelectrolyte complex formation. Conversely, fluorescence measurements of rhodamine-labelled polylysine show fluorescence quenching indicating that the polyion is encapsulated. By mixing quenched and unlabelled micelles, we show that both systems are immune to molecular exchange with their environment and instead the cargo remains trapped in the quenched micellar cores. Measurement of the micelle fluorescence above the LCST, decreases indicating no substantial release for either system. However, the increase in micelle quenching above the LCST and its persistence after cooling may offer an additional protective environment for cargo that can be triggered by temperature.

Bulk and nanoscale polypeptide based polyelectrolyte complexes.

Polyelectrolyte complexes (PECs) formed using polypeptides have great potential for developing new self-assembled materials, in particular for the development of drug and gene delivery vehicles. This review discusses the latest advancements in PECs formed using polypeptides as the polyanion and/or the polycation in both polyelectrolyte complexes that form bulk materials and block copolymer complexes that form nanoscale assemblies such as PEC micelles and other self-assembled structures. We highlight the importance of secondary structure formation between homogeneous polypeptide complexes, which, unlike PECs formed using other polymers, introduces additional intermolecular interactions in the form of hydrogen bonding, which may influence precipitation over coacervation. However, we still include heterogeneous complexes consisting of polypeptides and other polymers such as nucleic acids, sugars, and other synthetic polyelectrolytes. Special attention is given to complexes formed using nucleic acids as polyanions and polypeptides as polycations and their potential for delivery applications.

Chirality-selected phase behaviour in ionic polypeptide complexes.

Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a ß-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

Inhibition of atherosclerosis-promoting microRNAs via targeted polyelectrolyte complex micelles.

Polyelectrolyte complex micelles have great potential as gene delivery vehicles because of their ability to encapsulate charged nucleic acids forming a core by neutralizing their charge, while simultaneously protecting the nucleic acids from non-specific interactions and enzymatic degradation. Furthermore, to enhance specificity and transfection efficiency, polyelectrolyte complex micelles can be modified to include targeting capabilities. Here, we describe the design of targeted polyelectrolyte complex micelles containing inhibitors against dys-regulated microRNAs (miRNAs) that promote atherosclerosis, a leading cause of human mortality and morbidity. Inhibition of dys-regulated miRNAs in diseased cells associated with atherosclerosis has resulted in therapeutic efficacy in animal models and has been proposed to treat human diseases. However, the non-specific targeting of microRNA inhibitors via systemic delivery has remained an issue that may cause unwanted side effects. For this reason, we incorporated two different peptide sequences to our miRNA inhibitor containing polyelectrolyte complex micelles. One of the peptides (Arginine-Glutamic Acid-Lysine-Alanine or REKA) was used in another micellar system that demonstrated lesion-specific targeting in a mouse model of atherosclerosis. The other peptide (Valine-Histidine-Proline-Lysine-Glutamine-Histidine-Arginine or VHPKQHR) was identified via phage display and targets vascular endothelial cells through the vascular cell adhesion molecule-1 (VCAM-1). In this study we have tested the in vitro efficacy and efficiency of lesion- and cell-specific delivery of microRNA inhibitors to the cells associated with atherosclerotic lesions via peptide-targeted polyelectrolyte complex micelles. Our results show that REKA-containing micelles (fibrin-targeting) and VHPKQHR-containing micelles (VCAM-1 targeting) can be used to carry and deliver microRNA inhibitors into macrophages and human endothelial cells, respectively. Additionally, the functionality of miRNA inhibitors in cells was demonstrated by analyzing miRNA expression as well as the expression or the biological function of its downstream target protein. Our study provides the first demonstration of targeting dys-regulated miRNAs in atherosclerosis using targeted polyelectrolyte complex micelles and holds promising potential for translational applications.

Interfacial Tension of Polyelectrolyte Complex Coacervate Phases.

We consider polyelectrolyte solutions which, under suitable conditions, phase separate into a liquid-like coacervate phase and a coexisting supernatant phase that exhibit an extremely low interfacial tension. Such interfacial tension provides the basis for most coacervate-based applications, but little is known about it, including its dependence on molecular weight, charge density, and salt concentration. By combining a Debye-Hückel treatment for electrostatic interactions with the Cahn-Hilliard theory, we derive explicit expressions for this interfacial tension. In the absence of added salts, we find that the interfacial tension scales as N-3/2(η/ηc-1)3/2 near the critical point of the demixing transition, and that it scales as η1/2 far away from it, where N is the chain length and η measures the electrostatic interaction strength as a function of temperature, dielectric constant, and charge density of the polyelectrolytes. For the case with added salts, we find that the interfacial tension scales with the salt concentration ψ as N-1/4(1-ψ/ψc)3/2 near the critical salt concentration ψc. Our predictions are shown to be in quantitative agreement with experiments and provide a means to design new materials based on polyelectrolyte complexation.